production of caproate from undiluted thin stillage · [type here] - 3 - abstract the search for...

PRODUCTION OF CAPROATE FROM

UNDILUTED THIN STILLAGE

Aantal woorden: 22.692

Evert Vincent Stamnummer: 01070017

Promotor: Prof. dr. Leen De Gelder

Copromotor: Prof. dr. Korneel Rabaey

Tutors: José Arroyo, Stephen J. Andersen

Masterproef voorgelegd voor het behalen van de graad master in de richting Master of Science in de

industriële wetenschappen: biochemie

Academiejaar: 2016 – 2017

[Type here]

- 1 -

[Type here]

- 2 -

Acknowledgements

I would like to take this time to express my utmost and most deepfelt gratitude to my tutors; José Arroyo

and Stephen J. Andersen, my promotors Leen De Gelder and Korneel Rabaey, and all the other

wonderful people, the masterstudents, the PhD students, the postdoctorates and the professors of Cmet,

formerly known as Labmet who have made the past year, one of the best years in my life, and certainly

the best year of my academic record.

To my tutors I give an apology, for the amount of work and energy that was invested in me was

disproportionate to what I was able to give back. I cannot pay you back for what you have done, but I

do not forget the debts I owe. I am 95% sure, that if I had ended up with another thesis, with another

tutor, at another place, I doubt that the work I would be able to achieve would be even half of what I

managed to do this past year.

To my fellow masterstudents, to those who could laugh with everything and everyone, who played

pranks with and on me, and got pranked in return, I will miss those days we worked together side by

side.

To the PhD students and postdoctorates, I thank you for keeping me sane, grounded and telling me I

would succeed, even when I was convinced I would never make it to the end.

I would like to express special thanks to my family and loved ones, who did their best to make me

comfortable during sleepless nights, who believed in me, and spent so much effort in order to help me

get where I am today.

Thank you, Thank you, and thank you yet again,

Evert Vincent

[Type here]

- 3 -

Abstract

The search for green and ecological alternatives to products of petrochemical origins and

products with a large carbon footprint has been thriving for the past few decades, and is still

ongoing, more and more with every day that passes. Stillage, a side-stream from bio refineries

and distilleries is not used as a waste. Through an energy-intensive process it is converted into

a small amount of feed, suitable for ruminants and other livestock. The carboxylate platform,

which forms the basis of this thesis, was formed after the realization that biomass, for example

thin stillage, can be fermented into bio-fuels, chemical building blocks and medicinal

components. Through a process known as chain elongation, substrates such as carbohydrates,

ethanol and lactate, to name a few, can be converted to MCFAs. There are still many barriers

before this technology spreads around the world.

Through a number of experiments, from fed-batch tests to simple batch tests and continuous

fermentation tests, several areas of investigation were explored and studied, from the effect of

hydrogen partial pressure to the influence of hydrolyzation products to in-line extraction. It was

discovered that the presence of solids in fermentation medium lowered ethanol consumption

rates by a factor of five, whilst the solid-free broth of an anaerobic fermentor fed with thin

stillage for the production of caproate almost completely inhibited ethanol consumption. It was

observed that even though at lower pH, as claimed by others, MCFA production is greater, there

is no net-biomass growth in these experiments at a pH of 5.5 or below. Hydrogen partial

pressure was found to inhibit the formation of odd-chain VFAs, whilst simultaneously pushing

chain elongation to the production of MCFAs. And last but not least, there was confirmation

that MCFA production was possible when using thin stillage which did not contain solids.

[Type here]

- 4 -

Table of Contents Introduction ............................................................................................................................................. 1

Biomass & forms of biological waste ................................................................................................. 1

Biomass side stream: Stillage .......................................................................................................... 2

Carboxylate platform ....................................................................................................................... 4

Undefined mixed cultures................................................................................................................ 4

Production of SCCAs (C2-C5) ........................................................................................................ 5

Production of MCFAs (C6-C12) ..................................................................................................... 6

Extracting MCFAs ........................................................................................................................... 10

Precipitation ................................................................................................................................... 10

Membrane distillation .................................................................................................................... 11

Liquid/liquid separation................................................................................................................. 11

Electrochemical separation ............................................................................................................ 11

Adsorption ..................................................................................................................................... 12

Context of this thesis ......................................................................................................................... 13

Challenges and hypothesis’s: ........................................................................................................ 13

Materials and methods ........................................................................................................................... 15

Influence of PH2 on ethanol consumption and MCFA production ................................................... 15

Inhibition of ethanol by the feed matrix ............................................................................................ 16

Continuous fermentation of solid-free undiluted thin stillage ........................................................... 17

Analytical methods ............................................................................................................................ 18

Calculations ................................................................................................................................... 19

Results ................................................................................................................................................... 20

Influence of PH2 on MCFA production and ethanol consumption ................................................... 20

Inhibition of ethanol oxidation by the feed matrix ............................................................................ 24

Continuous fermentation of solid free undiluted thin stillage ........................................................... 34

Phase 1: Feeding solid-free undiluted thin stillage ........................................................................ 34

Phase 2: sludge retention ............................................................................................................... 36

Phase 3: Direct electrochemical extraction ................................................................................... 41

Discussion ............................................................................................................................................. 46

Influence of PH2 on MCFA production and ethanol consumption ................................................... 46

Inhibition of ethanol oxidation by the feed matrix ............................................................................ 47

Continuous fermentation of solid-free thin stillage ........................................................................... 49

Phase 1: feeding solid-free undiluted thin stillage......................................................................... 49

Phase 2: Sludge retention .............................................................................................................. 49

Phase 3: Direct electrochemical extraction ................................................................................... 50

Bibliography .......................................................................................................................................... 52

[Type here]

- 5 -

List of Figures

Figure 1: metabolic pathway for chain elongation of acetate to caproate through ethanol [65] ............. 7

Figure 2: metabolic pathway for chain elongation of acetate to caproate through ethanol [65] ............. 8

Figure 3: Daily maximum hydrogen partial pressure in reactors .......................................................... 20

Figure 4: Concentrations of even-chain RBO products and lactate ...................................................... 21

Figure 5: Product distribution (%-COD) for all reactors ...................................................................... 22

Figure 6: Ethanol production rates (gCOD/L.d) for the four reactors ................................................... 23

Figure 7: average ethanol concentrations (gCOD/L) over time for all series ....................................... 24

Figure 8: maximum ethanol consumption rates (gCOD/L.d) for all series ........................................... 25

Figure 9: VFA concentrations (gCOD/L) shown for all series ............................................................. 26

Figure 10: initial acetate and propionate production rates for all series ............................................... 27

Figure 11: Maximum production and consumption rates after lag phase (gCOD/L.d) for all series .... 28

Figure 12: net consumption/production of substrates and products in gCOD/L ................................... 28

Figure 13: Average ethanol concentrations in each series (gCOD/L) ................................................... 29

Figure 14: Maximum consumption rates of ethanol (gCOD/L.d) in each of the series ........................ 30

Figure 15: Average VFA concentrations (gCOD/L) in each of the batch series for the duration of the

experiment ............................................................................................................................................. 30

Figure 16: Average initial production rates (gCOD/L.d) for acetate and propionate in each of the series

............................................................................................................................................................... 31

Figure 17: Average maximum consumption and production rates of the various VFAs and ethanol ... 32

Figure 18: Net consumption/production of the various VFAs and ethanol (gCOD/L) ........................ 33

Figure 19: Feed composition during Phase 1 ........................................................................................ 34

Figure 19: Even-chained RBO substrates and products concentrations in both reactors ...................... 35

Figure 20: Average Concentrations (gCOD/L) and production rates (gCOD/L.d) for both reactors

during days 20-28 .................................................................................................................................. 35

Figure 21: Concentration of suspended solids (gTSS/L) in both reactors ............................................. 36

Figure 22: Composition of the feed during phase 2 .............................................................................. 37

Figure 23: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates

(gCOD/L.d) in R1.................................................................................................................................. 38


(gCOD/L.d) in R2.................................................................................................................................. 38

Figure 25: Concentraction of soluble solids (gTSS/L) in both reactors during phase 2 ........................ 39

Figure 26: Concentrations (gCOD/L) of lactate, glyerol, 1,3 PDO and ethanol in the feed, R1 and R2

during phase 2. ...................................................................................................................................... 40

Figure 27: Composition of feed during phase 3 (gCOD/L) ................................................................... 41


(gCOD/L.d) in R1.................................................................................................................................. 42

Figure 29:Concentration of undissociated and dissociated even-chain VFAs and lactate in the middle

compartment .......................................................................................................................................... 43


(gCOD/L.d) in R2.................................................................................................................................. 44


during phase 3 ....................................................................................................................................... 45

List of Tables Table 1: COD and BOD values of whole stillage from various sources and feed stocks [15, 16] .......... 3

Table 2: Composition of the different waste streams derived from stillage [17] .................................... 3

[Type here]

- 6 -

Table 3: thermodynamic values and reaction stoichiometry for various SCCAs through multiple

pathways [49, 53, 54] .............................................................................................................................. 5

Table 4: thermodynamic balances for caproate formation through ethanol [37, 47] .............................. 8

Table 5: thermodynamic balances for caproate formation through lactate [65] ...................................... 9

Table 6: Theoretical dissociation concentrations of various VFAs at different pH values ................... 10

Table 7: Compositions of the various feed batches used in: Influence of PH2 on MCFA production and

ethanol consumption .............................................................................................................................. 16

Table 8: Composition of fermentation media of the various series used in: Inhibition of ethanol

oxidation by the feed matrix: primary run ............................................................................................. 16

Table 9: Composition of fermentation media of the various series used in: Inhibition of ethanol

oxidation by the feed matrix .................................................................................................................. 17

Table 10: Composition of SM used for inoculum preparation and fermentation medium in: Inhibition

of ethanol oxidation by the feed matrix ................................................................................................. 17

Table 11: SL-10 composition (1L) used for SM medium ..................................................................... 17

Table 12: 7vit (10x) composition (1L) used for SM medium ............................................................... 17

Table 13: overview of the timeline of experiment 4. Phase 1: days 1-30, Phase 2: days 30-60, Phase 3:

days 60-end ........................................................................................................................................... 18

Table 14: Average concentrations (gCOD/L) for both reactors during days 20-28 .............................. 35

[Type here]

- 7 -

List of acronyms

A.D. Anaerobic

Digestion

BES of 2-bromoethanesulphonate

BOD Biological Oxygen

Demand

COD Chemical Oxygen Demand

C.R. Complete Retention

CSTR Continuously Stirred Tank Reactor

DDG Dried Distillers’ Grain

DDGS Dried Distillers’ Grain with Solubles

EC Enrichment Culture (inoculum)

HI High hydrogen partial pressure

LCFA Long Chain Fatty Acid

LO Low hydrogen partial pressure

MCFA Medium Chain Fatty Acid

Pi Pilot culture (inoculum)

RBO Reversed-β-Oxidation

SCCA Short Chain Carboxylic Acid

SM Synthetic Medium

SRT Sludge Retention Time

TSS Total Suspended Solids

VFA Volatile Fatty Acid

VLCFA Very Long Chain Fatty Acid

X%S medium with Solids

1

Introduction Each year, over 70000 square kilometers of forest are cut down in order to fuel the global farming economy,

heating and cooking, the paper and the wood industry. In 2015, 7,861 million tons of coal were produced,

and by 2030, the global consumption of coal is estimated to increase to 9.05 billion tons [1]). In Belgium, in

2011, almost 185 million m³ of water were used by various industries, and usage is projected to grow

[2].Global consumption increases in all areas, whether we are speaking about food, land, water, energy or

fuels. Day after day, the needs of the global community increase, and the lifespan of our natural resources

decreases. In order to address this issue, increasingly worldwide, countries have started pushing towards

more green alternatives, such as renewable energy, bio-fuels, and recycling. Over the years, these adaptations

have increased in quantity, but the world is still lacking in ability to close the cycle and be able to recover as

much from waste as is necessary for consumption. The drive for recovery of resources from waste includes

(focuses on?) the fact that there is a rapid depletion of sources of minerals, some estimated to be fully depleted

within the next 50 years [3], the need to provide clean water for drinking and domestic use, and many more.

Now, research on resource recovery from waste is being done across the world, not only for reducing

pollution, but because advances in science and technology now have changed waste management from being

a cost, to being a potential additional source of income for those industries who produce a waste stream which

is not being used to the utmost of its capabilities. Companies are now actively searching and funding research

for the technology and application of product recovery, whether it is stripping spare circuit boards for gold,

silver and copper [4], harvesting biogas from organic waste [5], or filtering proteins out of waste water from

the food and drink industry [6].

The valorization of a resource is defined as the increase of value of a substance or product through artificial

means. Of the many valorizing techniques used in the world, perhaps the resource with the widest arrange of

applications is biomass. Valorization of biomass does not follow the same line of thought as the treatment of

biomass, even though these two often go hand in hand. The largest difference between the two is the

difference in economic value. Treatment is a cost which is a necessity, whilst valorization is a source of

profit. When talking about the valorization of biomass, one typically speaks about valorizing a biomass waste

stream, i.e. a byproduct of an industrial process in which bio-based goods are made or processed. Such

streams which are readily valorized include, amongst others, whey to produce proteins and lactate, sludge

from wastewater treatment plants for biogas, cellulose and chitin-rich biomass for bio-polymers [7]. One of

the key factors that plays a driving role in developing more bio-based valorization is the need to reduce

fossil fuel usage, due to the costly nature of fossil fuels, the non-renewable aspect of it, as well as the

environmental impact.

Biomass & forms of biological waste Biomass is defined as the sum total of organic fuel derived from living or deceased organisms[8]. Industries

that produce raw biomass are typically logging industries and agricultural industries, the latter which can be

sub-divided into different branches, such as industries for the production of food, feed, textiles and oils.

Processed biomass is most often in the form of food or drink, but also in the form of furniture, bio-fuels,

bioplastics, and more recently, a whole range of bio-based chemicals. And then there is the biomass waste,

which is produced by the industries which produce or process raw biomass. Biomass waste has as many

different forms as there are companies in the various branches of the bio-based world. It can range from

specific aqueous streams, such as whey from the cheese industry, stillage from bio-refineries, sludge from

2

treatment plants and sawdust from the paper industry. Each of these different streams have different qualities

and properties, which largely determine what can be done with them.

Municipal solid waste (MSW) is a complex type of waste, produced by individuals, families, governments

and public institutions, as well as commercial industries. MSW contains, amongst other things, food wastes,

paper, wood plastics, glass and metal [9]. MSW in the United States consists of 29.6% organics, 35.5% paper,

13.2% plastics, and an assortment of other, nonorganic wastes. On average, the potential for MSW with this

composition to produce methane was measured to be at 78.2m³ per ton [10]. In the end, apart from sorting

MSW into the different components and recycling, the tendency remains to simply landfill the organic

fractions, and harvest the methane produced during the anaerobic fermentation that spontaneously occurs

within said landfills [11].

Another biomass waste stream is agricultural waste. The waste streams from agriculture have a tendency to

be completely used as either animal, fertilizer, or other direct applications. Because of the organic nature of

this waste stream, no or little treatment is needed prior to use in the various areas. However, to say every bit

of the waste stream is used in such manners would be false. In fact, there are many parts to the agricultural

waste streams that are not reused. In 1996 in the Netherlands, more than 73% of agricultural wastes did not

find its way to recycling [12].

Wood waste streams, which are mainly produced as the results of commercial forestry, pruning of public

parks, or industrial wood processing. In 2012, 52.9 million tons of wood were treated in the EU. 46% of this

waste stream was incinerated with recovery of energy, whilst 51% was recycled [12]. The remainder is either

landfilled, or incinerated without energy recovery [13].

Sludges and liquid waste, derived from waste treatment, are mainly landfilled (over 50%) or recovered in

other ways (42.6%). Typical sludge recovery consists of elemental recovery, such as of phosphor (0.5-0.7%

total solids), nitrogen (2.5-5% total solids) and carbon, the latter being extracted in the form of energy through

incineration or more commonly, anaerobic digestion. Other than elemental recovery, sludge can also be used

to produce organic molecules such as polyhydroxyalkanoates, proteins, enzymes and volatile fatty acids

(VFAs). Although 50% of sludge produced in Europe is landfilled, this mainly occurs in countries where

sludge is treated as waste. In other countries, such as Belgium and the UK, sludge is more often seen as a

valuable resource, due to its nutritional value [14].

Biomass side stream: Stillage

Stillage is an organic side stream produced as a by-product by the distillation of fermented grains from both

the bio-ethanol industry as well as the food and drink industry. Stillage is produced regardless of which

substrate is fermented, whether it is rice, wheat, corn or sugar crops such as beet. When the substrates, after

the starch is broken down into small saccharides by enzymes, are fermented, the resulting fermentation beer

is then distilled. Thus, the fermentation beer is split into two different fractions; a fraction with a high ethanol

concentration (the desired product), and the stillage. As a result, the stillage is mainly composed of leftover

saccharides, husks and other unused components. A rough estimate indicates that up to 20L of stillage may

be produced for every liter of ethanol produced through distillation [15]. The fact that produced stillage can

have chemical oxygen demand (COD) concentrations as high as 100g/L means that this side stream must

undergo a strenuous treatment to reduce its impact on the environment in the case of disposal or reuse in

other sectors. However, the actual composition of stillage differs greatly depending on the feedstock, the

3

process used and the final product. In table 1 a representation of the biological oxygen demand (BOD) and

COD of a few sources of stillage is shown.

Component Brewery

Guinness

Brewery

fermencam

Winery

Sofavinc

Corn

distillery

Beet

fermentation

Beet

molasses

COD (mg/L) 1118 26907.33 457.67 56000 65000 91100

BOD (mg/L) 337.67 25044.67 40 37000 38000 44900

Table 1: COD and BOD values of whole stillage from various sources and feed stocks [15, 16]

The first and most influential way in which the BOD/COD can be decreased from the stream, is by

centrifuging the whole stillage into two fractions called thin stillage (aqueous), and wet distillers’ grain

(WDG). Thin stillage can then undergo evaporation after which it becomes known as syrup. Both WDG and

syrup are used in animal feed, separate or together, in which case they combine to form dried distillers grain

with solubles (DDGS). DDGS is seen as a high-quality feedstock for animals, and is heavily scrutinized in

terms of nutritional value, digestibility and other important characteristics. In table 2, a summary is shown

of some of the average characteristics of the three fractions which are obtained from whole stillage.

DDGS (g/L) WDG (g/L) Thin stillage (g/L)

Dry mass 888 353 77

Soluble components 219 31

Hydrophobic

components

103 34

Proteins 221 129 1

Carbohydrates (starch) 308 (46) 139 (21) 2 (0.4)

Glucose 0.7

Lactic acid 13

Glycerol 11

Ethanol 0.5

Table 2: Composition of the different waste streams derived from stillage [17]

From table 2 it is possible to see that the composition of these three side products is very different, and due

to these inherent differences, different fields of application are open for further processing of these streams.

Starting from whole stillage, studies have been made to use this as feed for the production of lactate, after

which the remnants could be valorized to the form of animal feed [18, 19] or for the production of biogas by

manner of anaerobic digestion (A.D.) [20]. WDG only sees use as an animal feed[21]. DDGS, like WDG, is

mostly used for animal feed, and depending on its source it is supposedly better for some animals than others

[22, 23]. The difference is that there also exist novel adaptations of DDGS, more specifically for the

production of various solvents such as butanol and acetone [24], and more interesting approaches such as

reusing DDGS for a second fermentation cycle to produce more ethanol [25]. For whole stillage, DDGS and

WDG, before any fermentation for the production of new chemicals or products can take place, there is an

intense need to hydrolyze the leftover carbohydrates into a fermentable state, in the form of physical,

chemical or enzymatic processing, which adds to the cost of production. In the end, it is the thin stillage that

has the widest field of applications in industry. These applications include the more mundane such as

production of biogas [26, 27] or recovery of energy, but a large spread of uses based on the fact that lactate

4

and glycerol can be produced in thin stillage. Glycerol and lactic acid can be produced in quantities up to

47g/L of glycerol and 4g/L of lactic acid through repeated ethanol fermentation [28], although more

optimized fermentation processes can produce lactic acid at concentrations of above 42g/L [29]. A simple

method of extraction for example is to harvest the lactic acid and glycerol through filtration [30], but by using

the present components, being lactate and glycerol, amongst others, as substrate for fermentation, it is

possible to produce a wide array of economically interesting products including but not limited to, ethanol,

single cell oils and general chemicals [31]. For example, the glycerol present in thin stillage can easily be

fermented to butanol, with higher production rates compared to using synthetic media [32, 33]. It has also

been shown that by using genetically engineered strains of bacteria, it is possible to convert the vast amount

of glycerol present into more ethanol, thereby increasing the yield [34]. In another study, G. lucidum, a

fungus, was used to convert the soluble and remaining substrates in thin stillage into easily harvestable

polysaccharides[35]. From these examples, it can be shown that thin stillage is an excellent substrate

provider for many different fermentation processes, and it is for this reason that thin stillage was the waste

stream chosen to produce the desired products of this study.

Carboxylate platform

The carboxylate platform is the term given to mixed-culture fermentation of biomass where the desirable end

products are carboxylates, which can be further modified to bio-fuels [36]. Currently, success has been made

in producing carboxylates with a 2-8carbon chain [37] from waste streams, although further accomplishments

in the field could allow for the production of carboxylates with longer chain lengths. The carboxylate platform

is still in development, but could eventually reach the same degree of importance as the sugar platform and

the syngas platform. The carboxylate platform is a process identical to anaerobic digestion, in which biomass

is anaerobically fermented to a methane/carbon dioxide mixture, with the aim being to produce as much

methane as possible. The difference is that the carboxylate platform does not aim for the production of

methane, but cuts off at the previous step, the acetogenesis step. From biomass waste, the complex polymers

are first hydrolyzed, producing monomers. After this come the acidogenesis and acetogenesis steps, where

carbohydrates, proteins and lipids function as electron donors, and internal molecules serve the purpose as

electron acceptors, with the electron-accepting molecules, the carboxylates, being the desirable end-product

of the carboxylate platform. Carboxylates are molecules with at least one COO- tail, and although there are

many different substances which contain one or more carboxyl groups, the carboxylate platforms focus on

the simple straight chain molecules whose main defining characteristic is the carboxyl group. The length of

the carbon chains of all carboxylates vary from 2 (acetate) to 26 (hexacosanoic acid). The collection of

carboxylic acids with a carbon chain length of 2-5 are referred to as short chain carboxylic acids (SCCA), a

carbon chain length of 6-12 is called a medium chain fatty acid (MCFA), and for longer carbon chains the

terms can be long chain fatty acids (LCFA) or very long chain fatty acids (VLCFA). All of these molecules

can be produced by organisms, although more complex organisms are (currently) required to produce LCFAs

and VLCFAs [38].

Undefined mixed cultures

A major advantage of the carboxylate platform is the possibility of applying undefined mixed cultures. This

is in stark contrast to for example the sugar platform, which requires sterilized feed substrate, and

fermentation reactors with pure cultures. The various advantages that mixed cultures bring with them are

plentiful. For one, it removes the need for sterilization, which saves time, energy and capital. This also

includes the feed, which can be raw, non-heat-treated materials, except in the case that pretreatment is

required to hydrolyze the larger and more stable compounds into fermentable monomers. Then there is also

5

the ability to utilize impure streams, such as waste. Mixed cultures are more stable in response to shifts in

organic loading rate, composition of feed or other such shifts in parameters.. Furthermore, the efficiency with

which the available organic matter is converted to useful products is greater in the carboxylate platform when

compared to the sugar platform, or indeed, compared to using pure cultures due to a reduction of the amount

of oxidation to gaseous substances which occurs in the aerobic sugar platform [39], and due mixed cultures

suffer less from inhibitory components in mixed streams, which means a greater organic matter conversion

efficiency when fermenting mixed feed streams [40].

Production of SCCAs (C2-C5)

SCCAs are commonly produced products, with applications ranging from preservatives [41], production of

industrial polymers such as polyvinyl alcohol [42] and cellulose acetate butyrate [43] and pharmaceuticals

such as betamethasone valerate [44]. Typically, acetate and propionate are produced through chemical

means [45], but can also be produced by fermentation of sugars [46] or glycerol [47]. SCCAs are water

soluble components, with no upper limit on their solubility. Apart from the solubility, each SCCA has

another difficulty which prevents low cost extraction. Acetate, for example, is produced as a salt, in which

form it is difficult to separate from fermentation broth. Propionate has low added value when produced

through fermentation, where the used substrate can account for up to 50% of the product cost, which means

in order for an economically viable production, high yields of propionate are required. As for butyrate and

valerate, in order for down-stream processing techniques to have a lower financial impact, high titers of

product are required. However, due to the fact that acetate and propionate are produced as by-products

during the fermentation of butyrate and valerate respectively, it is difficult to produce high purity products

due to their similar nature. One way of overcoming this problem is through the use of artificial electron

carriers such as methyl viologen [45]. SCCAs can be produced both through anabolic (Table 3) and

catabolic metabolism, where in the case of the latter, carboxylic acids can be formed through diamide

pathway fragmentation of proteins after acid hydrolysis [48]. Examples of anabolic production of SCCAs

include the autotrophic production of acetate and propionate from biogas containing either a CO/H2

mixture [49, 50] or a CO2/CH4 mixture [51]. As for the larger SCCAs, table 3 shows that butyrate is

produced mostly by using other organic molecules as a substrate, mainly ethanol, lactate and acetate. One

study has shown that butyrate can also be produced using only CO2 as the carbon source with a microbial

electro synthesis technique [52].

Substrate Product ΔGr0 (kJ/mol)

Glucose + H2O Acetic acid + Ethanol + 2H2 + 2CO2

ethanol + H2O Acetic acid + H+ + 2H2 +10.11

lactic acid + H2O Acetic acid + 2H2 + CO2 28.51

4H2 + 2CO2 Acetic acid + H+ + 2H2O -86.78

4CO + 2H2O Acetic acid + 2CO2 -154.6

2CO + 2H2O Acetic acid -114.5

Acetic acid + H2O + CO2 + 3H2 Propionic acid + 3H2O -76.5

3HCO3- + 2H+ + 7H2 Propionic acid + 7H2O -181.1

Lactic acid + H2 Propionic acid + H2O -43.32

ethanol + acetate n-butyrate_ + H2O -40.34

2 acetate + H+ + 2H2 n-butyrate_ + 2H2O -47.55

lactic acid + acetic acid n-butyric acid + H2O + CO2 −57.52

Table 3: thermodynamic values and reaction stoichiometry for various SCCAs through multiple pathways

[49, 53, 54]

6

Production of MCFAs (C6-C12)

MCFAs are carboxylic acids where the aliphatic tail has between 6-12 carbons. The longer the tail is, the

lower the oxygen: carbon ratio is, which means that the hydrophobicity is increased, but also their usefulness

as fuels increases as shown in table 2. Currently, caproate (C6), caprylate (C8) and capric acid (C10) are

mainly produced from palm oil and other animal fats[53, 54]. This is inefficient, as can be seen from the

composition of palm oil (caproate: 0.2%, caprylate: 3.3%, capric acid: 3.5%) [55]. Caproate can, however be

extracted or produced through a few non-biological pathways, such as conversion of ethylene, and from

propanal or other hydrocarbons through oxidation [56]. However, due to the rise in petroleum costs, and the

environmental impact of petroleum, fermentation is rapidly becoming the preferred manner of production

over the petrochemical route. Caproate not only has potential as a possible bio-fuel, but it also has an

antimicrobial effect and is as such used as an antimicrobial agent [57, 58]. It is also used as a drug to lower

the chances of preterm delivery in pregnancy [59]and for the production of organic chemicals. But these are

not the main reasons why producing caproate is seen as a high-worth fermentation product. The crux of the

matter is that extraction of caproate from fermentation broth or other aqueous solutions is remarkably easy.

This is due to the low solubility of caproic acid (and caprylic acid). As a consequence, an oil phase separation

takes place when the concentration reaches 10.08g/L (0.79g/L for caprylic acid)[60]. This means that any

produced MCFAs do not need to be extracted by means of extensive downstream processing, but can be

extracted with simple procedures.

MCFAs are produced by micro-organisms under strictly anaerobic conditions as a sink for reducing

equivalents, which are formed when, for example, ethanol is oxidized to acetate for ATP production, where

the reducing equivalents would otherwise accumulate. The process by which medium chain carboxylates are

produced is the reverse-beta oxidation (RBO), also known as the chain elongation cycle. During this cycle,

a two carbon atom, acetyl CoA (reducing equivalent), is added to one of the SCCAs with each cycle. This

way, acetate is elongated to butyrate. Butyrate is elongated to caproate, and caproate to caprylate. If the

starting SCCA is propionate, then similarly, the elongated products will also have odd-numbered chains.

Because the production of even-chain elongation and odd-chain elongation depends mostly on the starting

substrate, the focus will be placed on the production of even-chained MCFAs, primarily due to the fact that,

when starting from sugars, more acetate is formed rather than propionate, where propionate is the starting

molecule for odd-chain RBO. Seeing as how thin stillage contains a large amount of sugars and

carbohydrates[61] as well as ethanol, thin stillage forms an almost ideal feed for even chained RBO.

However, acetate and ethanol can also be provided from an external source, produced by autotrophic

organisms, or obtained by the catabolism, hydrolyzation or oxidation[62] of larger molecules, as stated in

Production of SCCAs. An example of alternative RBO-substrate supply is when a reactor was fed with

hydrogen and carbon dioxide, and successfully produced MCFAs[63, 64]. The hydrogen and carbon dioxide

are fixated by homoacetogenic bacteria to form acetate, and then other acetogenic bacteria can further convert

the acetate to ethanol, using hydrogen as the electron donor. However, this type of setup is subpar compared

to simply feeding a reactor directly with ethanol. In terms of anabolic production of acetate, the pathway

taken varies depending on the type of substrate, and the thermodynamic and stoichiometric models have

already been represented in table 3. Below we will discuss the RBO of even chained MCFAs from ethanol,

lactate and acetate.

7

The figure below is a representation of the double cycle required to go from acetate to caproate using ethanol

as the source for reducing equivalents and energy.

Figure 1: metabolic pathway for chain elongation of acetate to caproate through ethanol [65]

From figure 1, it can be seen that the only place where ATP is produced is when ethanol, is oxidized to acetic

acid. This conversion produces 2 NADH molecules, which would normally accumulate and cause the

metabolism to enter dormancy. In order to continue growing and metabolizing, an extra 5 molecules of

ethanol are oxidized to acetyl CoA for every ATP molecule generated through oxidation of ethanol to acetate.

In order to stop these acetyl CoA molecules from accumulating, they either combine with only each other to

form butyrate, or combine with acetate to the same effect. During the conversion of crotonyl CoA to butyryl

CoA, a ferredoxin redox complex, the bifurcation system (not pictured above) allows for the regeneration of

NADH to NAD+, which also produces molecular hydrogen from protons. In this manner, the microorganisms

can sustain their metabolic activity by excreting their waste product, namely butyrate and the longer-chained

carboxylic acids [65].

Substrate Product ΔGr0 (kJ/mol) Repetitions Total ΔGr

0 (kJ/mol)

ATP generation Ethanol + H2O Acetate + H+ +

2H2

+10.11/+7.22 1 +10.11/+7.22

Cycle 1 Ethanol +

Acetate

n-Butyrate + H2O -38.62/-40.32 5 -193.10/-201.68

Cycle 2 Ethanol + n-

Butyrate

n-Caproate + H2O -38.74/-38.00 5 -193.70/-190.00

8

Overall caproate

formation

12 ethanol + 3

Acetate

5 n-caproate +

4H2 + 8H2O

-30.55 -183.59/-182.78

Table 4: thermodynamic balances for caproate formation through ethanol [37, 47]

The RBO cycle for production of caproate through lactate (Fig. 2) is very similar RBO through ethanol. The

two chief differences are for one, that for every lactic acid molecule converted to acetate, a CO2 molecule is

produced, resulting in a lower carbon efficiency. The other problem with using lactate as the source for

energy and reducing equivalents however, is the existing competing pathway, the acrylate pathway, through

which a shift from even-chained RBO to odd-chained RBO is caused [65, 66].

Figure 2: metabolic pathway for chain elongation of acetate to caproate through ethanol [65]

Substrate Product ΔGr0 (kJ/mol)

ATP generation Lactate + H2O Acetate + CO2 + 2H2 -8.79

Cycle 1 [65] Lactate + Acetate n-Butyrate + H2O + CO2 -37.52

9

Cycle 1 [36] 2 Lactate + H+ n-Butyrate + 2H2O -83.74

Cycle 2 Lactate + n-Butyrate n-Caproate + H2O + CO2 -57.65

Overall caproate formation 15 Lactate 5 n-caproate + 10H2 +

5H2O + 5CO2

-41.32

Table 5: thermodynamic balances for caproate formation through lactate [65]

From here, it can be seen that the chain elongation through ethanol is a thermodynamically more appropriate

pathway for the production of caproate when compared to the use of lactate, but this difference can be

attributed to the formation of a large amount of CO2, which implies a much lower carbon efficiency. It is also

not clear which conversions actually take place with lactate fermentation, seeing as how there is a higher

probability of odd-chained carboxylates being formed. As illustrated above, Cavalcante et al assume that

lactate and acetate are both required for butyrate production, whilst Zhu et al. [67] have shown that butyrate

production using only lactate is also feasible, and that the preferred substrate for chain elongation depends

on the inoculum.

As stated before, medium chain length carboxylic acids can only be produced by bacteria under strictly

anaerobic circumstances. This is due to the fact that, when oxygen is present, any present SCCAs and MCFAs

are consumed as an energy rich food source, and are oxidized to form CO2 and H2O. Not only does

fermentation with the intent of producing MCFAs require an anaerobic atmosphere, it is crucial for there to

be a minimal hydrogen partial pressure, PH2, of 10-2kPa. This minimal PH2 is required in order for the chain

elongation process to be thermodynamically feasible, more specifically, the bifurcation system which

regenerates NAD+ from NADH, which conserves energy and electrons[68, 69]. The PH2 also has an effect on

the metabolic pathways of homoacetogens and hydrogenotrophic methanogens. Homoacetogens use CO2 and

H2 present in the atmosphere to produce acetate, which can as a result be used by the chain elongating bacteria

to produce more MCFAs. To this end, the presence of homoacetogens is not a negative factor, providing that

they do not bring the hydrogen concentration to a level that is too low. The same goes for methanogens,

which also consume hydrogen, however, in contrast to homoacetogens, steps are taken in order to remove

methanogens from the fermentation culture. The reasoning behind the discrimination between the different

hydrogen consumers is the fact that amongst methanogens, there are acetoclastic methanogens which not

only consume hydrogen but also acetate. The consumption of acetate by acetoclastic methanogens would

cause the chain elongation to halt at the first step, which is why, in order to be able to produce MCFAs, a

method of inhibiting methanogens is needed. There are two widely used measures. The first is the use of 2-

bromoethanesulphonate (BES), an inhibitor with a greater selectivity for methanogenic bacteria when

compared to other inhibitors such as propionic acid or sodium nitrate which also inhibit the production of

MCFAs [70], and BES dosage is recommended to be used at concentrations of 0.5mM, for the inhibition of

methane producers [71]. However, to use BES at a large scale requires a large amount of BES, inflating the

cost of production, and although it is an inhibitor of methanogens, other bacteria are also influenced, which

would mean decreased production rates of MCFAs. The other option, which is cheaper and less intensive, is

to lower the pH of the reactor to a point where the growth of methanogens is inhibited. A pH of 6 is already

sufficient to completely limit methane production [72], but many studies that rely on pH to inhibit

methanogenic bacteria use lower pH values, ranging from 5 to 5.5 [73, 74].

10

Extracting MCFAs MCFAs have a low solubility. This particular quality means that evaporation, distillation, precipitation or

other energy-intensive extraction techniques normally reserved for organic molecules are not necessary,

unless the MCFAs have to be separated from other oily substances. The core of caproate extraction lies in

somehow increasing the concentration of undissociated caproic acid to the point at which phase separation

occurs, after which the oily layer can easily be harvested. The trouble lies in producing such a concentrated

stream when working with a biological production system, namely fermentation. Due to the long aliphatic

tail of MCFAs, these molecules can diffuse easily across the cytoplasmic membrane of cells. Once inside

they can interfere with cytoplasmic functioning [75]. The concentration of carboxylic acids needed to inhibit

growth and/or metabolism strongly depends on which carboxylic acid is taken into account, as well as the

organisms present in the fermentation culture. Microorganisms which produce MCFAs will naturally be able

to endure higher concentrations of the same MCFAs, but unless the fermentation is carried out in two stages

as opposed to single stage fermentation, presence of MCFAs will cause problems. In two stage fermentation,

with the first stage being a fermentor where sugars are oxidized to ethanol and acetate and the second being

the chain elongation fermentor, the non-MCFA producing bacteria are kept separate from the fermentor with

MCFA production. In a single-stage fermentation tank however, there is no separation of the primary

producers and the chain elongating bacteria. This means that there is a need to keep MCFA concentrations

as low as possible, so that production rates can be kept as high as possible. Undissociated butyric acid, for

example, can inhibit growth at levels of 47.9 mmol/l [76], whereas MCFAs such as caproate and caprylate

have a critical concentration of their undissociated forms at 10.5mmol/L and 0.62mmol/L [77]. At neutral or

high pH, this means that the concentration of caprylate in both dissociated and undissociated forms needs to

be higher than 78 mmol/L. However, if there is a need to work at pH values as low as 5, the maximum

allowable total concentration of caproate and caprylate is 25.3 and 1.4mmol/L (calculated using table 6)

respectively. To this end, an investigation of various extraction techniques will be discussed here.

pKa %Dissociated

form at pH 3

%Dissociated

form at pH 4

%Dissociated

form at pH 5

%Dissociated

form at pH 6

%Dissociated

form at pH 7

Acetate 4.756 1,72 14,92 63,69 94,61 99,43

Propionate 4.87 1,33 11,89 57,43 93,10 99,26

Butyrate 4.83 1,46 12,89 59,66 93,67 99,33

valerate 4.83 1,46 12,89 59,66 93,67 99,33

Caproate 4.85 1,39 12,38 58,55 93,39 99,30

Caprylate 4.89 1,27 11,41 56,30 92,80 99,23

Capric acid 4.9 1,24 11,18 55,73 92,64 99,21

Table 6: Theoretical dissociation concentrations of various VFAs at different pH values

Precipitation

Through the application of salts such as calcium carbonate or ammonium bicarbonate directly to the

fermentation tank, carboxylic acids in solution are converted to calcium- or ammonium carboxylate salts.

The reactor effluent can then be concentrated by means of dewatering processes to the point where crystals

of salt carboxylates are left behind [78]. However, this type of process is not an in-line extraction technique,

which means it would not serve to fulfill the goal of reducing MCFA concentrations inside the reactor in

order to limit product inhibition and toxicity.

11

Membrane distillation

Membrane distillation is a low-cost, low energy process in which fermenter liquid is heated up to 55C. On

the other side of the membrane, the permeate is kept cool, with the temperature gradient being the driving

force of MCFA diffusion. The end result of this membrane step is a mixture with up to 10% MCFA by

weight. This concentrated stream is then further processed using an extraction column, rectification and

water-stripping column. In the extraction column, solvents such as ethanol or methyl tert-butyl ether are

used, which can be recovered in a different down-stream process[78]. This type of process requires

multiple steps, which come at a cost and environmental impact. This process can be performed in-line with

a reactor, however, the recycle stream of the membrane separation step will be changed in some way, due

to having been heated up and cooled down again.

Liquid/liquid separation

This technique relies on the affinity that carboxylic acids have between two liquid media, the first being the

fermentation broth, and the other a solvent which has properties that would attract carboxylic acids across a

membrane, without the loss of water or other components. These liquids can include alcohols, ketones, ethers,

aliphatic hydrocarbons, organophosphates, and aliphatic amines [79].

A specific application of liquid/liquid separation is pertraction. A pertraction system consists of a

hydrophobic membrane, often in the form of a tube, through the inside of which which an extractant product

flows, whilst the feed consisting of fermentation broth flows around the membrane. The extractant product

used for extracting organic compounds is typically an oil based solvent, such as mineral oil [77, 80]. In these

studies, a second pertraction unit was applied to extract the MCFAs from the mineral oil to an alkaline

solution. Typically, once the alkaline solution has a high enough concentration of MCFAs, acid is added until

almost all of the dissolved caproate undergoes phase separation. The caproic acid can then be siphoned off.

Another method is by keeping the extractant alkaline, and allowing the MCFAs to precipitate as salts, which

can be recovered after filtration. In this type for dual pertraction system, the membrane which extracts the

desired component from the feed is called the forward module, and the membrane which extracts the

components from the first extractant product into another aqueous solution is called the backward module.

Whilst the first extraction phase relies on the hydrophobicity of MCFAs, the second is simply based on a

concentration gradient. In order to keep the gradient steep enough, the concentration in the alkaline solution

had to be kept low, which is achieved through continuous inline extraction using membrane electrolysis.

Electrochemical separation

There are two different kinds of electrochemical separation, with the first being membrane electrolysis, and

the other being electrodialysis.

Membrane electrolysis

Membrane electrolysis is an extraction where two liquids, namely the anolyte and catholyte, are separated by

a membrane, and depending on the type of membrane, only anions or cations can cross over. Anion exchange

membranes are used for the extraction of MCFAs, and the fermentation broth is recycled over the cathode

compartment. The dissociated MCFAs have a negative charge, and will thus cross over the membrane to the

anode compartment. The potential across the membrane is heavily affected by the concentration gradient

across the exchange membrane (among others, conductivity, distance between electrodes, resistance of the

membranes, pH gradient, etc.). When the concentration of dissolved molecules in the anolyte compartment

is too high, the power that is required to extract additional amounts from the fermentation broth/catholyte

increases rapidly. This explains why this technique is especially useful for extracting MCFAs such as

12

caproate. When the molecules reach the anode, they become protonated by the protons produced at the anode,

due to the protonation, are only present in their undissociated form. As seen before, the undissociated form

has a very low solubility, due to their long hydrophobic chains and their neutral charge, and from a certain

concentration (10g/L for caproate), they undergo phase separation. By recycling the anolyte over an external

anolyte chamber, oils phase droplets are removed from the inner workings of the electrochemical cell and

can accumulate in the external receptacle. Due to this, the concentration of MCFAs dissolved in the anolyte

liquid will never surpass the solubility limit, and cause an exponential increase in power usage/ potential

across the exchange membrane.

The use of membrane electrolysis has shown positive results in multiple studies, both in terms of extraction

efficiency, and in terms of increased productivity of MCFAs. This is achieved on one hand thanks to

increased hydrogen partial pressure[73, 81], and on the other by the removal of the RBO products which

indirectly inhibits RBO due to inhibiting the microorganisms responsible for the production of ethanol and

acetate from sugars and carbohydrates [81].

Another added advantage of using an electrochemical cell is that the hydroxide ions created by hydrolysis at

the cathode [82] also reduce the need for pH control in the fermentor through adding of caustic. In fact,

electrochemical cells are regularly used primarily for pH control, and can easily be set up to only become

active when the pH of the fermentation broth drops below a certain value, as determined by the operator.

This represents a decrease in operational costs, as well as removing an environmentally hazardous parameter,

increasing the sustainability of the project.

Electrodialysis

This is a setup composed of two anion and one cation exchange membranes arranged in parallel. The anode

is submerged in an acidic solution (or distilled water which becomes acidified due to hydrolysis at the anode

producing protons). Next is a cation exchange membrane, which allows protons from the acidic solution to

cross over into the next compartment, through which a distilled water stream, serving as an extractant, flows.

The extractant is separated from a third compartment by an anion exchange membrane. The third

compartment is the compartment through which a mixed stream containing the to-be-extracted carboxylic

acids flows, from which the carboxylic acids need to be extracted. Separating the third and fourth

compartments is another anion exchange membrane. In the fourth compartment, the cathode is submerged in

an alkaline solution (or distilled water which becomes alkaline due to hydrolysis at the cathode producing

hydroxide ions). The hydroxide ions formed at the cathode cross over the anion exchange membrane, and

force the dissociation (also deprotonation) of carboxylic acids in the mixed stream. Due to their dissociation,

the carboxylates gain a negative charge. Due to the subsequent electrostatic attraction between the negatively

charged carboxylates and the positively charged anode, they are pulled across the next anion exchange

membrane into the extractant, but cannot continue on to the anode due to the cation exchange membrane

limiting movement of carboxylates. However, the cation exchange membrane does allow for the protons

generated at the anode to pass over into the extractant. Once the protons come into contact with the

carboxylates, the carboxylates once more attain their undissociated carboxylic acid state. The carboxylic

acids at this point do not have a net charge anymore, and thereby remain unaffected by electrostatic

interactions so long as there are sufficient protons in the extractant stream [83].

Adsorption

Carboxylic acids can be adsorbed onto ion exchange resins, and provide a clean and otherwise unchanged

stream that can be recycled to the reactor. The problem lies in the high capital and expenditure costs, as well

13

as the need for continuously cleaning the resins or exchanging them for fresh resins. No references to

industrial application of this extraction method were found.

Context of this thesis This thesis was meant to play a supporting role in a demonstration project ongoing at the Center for Microbial

Ecology and Technology. In this project a pilot scale fermentation and extraction system has been built that

produces a mixture of MCFA from undiluted thin stillage. The pilot scale installation is comprised of a

fermenter where MCFA are produced at a pH of 5-6, and an electrochemically assisted pertraction unit that

extracts MCFA from the filtrate of the fermentation broth into an alkaline solution. The MCFAs are then

subsequently removed from the alkaline solution through electrochemical membrane separation, and are

transferred into an anode compartment. Once the concentration of undissociated MCFAs reaches their

respective maximum solubility points, an oil layer will form in the anolyte. This oil layer can then be siphoned

off. Through the operation of the pilot plant, a number of challenges have surfaced, and an investigation of

possible answers or alternative solutions was needed, which resulted in this thesis.

Challenges and hypothesis’s:

In the pilot, fed with a mixture of fermentation beer (rich in ethanol) and thin stillage from wheat

fermentation, there was no net ethanol consumption. There were two hypothesis’s which could explain this

occurrence. The first hypothesis was that PH2 affected the rate at which ethanol was consumed, as well as the

production rate of chain elongation products. From literature, it has been seen that a minimum PH2 is required

in order for chain elongation to start, although it has also been shown that high PH2 cause buildups of butyrate

and propionate, leaning towards propionate which is not a substrate for even-chain elongation but for odd-

chain elongation. At PH2 pressures above 0.098atm, ethanol oxidation to acetate coupled with ATP formation

is thermodynamically unfavorable, as discussed earlier in the carboxylate platform. To see whether the PH2

truly is a cause of reduced ethanol consumption, a first experiment was performed to evaluate the effects of

high and low PH2 on reactors fed with an ethanol enriched stream. A second hypothesis for the decreased

ethanol consumption rates can be attributed to the presence of solids, or more specifically, the presence of

products that originate from the hydrolization of solids. Although the exact mechanisms are not known, it

has been shown that increasing the solids loading rate causes increased ethanol titers [84]. The increased

ethanol titers in the given example are desired, however reports have been made on the inhibitory result that

hydrolyzation of cellulosic material could potentially cause [15].

A second goal is to investigate the application of membrane electrolysis as means to extract MCFAs from

fermentation medium with high extraction rates, and lower costs without liquid-liquid extraction. At an early

stage, the pilot used a pertraction system to deliver stream of dissolved MCFAs to an electrochemical cell.

The major disadvantages are firstly, the fact that the flowrate of MCFAs from fermentation medium across

the pertraction system and into the catholyte of the electrochemical cell is heavily limited by the surface area

of the membrane. This means that, if it is desired to increase the speed at which MCFAs are extracted, it is

necessary to apply larger separation membranes. A second disadvantage is the fact that organic solvents are

used in the pertraction process. These organic solvents do not only represent an additional expenditure, but

are also not environmentally friendly. From a life cycle assessment[85], the three largest contributors to

pollution and global warming were the addition of ethanol to stimulate chain elongation, solid waste

management during an acidification step, and the use of organic solvents in the liquid-liquid extraction

system. The third major disadvantage is the fact that a pertraction system consists of numerous parts, these

being the membrane modules, but also the organic solvent and alkaline solution. Pumps are needed to pump

the organic solvent and alkaline solution. In short, the inclusion of a pertraction system not only limits the

14

rate at which MCFAs can be extracted from the reactor, but also increases operational expenditures, due to

the number of pumps necessary for recirculation of the various liquids

As for the third area of investigation, the hypothesis was that by using solid-free undiluted thin stillage, chain

elongation would still be possible. Not only because of the mechanical issue with clogging that was explained

before, but also because there is reason to believe that hydrolyzation of the solids present in the feed are

partially responsible for decreased ethanol consumption and MCFA production rates. When solids derived

from plant matter are hydrolyzed, compounds named tannins are released into the surrounding broth. Tannins

are polyphenols which can comprise up to 50% of dry matter in leaves and stems of floral organisms, and

inhibit the growth of a large spread of organisms, including yeasts, bacteria of the genus Clostridium and

fungi[86]. Tannins are concentrated in the husks of spent grains, which form the vast majority of the solids

present in thin stillage. The presence of tannins has led many studies in chain elongation on thin stillage or

similar streams to be performed with diluted thin stillage. In this way, the inhibitory effects of tannins and

other such compounds are lessened in impact. However, in this thesis and the pilot project, efforts are made

to keep the whole process as environmentally friendly as possible, and using large amounts of clean water to

dilute a dirty stream is the opposite. But there is another perspective, that there might still be a use for the

solids. At the pilot, the culture used for RBO is a mixed culture, meaning that there are many different species

of bacteria, each with their own metabolic part in the grand scheme of things, and each with their own

optimum survival strategy. There is a case to be made for the possibility that there are certain bacteria which

require carrier materials in order to thrive. In the case that bacteria that fit this description are the primary

producers, or in other words are responsible for primary conversion of dissolved carbohydrates and other

compounds to RBO substrates, then removing of solids from the process might prove detrimental, because

chain elongating bacteria are unable to process larger polymers. To test once and for all whether or not the

solids are of importance to the chain elongation process, a continuous experiment was set up where undiluted

solid-free stillage was fed to duplicate reactors.

15

Materials and methods

Influence of PH2 on ethanol consumption and MCFA production In this experiment, four batch reactors were implemented. Two had a volume of 300ml, and two had a volume

of 1200ml. the smaller reactors were used to simulate environments with a higher PH2, hence forth referred

to as HI reactors, and the two large reactors were used to simulate an environment with low PH2, referred to

as LO reactors from this point onwards. One of each type of reactors (one HI and one LO) were inoculated

with inoculum obtained from the pilot, given the prefix PI, whilst the other two reactors were inoculated with

an inoculum derived from a continuously running reactor where an enrichment culture (EC) performing chain

elongation was grown anaerobically on synthetic medium (see material and methods: experiment 3) enriched

with ethanol and acetate, at pH 7. The latter inoculum is positive for chain elongation through ethanol, and

is used in this experiment as the positive control. The reactors inoculated with the known chain elongator are

given the prefix EC. Both inocula were obtained by centrifuging medium from respectively the pilot and

from the EC, and washing the pellets three times with substrate-free synthetic medium (SM), the composition

of which is described in tables 10, 11 and 12. The starting conditions of the experiment were 20ml of

respective inocula, and 180ml of tap water. Every day, 20 ml was removed and 20ml of feed (reduced by

volume needed for pH balancing) was fed to each of the reactors. Stillage was obtained from Tereos Starch

and Sweeteners, Aalst (Belgium), where bio-ethanol is produced from the fermentation of wheat and

subsequent distillation. After distillation, the remaining biomass left after the removal of most of the ethanol

is separated into a light fraction (thin stillage) and a heavy fraction (DDGs). The DDGs are sold as an animal

feed additive, whereas the thin stillage, rather than being concentrated to stillage syrup, is simply send to an

A.D. facility, for energy recovery in the form of biogas. Beer was also used in this experiment. The

fermentation beer is, like the thin stillage, obtained from Tereos Starch and Sweeteners. Rather than being a

waste stream, this is the pre-distillation product, which would normally have the ethanol extracted from it.

The difference between the two waste streams is that fermentation beer still contains a high concentration of

ethanol, as well as the other components found in thin stillage. Because this product is not separated into a

heavy and light fraction, it has a higher amount of solids compared to the thin stillage. The reactors were

constantly stirred using magnetic stirring devices, and were kept at a constant temperature of 28˚C. The

headspaces of the LO reactors were flushed (20 cycles) with N2 gas every day after sampling and oxygen

free for anaerobic conditions, after which the headspace pressure was relieved in all reactors. The pH of all

reactors was adjusted to pH6 on a daily basis. Because the duration of this experiment was scheduled to be

longer than 30days (>3HRTs), no biological triplicates were implemented for this experiment. In the

beginning, a mix of beer and thin stillage was used as feed, but was later changed to thin stillage with ethanol

dosed. In both feeds, ethanol concentrations varied around 12g/L, with the compositions of the three different

feed batches shown in table 7.

From the daily discharge of effluent, 10ml was frozen for total soluble solids (TSS)/volatile soluble solids

(VSS) measurements. 5ml was centrifuged for 10 minutes ate 7800 rpm and filtered using 0.45µm filters

(Chromafil® Xtra PA-45/25, Macherey-Nagel, Germany). 4ml was kept for pH measurement, and for

calculating (equation 1) the amount of 1M NaOH needed to bring each of the reactors back to pH 6. The last

1ml was centrifuged for 10 minutes at 14000rpm, and the pellet was kept in frozen state for potential DNA

analysis of the culture.

𝑌 = ((

𝑋0.1𝑀4 ) ∙ 180

10) + 1.3

With Y being the volume (ml) of 1M NaOH needed to bring the reactor back to pH 6, X0.1M being the amount

of 0.1M NaOH needed to bring 4ml of sample to pH6, and 1.3 being the volume of 1M NaOH needed to

16

bring 20 ml of feed to pH6. Every day, prior to sampling and feeding, pressure in the headspace was measured

using a tensimeter (INFIELD 7, UMS, Germany) and a sample of the headspace was analyzed for each

reactor.

Inhibition of ethanol by the feed matrix This experiment is separated in two runs, a primary run and a rerun. In the primary run, 5 series of small

batch reactors (penicillin bottles) in triplicates with a working volume of 15ml were used. For fermentation

medium, each triplicate group had a different ratio of SM:Reactor effluent without solids, as shown in table

10, with SM compositions shown in tables 12,13 and 14. The reactor effluent without solids was obtained by

siphoning off permeate from the solid-liquid separation membrane installed at the pilot. Prior to mixing the

two media in the corresponding ratios, the ethanol concentration in both media was adjusted to 12g/L, and

acetate to 6g/L, while the pH was adjusted to pH6. After preparation of the batch reactors, each reactor was

inoculated with EC inoculum. The reactors were kept on a shaker in a 34˚C room, and were flushed 20 times

with N2 gas at the start of the experiment. 0.6ml samples were taken on days 0,1,3,5,7,9 and 12. Samples

were centrifuged for 10 minutes at 14000rpm and the supernatant collected whilst the pellets were discarded.

pH was measured again at the end of the experiment.

Percentage broth (series #) 0% (1) 10% (2) 25% (3) 50% (4) 100% (5)

Volume Broth (mL) 0 1.35 3.375 6.75 13.5

Volume SM (mL) 13.5 12.15 10.125 6.75 0

Volume inoculation (mL) 1.5 1.5 1.5 1.5 1.5

Total (mL) 15 15 15 15 15

Table 8: Composition of fermentation media of the various series used in: Inhibition of ethanol oxidation

by the feed matrix: primary run

In the rerun, 6 series of triplicates were run, also in penicillin bottles, but this time with working volumes of

50ml. The sixth series was added so as to observe the effects of solids on ethanol consumption and MCFA

production. Solids from 45 ml of pilot effluent were separated from the broth by centrifugation. The resulting

pellet was then washed three times in SM without substrates. The solids were then resuspended in 45ml of

SM with substrates, and added to the series 6 batch reactors. Each batch was also inoculated with 5ml EC

inoculum, which has been prepared according to what was seen in experiment one and in the primary run of

this experiment. Other changes were also implemented. The beginning acetate concentration was reduced

from 6g/L to 4g/L, sampling was done every four hours as opposed to daily sampling, and headspace

composition was analyzed on a daily basis as well.

Percentage broth (series #) 0% 10% 25% 50% 100% 0%S

Volume Broth (mL) 0 4.5 9 22.5 45 0

Volume SM (mL) 45 40.5 36 22.5 0 45

Volume inoculation (mL) 5 5 5 5 5 5

Total (mL) 50 50 50 50 50 50

Feed batch 1 (mix of beer and

thin stillage) (1:5)

Feed batch 2 Feed batch 3

Ethanol 20.9 ±2.24 12.7 ± 0.77 14.3 ± 1.84

Lactate 0.32 0.40 0

Acetate 0 0.67 3.0

Propionate 0.02 0.10 0.8

Butyrate 0 0 1.4

Table 7: Compositions of the various feed batches used in: Influence of PH2 on MCFA production and

ethanol consumption

17

Table 9: Composition of fermentation media of the various series used in: Inhibition of ethanol oxidation

by the feed matrix

Component Concentration (g/L)

K2HPO4 0.31

KH2PO4 0.23

NH4Cl 0.25

MgCl2 0.06

MgSO4•7H2O 0.05

NaHCO3 2.5

Yeast extract 1

Se-W 1mL

SL-10 1mL

7Vit(10x) 0.1mL

Table 10: Composition of SM used for inoculum preparation and fermentation medium in: Inhibition of

ethanol oxidation by the feed matrix

HCl (25%; 7.7 M) 10.00 ml

FeCl2 x 4 H2O 1.50 g

ZnCl2 70.00 mg

MnCl2 x 4 H2O 100.00 mg

H3BO3 6.00 mg

CaCl2 x 6 H2O 190.00 mg

CuCl2 x 2 H2O 2.00 mg

NiCl2 x 6 H2O 24.00 mg

Na2MgO4 x 2 H2O 36.00 mg

Distilled water 990.00 ml

Table 11: SL-10 composition (1L) used for SM medium

Vitamin B12 100.00 mg

p-Aminobenzoic acid 80.00 mg

D(+)-Biotin 20.00 mg

Nicotinic acid 200.00 mg

Calcium pantothenate 100.00 mg

Pyridoxine hydrochloride 300.00 mg

Thiamine-HCl x 2 H2O 200.00 mg

Distilled water 1000.00 ml

Table 12: 7vit (10x) composition (1L) used for SM medium

Continuous fermentation of solid-free undiluted thin stillage Two 5L continually stirred tank reactors (CSTR) (FLC-6, BelachBioteknik) were fed with solid free thin

stillage for an HRT of 10days, with one serving as a control (R2) and the other being an experimental reactor

(R1) during phase 1 of this experiment. The CSTRs had a built in pH probe (EasyFerm Plus, Hamilton,

Bonaduz, Switzerland), 2 pumps with pumpheads (Watson-Marlow, USA), 2 small pumps for acid and base

dosage, a level probe, a gas production column, a temperature probe and heating blanket which kept the

18

temperature of both CSTRs at 30ºC, and a stirrer with a helix blade with stirring kept at 100rpm, with reversal

of stirring direction. The solid-free thin stillage was obtained by allowing the thin stillage which was kept in

a 1000L cubic container to settle over the course of a few days, and collecting the supernatant, which had a

TSS of less than 0.1g/L. Feeding was done once an hour, and effluent was automatically discharged when

the level in the reactor exceeded 5L, as detected by the level probe. Both reactors were kept at a pH of 5,

with 5M NaOH dosage for pH control. The pH set point was later increased to 5.5, and then later again, to 6.

After 30 days, in phase 2, a ceramic membrane was attached to the R1, where a sludge retention time (SRT)

of 20 days was achieved, although later the SRT was changed to complete retention (C.R.). A pump capable

of high flowrates (600 series mid-flow process pump, Watson-Marlow, USA) was used to run the reactor

broth through the ceramic membrane with an rpm of 65. On day 60, at the commencement of phase 3, a direct

electrochemical extraction was performed on R1 using a three-chamber electrochemical cell, using a current

of 0.710 A. The electrochemical cell was built locally, using plexiglass (PMMA) frames (Vlaeminck A. NV,

Belgium). The internal chamber area was 0.0002m2 for each compartment (5*2*20cm). The outer plates

(14*2*24) were held together with bolts and wingnuts. Between each compartment, a rubber sheet was used

to prevent leaks. For the parts of the middle compartment, thicker versions of the same rubber sheets were

used, due to the corrosive nature of the middle compartment. The end plate of the anode compartment

contained an extra hole, through which the anode could be linked to a power bank. For the anode, a 5*20cm

titanium electrode coated with iridium oxide was used, whilst the cathode was a 5*15cm stainless steel gauze

with 0.5cm mesh. The anode was connected to an external anolyte reservoir, filled with 10% sulphuric acid,

which was continuously recirculated across the anode compartment. The middle compartment was also

connected to an external reservoir, where the solution in the middle compartment was recirculated from the

bottom of the reservoir to the bottom of the middle compartment, so as to avoid any oil-phase layer formed

in the middle compartment from flowing through and damaging various parts. The cathode compartment was

fed directly from R1, and returned to the feed port of the reactor.

Sampling during phases one and two consisted of taking 16ml samples from both CSTRs, 8ml kept separate

in a frozen state for TSS/VSS analysis, and 8ml was centrifuged for 10minutes at 7800rpm, and filtered using

a 0.45µm filter, before storage in a frozen state. From phase three onwards, samples from R1 were taken

from a sampling port placed after the cathode compartment of the electrochemical cell. Increased sample

volume from R2 was used, 25ml per sampling, in order to perform TSS/VSS from this reactor in triplicate.

8ml samples was also taken from the middle compartment by means of a sampling port.

day -8 0 31 47 56 60 67

Feed (R1, R2) TS SF-TS SF-TS SF-TS SF-TS SF-TS SF-TS

pH (R1, R2) 5 5 5 5.5 5.5 5.5 6

Sludge retention (R1) HRT HRT 2 X HRT 2 X HRT C.R. C.R. C.R.

Extraction (R1) X X X X X ✓ ✓

Table 13: overview of the timeline of experiment 4. Phase 1: days 1-30, Phase 2: days 30-60, Phase 3: days

60-end

Analytical methods

VFA analysis

C2-C8 fatty acids (including isoforms C4-C6) were measured by gas chromatography (GC-2014,

Shimadzu®, The Netherlands) with DB-FFAP 123-3232 column (30m x 0.32 mm x 0.25 µm; Agilent,

Belgium) and a flame ionization detector (FID). Liquid samples were conditioned with sulfuric acid and

sodium chloride and 2-methyl hexanoic acid as internal standard for quantification of further extraction

19

with diethyl ether. Prepared sample (1 µL) was injected at 200ºC with a split ratio of 60 and a purge

flow of 3 mL min-1. The oven temperature increased by 6ºC min-1 from 110ºC to 165ºC where it was

kept for 2 min. FID had a temperature of 220ºC. The carrier gas was nitrogen at a flow rate of 2.49 mL

min-1.

TSS/VSS analysis

TSS and VSS were performed according Standard Methods 2540D and E (APHA, 1997).

Organic Ion Analysis

Volatile fatty acids were analysed using a Dionex DX 500 ion chromatography system using an IonPac

ICE-AS1 column with 0.4 mM HCl as eluent and an ED50 conductivity detector. The lower limit of

detection was XX (here you fill in your calculated limit of detection, also taking care of any dilution

factors).

Inorganic Ion Analysis

Cloride, nitrite, nitrate, sulphate and phosphate were determined on a 761 Compact Ion Chromatograph

(Metrohm, Switzerland) equipped with a conductivity detector.

Headspace analysis

The gas phase composition was analyzed with a Compact GC (Global Analyser Solutions, Breda, The

Netherlands), equipped with a Molsieve 5A pre-column and Porabond column (CH4, O2, H2 and N2) and a

Rt-Q-bond pre-column and column (CO2, N2O and H2S). Concentrations of gases were determined by

means of a thermal conductivity detector.

COD analysis

COD was analysed with Nanocolor® kits (CODE; Macherey-Nagel)

Calculations

(1) Conversion g/L to COD/L

𝑋 𝑔(𝐶𝑎𝐻𝑏𝑂𝑐𝑁𝑑) 𝐿⁄ = 𝑋 ∙ (𝑎 + 𝑏

4−

𝑐

2−

3𝑑

4) 𝑔𝐶𝑂𝐷 𝐿⁄

(2) Organic loading rate

𝑂𝐿𝑅 = 𝑔𝐶𝑂𝐷 𝐿⁄ (𝑓𝑒𝑒𝑑)

𝐻𝑅𝑇

(3) Calculation of production rates

𝑔𝐶𝑂𝐷 𝐿⁄ 𝑑⁄ = 𝑔𝐶𝑂𝐷 𝐿⁄

𝐻𝑅𝑇

(4) Partial Pressure

𝑃𝑥(𝑎𝑡𝑚) = [1 + 𝑃𝑚𝑒𝑎𝑠𝑢𝑟𝑒𝑑 ∙ (

𝑉𝐻𝑆 + 𝑉𝑆𝑉𝐻𝑆

)

1013,25] ∙ 𝐶𝑥

With

▪ Px = The partial pressure of component x in atmospheric units (1atm = 1013.25 hPa)

▪ Pmeasured = The gas pressure measured in the reactor after sampling in hPa

▪ VHS = the volume of headspace in the reactors = Lfull-L0.2L

20

▪ Vs = the volume of sample taken + the volume removed from reactor prior to

measuring

▪ Cx = the concentration of component x in the headspace

Results Influence of PH2 on MCFA production and ethanol consumption In figure 3, a general overview of the PH2 in all reactors is shown for the duration of the experiment. On day

5 and day 6, PH2 in PiHI and ECHI reached local maxima respectively 0.50 and 0.77atm, after which they

respectively dropped down to 0.01atm and 0.12atm on day 10 for PiHI and ECHI respectively. From day 10

onwards, PH2 in both reactors increased gradually until day 21 in PiHI and day 29 in ECHI. Following this

gradual increase is a jump in PH2, where PH2 in PiHI remained relatively stable at 0.90 ±0.05atm between

days 25 and 35. In ECHI, a similar peak was observed, with a PH2 of 0.76 ±0.07 between days 30 and 35.

From day 35 onwards, in both of the HI reactors, PH2 then starts decreasing, at which point the experiment

ended. The PH2 in PiLO and ECLO remained at a low value, with maximum daily PH2 levels of 0.10±0.01

and 0.10±0.03atm, respectively.

Figure 3: Daily maximum hydrogen partial pressure in reactors

Figure 4 shows the evolution of component concentrations of the various VFAs and. Both of the EC reactors

show a lag phase for caproate production, which started to accumulate from day 7 onwards. Caproate

production in PiHI started immediately, whilst PiLO showed a 7-day lag-phase. In both of the LO reactors,

a slight buildup of lactate accumulated in the beginning of the experiment until day 8 for PiLO and day 10

for ECLO. In contrast, in treatments with high PH2 lactate buildup only occurred from day 23 in PiHI and

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 5 10 15 20 25 30 35 40 45

Pre

ssu

re (

atm

)

Time (days)

PH2

PiHI PiLO ECHI ECLO

21

from day 28 in ECHI, with a larger amount in PiHI than in ECHI. Higher acetate concentrations accumulated

at low PH2, averaging 5.64±0.24gCOD/L from day 28 onwards in PiLO and 5.15±0.15gCOD/L in ECLO,

whilst in PiHI and ECHI, acetate concentrations averaged 3.40±0.12g/L from day 32 onwards and 3.67±0.18

from day 28 onwards respectively. Propionate concentrations remained low in all reactors throughout the

experiment, although concentrations rose slowly over time, most notable in the LO reactors. Looking at

butyrate, PiLO maintained a concentration 2g/L higher than PiHI from day 9 onwards. This trend is not seen

in the EC reactors, although towards the end of the experiment, from day 30 onwards, ECLO sees a slow

accumulation of butyrate, which is not seen in ECHI, with a difference of 1.3g/L on the last day. For valerate,

a similar trend is seen as with propionate. Low concentrations were maintained throughout the experiment,

although concentrations did increase slowly over time in both of the LO reactors. Looking at caproate, stable

concentrations were reached on days 28-38, 30-35, 25-34 and 27-38 for PiHI, PiLO, ECHI and ECLO

respectively. After the stable phase seen in PiLO and ECHI, caproate concentrations dropped to 8.90±0.36

and 9.91±1.10gCOD/L respectively. In both the HI reactors, octanoate concentrations reached up to 0.66 and

0.59gCOD/L in PiHI and ECHI, whilst octanoate concentrations in PiLO and ECLO barely reached

concentrations of 0.33 and 0.14gCOD/L.

Figure 4: Concentrations of even-chain RBO products and lactate

22

Figure 5: Product distribution (%-COD) for all reactors

In figure 5, the relative proportions of VFA COD production are shown. This figure allows for a clearer view

of how the difference in PH2 affected VFA production performance in the four reactors. Apart from the

difference in the lag time which is required for production of MCFAs, the distribution of even and odd length

VFAs is affected, as well as the total amount of MCFA production. In both of the HI reactors, from day 28

onwards, 83.82%±1.86 and 84.01%±2.71 of the VFA-COD is in the form of even chained VFA products for

PiHI and ECHI respectively. On the other hand, during the same period, even chained VFA products took up

only 79.96%±1.18 and 77.93%±1.27 of the total VFA COD in PiLO and ECLO respectively. When observing

the total amount of MCFAs produced in each of the reactors from day 28 onwards, 64.16%±2.28 of VFA-

COD was in the form of MCFAs in PiHI, compared to 52.88%±3.89 in PiLO. Similarly, 63.71%±3.73 of

VFA-COD in ECHI was in the form of MCFAs compared to 53.08%±3.47 in ECLO.

In the four reactors, the applied levels of PH2 did not have a significant effect on ethanol consumption (Fig.

6). Ethanol concentrations remained similar in all four reactors throughout the duration of the experiment,

with a few solitary outliers spread throughout the data. The data does show, however, that the ethanol

concentrations in the LO reactors had the tendency of being slightly lower than in the HI reactors.

23

Figure 6: Ethanol production rates (gCOD/L.d) for the four reactors

24

Inhibition of ethanol oxidation by the feed matrix An experiment was designed to assess if chain elongation, with ethanol as electron donor, was negatively

affected by the composition of the fermented thin stillage. An enriched culture, adapted to utilize ethanol for

chain elongation, was exposed to the liquid fraction of the pilot effluent diluted to different levels with

modified M52 medium. The impact of the broth on the microbial process was evaluated by comparing ethanol

oxidation rates under the different conditions tested. Figure 7 shows the average concentrations of ethanol

measured in each of the batch series for the duration of the experiment. All treatments showed a lag phase in

ethanol oxidation that lasted 0.9d. Despite this initial lag-phase, all the ethanol was oxidized in the control

treatment after 2.9 d, and the same occurred in the rest of the treatments after 5 days.

Figure 7: average ethanol concentrations (gCOD/L) over time for all series

Looking at the average maximum ethanol consumption rates, a clear progression in average rates at which

ethanol is consumed can be seen from figure 8, where with increasing broth concentrations, ethanol

consumption rate decreases. The exception to this trend is the series with 100% broth, which has an almost

as high ethanol consumption rate as the series with 0% broth, which has the highest.

25

Figure 8: maximum ethanol consumption rates (gCOD/L.d) for all series

A clear progression of VFA concentrations (Fig. 9) was seen in function of medium composition. VFA

production ends its lag phase at 0.9 days, with the exception of the 50% broth series, which has a lag phase

duration of 2.9 days. As seen in Figs. 8 and 9, trends can be seen in the series with 0% broth through to the

series with 50% broth, with the 100% broth series breaking the trends. The first trend is the production of

butyrate, where the concentration of butyrate present in each series increases in relation to the amount of

pilot fermentation broth present in the fermentation medium. Another major trend is the production of

caproate, where the attained concentrations at the end of the batch experiment decrease from the 0% broth

series to the 50% broth series. In order of increasing broth concentration, butyrate concentrations at the end

of the batch experiment were 5.99±0.75g/L, 7.26±0.66g/L, 8.63±0.77g/L and 11.08±2.48g/L. Caproate

concentrations, in the same order, reached final concentrations of 6.37±0.25g/L, 4.90±0.22g/L, 4.49±0.44g/L

and 1.53±0.21g/L. In both of these trends, butyrate and caproate concentrations in both of these series differed

from the trend in the 100% broth series, with final concentrations of 8.31±0.86g/L and 7.80±0.39g/L

respectively.

26

Figure 9: VFA concentrations (gCOD/L) shown for all series

In each of the batch series, during the initial stages of the experiment, acetate and propionate production

occurred. With the exception of the 50% broth series, this initial production lasted for approximately one

day, whilst in the 50% broth series the initial production lasted 3 days, as seen in fig. 10. The rates at which

acetate and propionate were initially produced in the batches show some trending nature as well, shown in

fig. 11, with decreasing acetate production rates from the 0% to 25% broth series, but increasing propionate

production rates. In these two trends, both the 50% and 100% broth series do not follow the same pattern,

where the 50% broth series shows a higher initial acetate production rate and a lower initial propionate

production rate than expected. On the other hand, the 100% broth series shows minimal initial production

27

rates for both acetate and propionate, and is also the only series in which propionate is not consumed, but

produced throughout the duration of the experiment, as seen in fig. 10.

Figure 10: initial acetate and propionate production rates for all series

The composition of the fermentation medium also affects the speed at which the substrates are consumed,

and the speed at which the RBO products are produced. The maximum consumption and production rates

then (fig. 12) are also impacted. Maximum acetate and ethanol consumption rates are highest in the 0%

broth series, and the consumption rates of the two substrates decreases with increasing Pilot fermentation

broth in the fermentation medium of the batches, to the point where in the 50% broth series, the maximum

caproate production attained during this experiment was only 0.36±0.07gCOD/L.d. On the other hand, in

the 50% broth series, a maximum butyrate production rate of 9.00±1.21gCOD/L.d.

28

Figure 11: Maximum production and consumption rates after lag phase (gCOD/L.d) for all series

When looking at the total change of concentrations of dissolved VFAs and ethanol in each of the series (fig.

12), in each series, more COD was produced than there was being consumed. However, the 50% broth series

shows the lowest net production of VFA-COD when compared to the other series, even though the same

amount of COD in the form of acetate and ethanol was consumed, suggesting a greater CO2 production. In

the 100% broth series, a far larger net production of total VFA-COD was observed compared to the other

series. This suggests that there were additional sources of carbon, energy and reducing equivalents present

in the pilot fermentation broth.

Figure 12: net consumption/production of substrates and products in gCOD/L

29

When looking back at the results obtained from this experiment (Fig 10.), it can be seen that the major

changes in composition of the fermentation broth in terms of production and consumption occurred within

the first three days. Due to the fact that samples were not consecutively taken during this transitionary

period, the resolution of the obtained data was not great enough for a more in-depth look into the manner in

which the fermentation matrix influenced ethanol oxidation. Furthermore, when looking at the difference in

ethanol consumption in the 100% broth series (Fig. 8) and the ethanol consumption in the PH2 testing (Fig.

7), a hypothesis was formed that the presence of solids could possibly be responsible for the inhibition of

ethanol oxidation. In order to perform a more close-up investigation, a second batch test was designed and

implemented. In this second batch test, the goals were to strengthen the findings from the first batch test, as

well as to investigate the possible influence of solids derived from thin stillage on ethanol oxidation using

an additional batch series, the 0% broth with solids (0%S) series.

Figure 13: Average ethanol concentrations in each series (gCOD/L)

The results of the second batch experiment were markedly different in certain aspects from the first batch

experiment, such as the ethanol consumption. From figure 13, it can be seen that the ethanol concentration

in the various series decreased a lot less over time when compared to figure 8 of the first batch experiment.

However, a trend can still be seen, where less ethanol was consumed in the batches with higher pilot

fermentation broth than in the series with less. In contrast to the first batch experiment, there was no batch

series in which all of the available ethanol was consumed.

0

5

10

15

20

25

30

35

40

0.0 0.8 1.2 1.8 2.2 2.8 3.2 3.9 4.2 5.8

Co

nce

ntr

atio

n (

gCO

D/L

)

Time (days)

0% 10% 25% 50% 100% 10%S

30

Figure 14: Maximum consumption rates of ethanol (gCOD/L.d) in each of the series

When looking at the maximum consumption rates observed in each of the batch series (Fig 14), a similar

trend can be seen as was seen in figure 9 of the first batch experiment. The difference lies in the fact that,

where in the first experiment the 100% broth series did not follow the trend of decreasing maximum ethanol

consumption rates with increasing pilot fermentation broth, here the 100% broth series does follow the trend,

with only 0.22±0.17gCOD/L as the maximum consumption rate. Lastly, the 0%S broth series also showed a

much lower maximum ethanol consumption rate, with 0.97±0.34 gCOD/L.d compared to

5.28±0.37gCOD/L.d in the 0% broth series, even though neither of these series have any pilot fermentation

broth.

Figure 15: Average VFA concentrations (gCOD/L) in each of the batch series for the duration of the

experiment

31

Apart from the trend in ethanol consumption, trends can also be seen in the production of the various VFAs

(Fig. 15). Looking at the progressions of acetate concentration, in the 0%, 0%S, 10% and 25% broth series,

acetate concentrations start decreasing from day 2.2 onwards, whilst the acetate concentration in the 50%

and 100% broth series does not seem to be consumed in function of time. In the series with 50% broth, a bare

minimum of change in each of the VFAs can be seen, whilst in the 100% broth series, based on the data

shown in figure 14, changes in VFA concentrations might as well be statistical errors, rather than a

quantitative shifting of medium composition over time. From the graphs in figure 16, it is also possible to

see that there was a considerable amount of caproate and octanoate present in the filtered reactor fermentation

broth, with initial caproate concentrations of 6.11±0.46gCOD/L at the start of the experiment in the 100%

broth series. Broth concentration is also reflected in the Initial octanoate concentrations, with the 100% broth

series containing 1.06±0.15gCOD/L, and the 50% broth series containing 064±0.17gCOD/L. by looking at

the graphs in figure 16, estimates can be made of the respective lag phases for some of the series. The 0%,

10%, 25% and 0%S broth series both end their lag phases between 2.8 and 3.2 days, and no end to the lag

phase can be identified for the 50% and 100% broth series. In each of the series, butyrate and caproate

production begins from the start of the experiment, but production rates do not increase drastically until the

lag phase is over.

As in the primary run, a brief period of acetate and propionate production was seen in every series, although

the total amount produced and the production rate changed from series to series. In figure 16, the average

initial production rates for acetate and propionate are given. From the figure, a general trend can be seen,

where the series with higher broth concentrations have greater acetate production rates than the batches with

more SM. The 0%S series shows itself to be the exception to this rule, with almost exactly the same average

initial acetate production rate as the series with 100% broth.

Figure 16: Average initial production rates (gCOD/L.d) for acetate and propionate in each of the series

0

0.2

0.4

0.6

0.8

1

1.2

0% 10% 25% 50% 100% 0%SPro

du

ctio

n r

ate

s (g

CO

D/L

.d)

acetate propionate

32

Figure 17: Average maximum consumption and production rates of the various VFAs and ethanol

Some trends similar to figure 13 can also be seen in figure 16, where the maximum consumption and

production rates of each batch series are shown for the various VFAs and ethanol. After the series with 0%

broth, the series with 25 and 10% broth have the next greatest ethanol consumption rates, with 3.8±1.84 and

3.3±0.76 gCOD/L.d each. The series with 50% broth comes in fourth place, with a maximum ethanol

consumption rate of 1.5±1.19 gCOD/L.d, followed by the 0%S series, with 1.26 gCOD/L.d and rounding of

is the series with 100% broth, where almost no ethanol consumption took place, with 0.22±0.17 gCOD/L.d

being consumed. Butyrate production rates are in the same order, with the exception of series with 0%S

broth, which takes the number 2 place, with 6.2±0.98 gCOD/L.d being produced as opposed to the series

with 0% broth, where a maximum butyrate production rate of 8.3±1.64gCOD/L.d being observed. Worth

noting is the fact that, whilst acetate was consumed in the series with 0, 10, 25 and 0%S broth, a continuous

production of acetate was observed occurring in the series with 50% and 100% broth.

-10

-5

0

5

10

15

0% 10% 25% 50% 100% 0%S

Pro

du

ctio

n a

nd

C

on

sum

pti

on

Rat

es

(gC

OD

/L.d

)

acetate propionate butyrate

valerate caproate heptanoate

octanoate ethanol

33

Figure 18: Net consumption/production of the various VFAs and ethanol (gCOD/L)

In figure 19, net changes in the COD present in the form of the various VFAs and ethanol is shown. The 0%

broth series experienced a net decrease of 1.01±1.14gCOD/L, the 10%broth series a decrease of

0.82±0.69gCOD/L, the 25% broth series an increase of 4.64±9.23gCOD/L (large variation due to highly

elevated butyrate concentration being measured in one of the triplicates of this series on the last day), the

50%broth series an increase of 2.25±4.28gCOD/L (large variation due to one triplicate in the series

effectively consuming more COD), an increase of 1.85±1.11gCOD/L in the 100% broth series, and finally

an increase of 2.77±1.40gCOD/L was observed in the 0%S broth series.

Looking at the changes in COD present in acetate, the 0% broth series consumed the most acetate,

2.70±0.03gCOD/L, followed by the 10% and 25% broth series, which consumed 2.28±0.74 and

1.63±1.20gCOD/L respectively. In the 0%S series, a net decrease of acetate COD of 1.52±0.45gCOD/L was

seen. In contrast, both the 50% and 100% broth series rendered a net increase in acetate COD, with a net

production of 0.96±1.25gCOD/L in the 50% broth series, and 0.99±0.64gCOD/L in the 100% broth series.

What can clearly be seen from figure 19 is that the net production of butyrate increases from the 0% to 25%

broth series, with 7.17±1.32, 9.55±0.55 and 18.69±9.25gCOD/L net production in the 0%, 10% and 25%

broth series respectively. As for the 50 and 100% broth series, only 2.42±0.73 and 0.75±0.44gCOD/L of

butyrate was produced. In the 0%S broth series, a net production of 7.90±0.69gCOD/L was seen.

In terms of net caproate production, highest production was observed in the 0% broth series, with

7.10±0.85gCOD/L. as the concentration of broth in each series increases, the concentration of caproate COD

decreases. In the 0%S broth series, 1.56±1.15gCOD/L was produced.

-20

-15

-10

-5

0

5

10

15

20

25

30

35

0% 10% 25% 50% 100% 0%S

Ne

t ch

ange

in c

on

cen

trat

ion

s (g

CO

D/L

)

acetate propionate butyrate valerate

caproate heptanoate octanoate ethanol

34

Continuous fermentation of solid free undiluted thin stillage As this experiment was a complex one, there are many different streams of data which have to be analyzed

and compared.

Phase 1: Feeding solid-free undiluted thin stillage

In phase one, solid-free thin stillage was fed to both the experimental reactor (R1) and the control reactor

(R2), for 3HRTs (30 days) in order to get a baseline for both reactors, and to have an acclimatized culture

for the following phases.

During phase 1, the feed, which is a complex real feed subject to changes in composition as can be seen

from figure 20 was the only experimental condition. Figure 19 shows an overview of the composition of

the feed during phase 1. Apart from lactate and the components which are not present in large amounts, the

remainder of substrates such as ethanol, glycerol and 1,3-propandil (1,3 PDO), which contain the remainder

of observable COD in the feed continuously vary in concentrations over time. Lactate had an average

concentration of 2±0.7gCOD/L during phase 1. Maximum acetate concentrations in the beginning (days 0-

5) were 0.277±0.039gCOD/L and towards the end of phase 1 (day 25,26) was 0.74±0.07gCOD/L. Glycerol

concentrations were 2.72±0.031gCOD/L during the first 5 days, and from day 10 onwards,

5.21±1.32gCOD/L. 1,3PDO varied as well, averaging 10.32±0.51 on days 11 till 17, an isolated peak on

day 21, with 14.10 gCOD/L., and overlapping with phase 2, 12.74±0.54 gCOD/L on days 28-32.

Figure 19: Feed composition during Phase 1

During phase 1, even chained RBO substrates and products showed similar trends in R1 and R2, as seen in

the comparative figure 19. In R1, there was a lag phase of 7days in caproate production and lactate

consumption, which was not seen in R2. In R1, ethanol was present throughout phase one, with an average

of 0.86±0.64gCOD/l, whereas in R2, ethanol concentrations from day 1-12 were around 0.23gCOD/l, after

which a period of 10 days of higher ethanol concentrations emerged with an average concentration of

1.85±0.39gCOD/L. However, as stated, the intention of phase 1 was to reach a stable baseline in both

0

2

4

6

8

10

12

14

16

0 5 10 15 20 25 30

Co

ncn

etr

atio

n

(gC

OD

/L)

Time (days)

lactate acetate glycerol 1,3 PDO ethanol

35

reactors. This was achieved towards the end of phase 1, for the last 4 days (days 20-28). In figure 20, the

average concentrations of even-chained RBO substrates and products are shown. From figure 20, it can be

seen that the two reactors show similar trends, though in R1, higher butyrate and caproate concentrations

and production rates are seen. On the other hand, R2 shows a higher octanoate concentration and

production rate, and a lower ethanol concentration and production rate.

Figure 20: Even-chained RBO substrates and products concentrations in both reactors

Figure 21: Average Concentrations (gCOD/L) and production rates (gCOD/L.d) for both reactors during days 20-28

The results from figure 20 are re-represented in numerical form in table 14. From these results, it can be

seen that there is a higher odd-chain RBO production in R2, whilst at the same time, total MCFA

production in R2 is higher with 47.32% as opposed to 44.83% in R1.

acetate propionate butyrate valerate caproate heptanoate octanoate

R1 2.91±0.66 0.13±0.17 47.18±6.10 4.95±1.11 36.87±4.57 1.02±1.19 6.94±2.16

R2 6.97±4.50 0.33±0.08 41.53±3.33 3.85±0.70 34.02±4.65 2.40±0.25 10.90±2.60 Table 14: Average concentrations (gCOD/L) for both reactors during days 20-28

A last important parameter which will be discussed during phase one is the progression of TSS in both of

the reactors, as shown in figure 21. In the beginning of phase 1, there were still solids in the reactors left

over from before the reactors were fed with solid-free thin stillage. As a result it takes until day 5

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25 30

Co

ne

ntr

atio

n

(gC

OD

/L)

Time (days)

R1

Lactate Acetate Butyrate Caproate Octanoate Ethanol

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25 30

R2

36

approximately before the remaining solids in the reactors are washed out. Even after the solids remaining

from the reactor startup were washed out, TSS continuously decreased, even during the period from day

20-28, although the concentrations were more stable compared to earlier in the experiment, with 3.7±0.89

and 3.0±0.16 gTSS/L in R1 and R2 respectively.

Figure 22: Concentration of suspended solids (gTSS/L) in both reactors

Phase 2: sludge retention

In phase 2, a ceramic membrane was first attached to R2 (originally the experimental reactor) and later to

R1 (originally the control reactor). SRT was set up to be twice the HRT, and was changed towards the end

of phase 2 to C.R.

In figure 22, the composition of the major constituents in the feed is shown for days 31-59. In the

beginning, until day 32, some 1,3 PDO is still present from phase 1, which was discussed in the previous

section. In phase 2, lactate, acetate and glycerol values remained relatively constant, with

3.85±0.52gCOD/L in the form of lactate, 0.64±0.20gCOD/L in the form of acetate and 7.47±1.03gCOD/L

as glycerol. On day 42, a sudden shift occurred in the composition of the feed, concerning ethanol and 1,3

PDO concentrations. From day 42 until day 56, average ethanol concentrations were 18.6±0.51gCOD/L,

and average 1,3PDO concentrations were 10.73±1.41gCOD/L.

37

Figure 23: Composition of the feed during phase 2

Due to the changes in the feed, the composition and production rates of both R1, shown in figure 23 and

R2, shown in figure 24 changed drastically. Until day 33, the stable phase which was accomplished in R1

maintained its composition. From day 34 onwards, an increase in ethanol concentrations was seen in R1, as

well as an increase in butyrate concentrations during the same time period. In the period of 34-40, ethanol

concentrations increased from 0.02gCOD/L to 3.47gCOD/L. In the same period, butyrate concentrations

increased from 3.56gCOD/L to a maximum of 7.23gCOD/L. After this period, a sudden increase in ethanol

concentration is apparent, jumping from 3.47 to an average of 11.60±0.56gCOD/L until day 54, after which

concentrations dropped down to 3.58±0.16gCOD/L for the remainder of phase 2. Looking at the

productivity values for the products of even-chained RBO, shown on the right in figure 23, caproate

production levels averaged around 0.37±0.16gCOD/L.d until day 52, after which a short period of

heightened caproate production appeared, with a maximum of 2.5gCOD/L.d. During this period, octanoate

productions also increased in production rates, from an average of 0.07±0.017gCOD/L.d before day 52 to

0.25±0.04gCOD/L.d.

38

Figure 24: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates (gCOD/L.d) in R1

In figure 24, the concentrations of substrates and products of even-chained RBO in R2 are shown on the

left, and the production rates on the right. On day 32, R2 was reinoculated after leakage with a mix of R1

effluent and water. During the following days, the system was still in the process of stabilizing, with a

momentary lapse due to unknown causes on day 40. After this lapse, a sudden increase in ethanol

concentration can be seen, with an average of 11.39±0.44gCOD/L during days 40-54, after which

concentrations decrease to 3.42±0.33gCOD/L. Looking at production rates, the average butyrate production

rate from day 39 until day 54 was 1.11±0.37gCOD/L.d, after which production rates decreased. Caproate

production restarted on day 42, with continuously increasing production rates until day 54, at which point a

peak in production rates occurred, with a maximum production rate reached of 3.7gCOD/L.d. After this

peak in caproate production, a decrease in production rates are observed, although the caproate

concentration in the reactor remained above 18gCOD/L.


(gCOD/L.d) in R2

39

Figure 26: Concentraction of soluble solids (gTSS/L) in both reactors during phase 2

From figure 25, which portrays the TSS in both reactors, it can be seen that the TSS in both reactors

remained low, averaging 3.22±0.95 and 2.78±1.60 in R1 and R2 respectively, with only a small amount of

fluctuation in R2, and almost none in R1.

In order to observe whether the elevated ethanol and 1,3PDO concentrations in the feed between days 42

and 56 had a different effect on R1 and R2, a comparative analysis is shown in figure 26. For lactate, the

concentrations in R1 remained low, with an average of 0.95±0.43gCOD/L from day 30 until day 47.

Between day 47 and day 56, no lactate was observed in R1, although afterwards, towards the end of phase

2, lactate concentrations increase. After the restarting of R2, a period of elevated lactate concentrations was

observed, with a maximum concentration of 7.46gCOD/L. This period lasted 5days, after which lactate

concentrations in R2 remained under the limit of detection. On day 40, a significant increase in

concentration in ethanol was observed in both reactors, going from 2.70gCOD/L in R1 and 0.96gCOD/L in

R2 to 11.61±0.56 and 11.39±0.44gCOD/L for R1 and R2 respectively. Between day 42 and day 45, ethanol

in the feed increased from 0 to 18.60±0.51gCOD/L. During the same period that increased ethanol

concentrations were observed in the feed, increased 1,3PDO concentrations were likewise observed, with

an average of 10.73±1.41gCOD/L. During this same period, elevated 1,3PDO concentrations were likewise

observed in R2, with an average of 3.30±0.50gCOD/L, but no concentration increase was observed in R1.

40


during phase 2.

41

Phase 3: Direct electrochemical extraction

In phase 3, a three-chamber electrochemical cell was added to R1. The current applied over the membranes

started at 0.511A, and was later increased to 0.711A. The application of electrochemical extraction caused

the pH in R1 to rise to a pH of around 6. The pH setpoint of R2 was then similarly increased from 5.5 to 6.

Figure 28: Composition of feed during phase 3 (gCOD/L)

After phase 2, at the end of which the ethanol and 1,3 PDO concentrations dropped down to 0gCOD/L as

seen in figure 27, there was a recurring situation as seen in phase 2, where elevated concentrations were

measured in the feed for both of these components. On day 63, 1,3PDO has a peak in concentration of

13.11gCOD/L, and is present for 5 days before not being present anymore until day 82, from which point

on, concentrations average at 0.92±0.65gCOD/L. As for ethanol, similar to 1,3 PDO, concentrations rose

again on day 63, and increased over time to a maximum of 8.88gCOD/L on day 77. After day 77, ethanol

concentrations decreased again to 3.39±0.44gCOD/L until day 96, where it dropped down to 0gCOD/L

once more. Lactate and acetate concentrations in the fee show a similar trend, with a slow increase in

concentration from 4.07 and 0.75gCOD/L to 6.12 and 2.96gCOD/L respectively on day 77. After day 77, a

constant in concentration of 5.57±0.28gCOD/L for lactate and 2.38±0.31gCOD/L was reached until day 98.

After day 98, lactate decreased over time to an end of 3.86gCOD/L, and acetate remained at

0.61±0.07gCOD/L from day 98 onwards. Looking at glycerol, there was a continuous increase in

concentration over time, 6.72 to 12.15gCOD/L.

42

Figure 29: Even-chained RBO substrates and products concentrations (gCOD/L) and production rates (gCOD/L.d) in R1

In order to observe the effects of the direct electrochemical extraction, the data from R1 has to be split into

multiple sections. In this first section, in figure 28, an overview is given of the complete concentration of

even-chained RBO substrates and products, compiled from the fermentation liquid, the middle

compartment of the electrochemical cell and the oil harvested from the electrochemical cell. A very

noticeable occurrence is the fluctuations of caproate concentrations and production rates during phase 3,

with local maximums of 10.3gCOD/L and 1.27gCOD/L.d on day 73, 14.70gCOD/L and 1.61gCOD/L.d on

day 89 and 1.44gCOD/L.d on day 98. During phase 3, lactate concentrations remained below 2gCOD/L,

except for a solitary peak on day 80, where the concentration was 2.53gCOD/L. Acetate showed a clear

trend, with increasing concentrations over time until a maximum concentration of 5.82gCOD/L is observed

at the end. Similar to the caproate concentrations and production rates, butyrate also has a fluctuating

characteristic, though not to the same extent. When looking at octanoate, for the first time during this entire

experiment, concentrations surpassed 2gCOD/L, with a maximum of 5.02gCOD/L and a maximum

production rate of 0.62gCOD/L.d on day 73. After this maximum, octanoate concentrations and production

rates steadily decreased over the course of the experiment until the end of phase 3, where a stable

concentration of 0.51±0.02gCOD/L was measured for the last three days. Ethanol concentrations were

relatively stable throughout phase 3, slightly decreasing over the course of phase 3 from 5.11gCOD/L until

a concentration of 3.71±0.16gCOD/L.

43

Figure 30:Concentration of undissociated and dissociated even-chain VFAs and lactate in the middle

compartment

The electrochemical cell experienced multiple mishaps, the frequency of which can be deduced from figure

29. These mishaps occurred on days 67, 76, 90, 98 and 101. The cause of these mishaps was due to

pressure buildup causing the middle compartment to be fouled as the result of a tear in the anion exchange

membrane, an error during attempted oil harvesting or spontaneous fouling of the middle compartment,

most likely due to leaks. Oil harvesting succeeded on two different occasions, once on day 101 and on day

105. In the first attempt at extracting, total caproate levels (both the dissociated form and the undissociated

form) reached a maximum of 11.22gCOD/L, or 5.09g/L. In the second attempt, total caproate

concentrations reached a maximum of 15.61gCOD/L, or 7.08g/L. In both of these cases, total caproate

concentrations stabilized before phase separation occurred. In the third attempt, total caproate

concentrations exceeded 22.87gCOD/L, or 10.38g/L and phase separation took place. Regrettably, in the

process of trying to harvest the formed oil layer, the middle compartment was fouled, and the extraction

had to recommence. In the fourth attempt, total caproate concentrations reached 27.7gCOD/L, or 12.58g/L,

and in the fifth attempt, 30.76gCOD/L or 13.96g/L. During the last week of phase 3, a more thorough

extraction was planned, with a daily follow up instead of sampling once every two days, but during this

period, most likely to the last fouling, R1 experienced heavy decreases in productivity of VFAs, and the

maximum total caproate concentration achieved during the last trial was only 15.23gCOD/L or 6.91g/L.

44


(gCOD/L.d) in R2

In order to determine the effect of the direct electrochemical extraction on production rates, a comparison

can be drawn between R1 and R2, using figures 28 and 30. By comparing the two figures, it can be seen

that for every analyzed component except for caproate, concentrations and production rates were a lot more

stable in the R2 reactor. Lactate concentrations remained low throughout phase 3 in R2, with a solitary

maximum of 2.45gCOD/L on day 84. For the remainder of phase 3, lactate levels remained below

1gCOD/L. Acetate concentrations were similarly low throughout phase 3 in R2, averaging

1.76±0.30gCOD/L. Butyrate concentrations showed a little more fluctuation when compared to acetate and

lactate, but even so the concentration remained relatively stable at 6.08±1.27gCOD/L during phase 3, with

a maximum of 8.59gCOD/L on day 88. As for caproate, the concentration at the start of phase 3 was

16.19gCOD/L. This high concentration was a remnant of the period during phase 2 where caproate

production was inflated due to the feed gaining a large amount of ethanol. From 16.19gCOD/L the

concentration decreased in a logarithmic manner until day 84, where a minimum caproate concentration of

3.53gCOD/L was measured. From this point onwards, the caproate concentration and production rate once

more increased to 16.87gCOD/L and 1.75gCOD/L.d on day 94. This was followed by a slight decrease

until day 103, after which, during the final week of the experiment, caproate concentrations increased once

more. Octanoate concentrations increased throughout phase 3, starting from day 49 during phase 2. At the

end of phase 3, a maximum octanoate concentration of 3.63gCOD/L was measured, with a production rate

of 0.37gCOD/L.d. finally, ethanol concentrations also remained stable throughout phase 3, with an average

concentration of 4.82±0.97gCOD/L.

In order to compare the whether the application of direct electrochemical extraction had an influence on the

consumption of the various components in the feed, the comparative study shown in figure 31 was

analyzed. With the exception of glycerol, the consumption behavior of R1 and R2 was similar for lactate,

1,3 PDO and ethanol. In the case of glycerol, however, it can be seen that more glycerol was consumed in

R1 when compared to R2.

45


during phase 3

46

Discussion

Influence of PH2 on MCFA production and ethanol consumption This experiment resulted in some interesting and novel observations. For one, the PH2 applied to the

reactors seems to have influenced the lag duration before lactate consumption began. As seen from figures

3 and 4, The reactors which were maintained at a lower PH2 showed some lactate accumulation during the

first HRT, whilst the HI reactors experience lactate accumulation during the latter part of the experiment. It

has been found that by decreasing the PH2 in a reactor would facilitate the consumption of lactic acid [87,

88], which the findings from the later part of the experiment support. However, the reason as to why the

LO reactors experienced a lag phase is not clear yet, however, it could be due to the fact that an elevated

PH2 allows for greater ethanol production from sugars [89]. Given that in the beginning of the experiment

the measured ethanol concentrations in all reactors was low, this endogenous ethanol production stimulated

by high PH2 could have caused a greater biomass production rate at the beginning of the experiment, which

translated into a faster start to lactate consumption.

Another finding which did not add up to what was found in literature, was the necessary PH2 required for

chain elongation, and therefore MCFA production, to occur. It is commonly accepted that, when the PH2 is

below 0.098atm, chain elongation is thermodynamically unfeasible [68, 69]. However, production of

caproate in the LO reactors started on day 6 and on day 8 for PiLO and ECLO respectively, whilst the

threshold of 0.098atm was not reached until day 13 and day 19 for PiLO and ECLO, respectively. Seeing as

how the headspaces of the reactors were sampled before feeding and flushing, this means that in 24 hours’

time, there was at no point an accumulation of H2 to the point that chain elongation was possible, according

to theory.

In literature, it was found that by maintaining a high PH2, ethanol oxidation is diminished, due to

unfavorable thermodynamics, but that this was not the case for MCFA producing bacteria, due to their

ability to get rid of excess reducing equivalents [90]. So according to this study, low increases in PH2 should

not have an effect on the ability of a chain elongating mixed culture to oxidize ethanol. This is in line with

what was seen in this experiment, where elevated PH2 up to 1atm did not influence the oxidation of ethanol,

but on the other hand, lowering the PH2 did not increase ethanol consumption rates either, which the same

source suggests it should. This finding lowered the expectation that the high PH2 achieved in the pilot

reactor was responsible for the decreased ethanol consumption.

Another difference which was observed between the HI and Lo reactors was the manner in which the VFA-

COD was distributed between SCCAs and MCFAs, but also in the distribution between odd- and even-

chain VFAs. In the HI reactors, more VFA COD was in the form of MCFAs compared to in the form of

SCCAs. In PiHI, 44.72±2.25% of the VFAs produced from day 24 onwards were in the form of SCCAs,

compared to PiLO, where 58.02±3.15% of VFA COD was in the form of SCCAs (p=0.005, paired t-test).

This is recurrent in the EC reactors, with ECHI possessing 47.73±5.76% of its VFA COD in the form of

SCCAs, and ECLO 56.74%±1.77. As for the odd-even distribution, 84.05±1.65% of COD in PiHI was in

the form of even-chained products, whilst in PiLO even-chained VFAs took up 79.91±1.01% of total VFA

COD. In ECHI and ECLO, a similar result is apparent, with 83.40±2.60 and 78.64±1.93% respectively of

VFA-COD in the form of even chained VFAs.

When looking at the effect of PH2 on chain length of produced VFA-COD, it has been established that at

higher hydrogen partial pressures, acetate is more readily converted to butyrate [91, 92], and butyrate in

turn to caproate, although in the case of the latter, it is only up to the point where caproate starts inhibiting

micro-organisms [93], after which hydrogen accumulation can be observed. In another study, it was

hypothesized that in order to maintain high caproate production rates, lower hydrogen partial pressures

47

would be advisable, but that an increase in PH2 would push the chain elongation process to longer chained

molecules [54].

When regarding the distribution between odd-and even chain VFAs, it is suggested that higher PH2 would

induce a greater propionate production, which would lead to more odd-chain VFAs [65, 66]. However, in

this experiment the opposite was found, where higher PH2 seemed to push the chain elongation more

towards even-chain elongation. After extensive research, a possible cause for this phenomenon was found.

As stated before, odd-chain VFAs are produced by chain elongation bacteria when propionate instead of

acetate is used as the starting molecule, which is then further elongated to larger odd-chained VFAs.

Propionate is a common hydrolization product of proteins, which are a major constituent in thin stillage,

where up to 19% of dry matter in thin stillage derived from corn consists of crude protein[94]. It has been

observed before, that at high hydrogen partial pressures, hydrolization of substrates including proteins is

inhibited [95]. This could be a plausible explanation for why more odd-chain VFAs were observed in the

LO reactors when compared to the HI reactors.

Conclusions:

• Lower or higher hydrogen partial pressure did not affect ethanol consumption. Under the

conditions, no ethanol consumption occurred even when the inoculum was derived from en

enrichment culture known to be positive for chain elongation through ethanol.

• Higher hydrogen partial pressures were beneficial for steering chain elongation towards the

production of MCFAs.

• Higher hydrogen partial pressures caused a greater amount of VFA-COD to be produced in the

form of even-chain products.

Inhibition of ethanol oxidation by the feed matrix After the first experiment, where the possibility of hydrogen pressure being responsible for the lack of

ethanol consumption was investigated, a second experiment was designed to investigate the second

hypothesis, namely whether there were components in the matrix of the feed which would inhibit ethanol

consumption such as phenolics, melanoidins and furfurals [15].

In the primary run of this experiment, reliable results were gained from the 0% broth through 4, which

showed a clear progression in parameters such as initial acetate and propionate production rates, subsequent

acetate and ethanol consumption rates as well as butyrate and caproate production rates. Because the series

with 100% broth broke the trend in every single one of these cases, and because a rerun of this experiment

was performed, for the discussion on the results of the primary run, the series with 100% broth will be left

out of consideration. The reason as to why the 100% broth series behaved in a different trend as opposed to

the other series is unknown, but could be worth investigating further, but in this case, the experiment was

simply done over.

Starting with the progression of ethanol consumption in the 0-50% broth series, figures 8 and 9 show

without a doubt that the higher the concentration of Pilot fermentation broth in fermentation medium is, the

lower the maximum ethanol consumption rate is. However, contrary to what was expected to be observed,

there was an (almost) complete consumption of ethanol in every batch series, whilst there was an

expectation of there being a much more inhibitory effect of the pilot fermentation broth on ethanol

consumption, along the lines of what was seen in the Influence of PH2 on MCFA production and ethanol

consumption experiment where no discernable amount of ethanol was consumed. Therefore, in the primary

run of this experiment, although we can say with relative assurance that the pilot fermentation broth does

inhibit ethanol consumption to some extent, the matrix of the pilot fermentation broth used was not the key

48

inhibitor. In the end, we came upon a single hypothesis, namely that it could be the presence of the solids in

the Influence of PH2 on MCFA production and ethanol consumption experiment which were not present in

the primary run of this experiment, which are the ones responsible for the inhibited ethanol consumption.

And thus the secondary run of this experiment was carried out, with an extra series where solids from the

pilot fermentation liquid were added to a SM fermentation medium. In the end, by comparing the ethanol

consumption rates and net consumption, it was seen that the presence of solids reduced net consumption of

ethanol by more than half, and reduced the maximum ethanol consumption rate by over fivefold. However,

this inhibitory effect caused by the solids was a lot less when compared to the inhibitory effect of the feed

matric in the secondary run. In the secondary run, the series with 50% broth had a maximum ethanol

consumption rate of 0.57gCOD/L.d and the series with 100% broth only 0.22gCOD/L.d. In the secondary

run of this experiment, net ethanol consumption and maximum consumption rates were a lot lower when

compared to the primary run. Seeing as how this phenomenon also took place in the 0%broth series, the

most likely cause is that the continuous reactor from whence the inoculum was derived was in a period of

stress, and therefore the mixed culture from there was less metabolically active.

As for the effects which the Pilot fermentation broth and solids derived from the Pilot had on the other

fermentation medium composites, figures 10 and 16 portray very different stories, and support the claim

that the inoculum for both runs, despite being obtained from the same source, had very different

characteristics. In the primary run, initial acetate and propionate production rates decreased as the

concentration of Pilot fermentation broth in the fermentation liquid increased, whilst the opposite was

observed in the secondary run, although in both runs the 50% broth series did not follow the trend, the other

series did. Combined with figures 9 and 15, it can be assumed that this difference in trends is due to the

lagphase during which the inoculum was forced to adapt to the new environment. In the first run, where

there was a healthy inoculum, the initial acetate and propionate production rates likely remained low in the

series with low Pilot fermentation broth due to the inoculum having the ability of producing MCFAs after

only 1 day. This is in contrast to the secondary run, where the lagphase had a duration of 2-3 days, which

means that during the lagphase, the only way to procure energy was by oxidizing ethanol to acetate. In both

cases, the evidence shown by figures 10 and 16 show that the 0%broth series had a significant advantage

over the 100%broth series.

Looking back once more at figures 9 and 15, it can be seen that the net production of caproate was highest

in the 0% broth series, followed by the series with less Pilot fermentation broth (leaving the 100%broth

series from the primary run aside). In contrast, butyrate accumulation occurred in the series with higher

Pilot fermentation broth concentrations, except for the 50% and 100% broth series in the secondary run,

which did not experience any major change in composition of the fermentation medium. The results from

the primary run show that although chain elongation is possible in the Pilot fermentation broth, this

primarily extends to the elongation of acetate to butyrate, and only a little bit of elongation from butyrate to

caproate, and in the secondary run, that chain elongation in Pilot fermentation broth requires either a strong

inoculum, or a modification of the matrix.

Conclusions:

• The presence of Pilot fermentation broth has a negative effect on ethanol consumption, confirming

the hypothesis that there are inhibitory components present in the thin stillage matrix.

• Production of MCFAs through chain elongation is also inhibited by components present in the Pilot

fermentation broth matrix, with accumulation of butyrate occurring in the series with higher broth

concentrations compared to caproate production in the series with lower broth concentrations.

49

Continuous fermentation of solid-free thin stillage

Phase 1: feeding solid-free undiluted thin stillage

During the first phase of this experiment, a heterogeneous and constantly changing feed was fed to the two

reactors. This meant that throughout the experiment, no truly stable state was achieved, however, from

phase 1, from day 21-28, an average butyrate and caproate production rate of 0.40±0.03 and

0.32±0.08gCOD/L.d was achieved in R1, whilst for R2, the values were 0.31±0.03 and

0.26±0.05gCOD/L.d were obtained for butyrate and caproate respectively. Maximum production rates of

caproate were 0.68 and 0.85gCOD/L.d for R1 and R2 respectively during the same period. When looking at

octanoate, a maximum production rate of 0.09gCOD/L.d was observed in R1, compared to 0.11gCOD/L.d

in R2. When compared to other studies where MCFAs were produced through chain elongation in a

fermenter fed with a complex “real” feed, we can see that this base-line which was obtained during phase 1

did not lack when compared to certain other studies. In an experiment as performed by Steinbusch et al

[49], a maximum caproate production rate of 1.09gCOD/L.d was achieved. Towards the end of phase 1,

octanoate concentrations increased to 0.89 and 1.17gCOD/L from days 10 and 7 onwards in R1 and R2

respectively, suggesting that the bacterial communities were acclimatizing to the environment and the feed.

However, as seen in the course of the TSS in the two reactors during phase 1, no increase in TSS was

observed during phase 1. These results combined indicate that at pH 5, where 58.55% of caproate is

theoretically present in its dissociated form, no significant metabolic inhibition takes place, however the

cellular growth might be inhibited to the point where an HRT of 10 days did not allow for a net increase in

biomass in the reactors.

Phase 2: Sludge retention

During this phase, a ceramic membrane was attached to R1, in the hope of seeing an increase in biomass

accumulation in R1 by increasing the SRT. When applying an SRT of 20 days (2xHRT), still no increase in

biomass was seen in R1. It was hypothesized that the pH would be the determining factor for increasing

biomass growth, both by reducing the ratio of caproate present in its undissociated form, as well as

improving the acidity of the fermentation broth to a more suitable level for chain elongating bacteria. On

day 47, the pH in both reactors was increased to 5.5, which still did not produce any result in increasing the

TSS in either reactor.

As illustrated in figure 22, on day 42, a large spike of ethanol concentration was registered in the feed.

After this time period, where 18.6±0.51gCOD/L of ethanol was fed into both reactors, spikes in caproate

production were achieved, with maximum caproate production rates of 2.50 and 3.70gCOD/L.d were

observed in R1 and R2 respectively. These production rates showed that, in contrast to the pilot reactor, the

mixed cultures in the two reactors were capable of consuming roughly a third of the ethanol introduced into

the reactors. This finding fits with what was seen in the secondary run of inhibition of ethanol oxidation by

the feed matrix, where the batch series with 0% broth with solids (0%S) showed a decreased ethanol

consumption, drawing a comparison between the 0%S broth series and the pilot, which was fed with

undiluted thin stillage with solids. When the caproate reached its maximum concentration in both reactors,

these were 13.46gCOD/L (6.11g/L) in R1 and 19.53gCOD/L (8.86g/L)in R2, with 77.6% being in its

dissociated form. This means that in R1, 1.37g/L and in R2, 1.98g/L of undissociated caproic acid were

present at these times. These values were already well above the theoretical inhibition/toxicity values, and

as such, direct electrochemical extraction needed to be implemented so as to avoid continuous inhibition.

Indeed, after these peaks were attained, caproate production in R2 dropped for more than 20 consecutive

days, hinting at a decreasing chain-elongating biomass presence in the reactor. In an effort to save the

experimental reactor R1 at least, from the excessive MCFA concentrations, Direct electrochemical

extraction was implemented.

50

Phase 3: Direct electrochemical extraction

In phase 3, the direct electrochemical cell suffered from multiple breakdowns, which affected not only the

extraction rates, but also the stability of the reactor R1 whenever the middle compartment mixed with the

cathode compartment, resulting in a steep increase in acidic, concentrated VFA solution entering the

reactor. The electrochemical cell had to be restarted 6 times in total. In the first trial(d60-67),

concentrations of VFAs stabilized at relatively low concentrations, for an unknown reason. In the second

trial(d67-76), VFA concentrations quickly reached higher levels when compared to the first trial, but

caproate concentration stabilized at around 5.5g/L, which, assuming everything was present in its

undissociated form, would still be well below the concentration needed to achieve phase separation (10g/L

undissociated caproate). In the third trial (d79-88), even higher concentrations were achieved, with

maximum caproate concentration of 8.49g/L. During this third trial, the formation of an oil layer was

observed, however, the middle compartment was fouled before harvesting, quantification and analysis of

the oil layer was possible. The fourth trial (d88-95) was when 15.1g of oil was able to be harvested for the

first time, at the end, after the oil was harvested, however, the middle compartment fouled once more due

to unknown reasons. In the fifth trial (d96-101), an oil phase layer developed at a more rapid pace than in

previous instances, and 6.7g of oil was harvested. After harvesting, the anion exchange membrane ripped,

causing a complete mixing of the middle compartment and the cathode compartment, which caused an

influx of organic acids and other anions into the reactor. This caused severe problems in the reactor itself,

where caproate concentrations in R1 dropped from above 4g/L to 2.24±0.10g/L, octanoate concentrations

dropped from above 0.3g/L to 0.20±0.01g/L.

During the course of phase 3, other observations were made. For instance, acetate concentrations in the

reactor (not accounting for the middle compartment) rose dramatically from day 60, the start of phase 3,

dramatically. Acetate concentrations during phase 2 remained below 1g/l, except for a solitary peak on day

52. After applying direct electrochemical extraction, acetate concentrations increased. During the first trial

of the electrochemical cell, acetate concentrations did not increase, but from day 68 onwards,

concentrations remained continuously above 1.5g/L, and went as high as 2.99g/L, and from day 94

onwards, concentration increased in an exponential manner from 1.88g/L to 4.63g/L on the final day.

Neither of these observations were seen in R2, where acetate concentrations remained below 2.26g/L

during phase 3. When taking into account the acetate extracted from R1 into the middle compartment of the

electrochemical cell, then it can be seen that the concentration of acetate in R1+MC is significantly higher

than in R2 throughout the duration of phase 3, with the opposite being the case throughout phase 2. This

indicates that the functionality of the electrochemical cell was an impediment to the consumption of

acetate, and, by extension, chain elongation.

Butyrate concentrations in R1 were between 2 and 2.5g/L during phase 2, and remained almost constantly

below 1.7g/L during phase 3, whilst butyrate concentrations in R2 increased (after a period of product

inhibition after the ethanol spike in phase 2) from above 2g/L to almost 4g/l, and even 4.7 on day 87. This

means that during phase 3, most likely due to the repeated failing of the electrochemical cell, butyrate

concentrations decreased in R1, supporting the earlier finding that the extraction limited chain elongation.

Caproate concentrations inside R1 fluctuated vigorously during phase 3, and no clear correlation between

this fluctuation and the direct electrochemical extraction can be seen. As with butyrate, caproate

concentrations and production rates were higher in R2 than in R1

Octanoate concentrations in R1 increased after the ethanol spike occurred during phase 2 from around

0.5g/L to just below 2.2g/L on day 73. Afterwards, during the remainder of phase 3, octanoate

51

concentrations continuously dropped until a final concentration of around 0.2g/L. This is in contrast to what

was seen in R2, where we see the concentration of octanoate increase for the duration of phase 3, from

1.55gCOD/L on day 60 to 3.63gCOD/L on day 105.

Conclusions:

• Caproate or MCFA production in a mixed-culture fermentation process fed with solid-free thin

stillage is possible. At a pH of 6, with an HRT of 10 days, no sludge retention and no in-line

extraction, production rates of 1.48±0.17gCOD/L.d caproate and 0.33±0.04gCOD/L.d of octanoate

were achieved.

• No net-growth of biomass was observed when the reactor was kept at 5 or5.5, regardless of

whether sludge retention was applied or not. This means that even if MCFA production is

stimulated under more acidic conditions, biomass growth will be stifled [97], and in danger of

washing out in continuous fermentation. Net biomass growth was only observed when the pH was

increased to 6.

• Ethanol consumption was observed during phase 2, where 37-38% of ethanol present in the feed

was consumed, increasing caproate production rates from 0.27 and 0.04gCOD/L.d to 2.50 and

3.7gCOD/L.d in R1 and R2 respectively. However, little no ethanol consumption was observed

during phase 3, even though there was 3.5-6.5gCOD/L present in the feed. This suggests that there

is a minimum ethanol concentration needed before it is used as a substrate for chain elongation.

• The fact that there was at most 37-38% consumption of ethanol means that there are inhibitory

components present in the solid-free thin stillage, reinforcing the findings from inhibition of

ethanol oxidation by the feed matrix.

• In the event that direct electrochemical extraction cannot be maintained well, and function without

failure for a long period of time, there can be severe impacts on the fermentative process,

decreasing production rates.

52

Bibliography 1. Agency, U.S.E.I., International Energy Outlook 2016 2016. 2. Eurostat. Water Statistics. 2015 [cited 2017 8/4]; Available from:

http://ec.europa.eu/eurostat/statistics-explained/index.php/Water_statistics. 3. Dodson, J.R., et al., Elemental sustainability: Towards the total recovery of scarce metals.

Chemical Engineering and Processing, 2012. 51: p. 69-78. 4. Das, A., A. Vidyadhar, and S.P. Mehrotra, A novel flowsheet for the recovery of metal values from

waste printed circuit boards. Resources, Conservation and Recycling, 2009. 53(8): p. 464-469. 5. Lastella, G., et al., Anaerobic digestion of semi-solid organic waste: biogas production and its

purification. Energy Conversion and Management, 2002. 43(1): p. 63-75. 6. Fabiani, C., et al., Treatment of waste water from silk degumming processes for protein recovery

and water reuse. Desalination, 1996. 105(1): p. 1-9. 7. Visakh, P.M. and S. Thomas, Preparation of Bionanomaterials and their Polymer Nanocomposites

from Waste and Biomass. Waste and Biomass Valorization, 2010. 1(1): p. 121-134. 8. YourDictionary, biomass, in Your Dictionary. 2017: Web. 9. Mor, S., et al., Municipal solid waste characterization and its assessment for potential methane

generation: A case study. Science of The Total Environment, 2006. 371(1): p. 1-10. 10. Staley, B.F. and M.A. Barlaz, Composition of Municipal Solid Waste in the United States and

Implications for Carbon Sequestration and Methane Yield. Journal of Environmental Engineering, 2009. 135(10): p. 901-909.

11. Noor, Z.Z., et al., An overview for energy recovery from municipal solid wastes (MSW) in Malaysia scenario. Renewable & Sustainable Energy Reviews, 2013. 20: p. 378-384.

12. Faaij, A., et al., Characteristics and availability of biomass waste and residues in the Netherlands for gasification. Biomass & Bioenergy, 1997. 12(4): p. 225-240.

13. Eurostat. Afvalstatistieken. 2017 [cited 2017 17-08]. 14. Healy, M.G.C., et al., Resource recovery from sewage sludge. Sewage treatment plants: economic

evaluation of innovative technologies for energy efficiency, ed. K.P.T. K. Konstantinos. 2015: IWA. 15. Wilkie, A.C., K.J. Riedesel, and J.M. Owens, Stillage characterization and anaerobic treatment of

ethanol stillage from conventional and cellulosic feedstocks. Biomass and Bioenergy, 2000. 19(2): p. 63-102.

16. Noukeu, N.A., et al., Characterization of effluent from food processing industries and stillage treatment trial with Eichhornia crassipes (Mart.) and Panicum maximum (Jacq.). Water Resources and Industry, 2016. 16: p. 1-18.

17. Kim, Y., et al., Composition of corn dry-grind ethanol by-products: DDGS, wet cake, and thin stillage. Bioresource Technology, 2008. 99(12): p. 5165-5176.

18. Djukić-Vuković, A.P., et al., Effective valorisation of distillery stillage by integrated production of lactic acid and high quality feed. Food Research International, 2015. 73: p. 75-80.

19. Markovic, M., et al., The Possibility of Lactic Acid Fermentation in the Triticale Stillage. Chemical Industry & Chemical Engineering Quarterly, 2011. 17(2): p. 153-162.

20. Eskicioglu, C., et al., Anaerobic digestion of whole stillage from dry-grind corn ethanol plant under mesophilic and thermophilic conditions. Bioresource Technology, 2011. 102(2): p. 1079-1086.

21. Anderson, J.L., et al., Evaluation of Dried and Wet Distillers Grains Included at Two Concentrations in the Diets of Lactating Dairy Cows1. Journal of Dairy Science, 2006. 89(8): p. 3133-3142.

22. McKinnon, J.J. and A.M. Walker, Comparison of wheat-based dried distillers’ grain with solubles to barley as an energy source for backgrounding cattle. Canadian Journal of Animal Science, 2008. 88(4): p. 721-724.

23. Cromwell, G.L., K.L. Herkelman, and T.S. Stahly, Physical, chemical, and nutritional characteristics of distillers dried grains with solubles for chicks and pigs. Journal of animal science, 1993. 71(3): p. 679-686.

24. Ezeji, T. and H.P. Blaschek, Fermentation of dried distillers’ grains and solubles (DDGS) hydrolysates to solvents and value-added products by solventogenic clostridia. Bioresource Technology, 2008. 99(12): p. 5232-5242.

25. Lau, M.W., B.E. Dale, and V. Balan, Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation. Biotechnology and Bioengineering, 2008. 99(3): p. 529-539.

http://ec.europa.eu/eurostat/statistics-explained/index.php/Water_statistics

53

26. Nasr, N., et al., Comparative assessment of single-stage and two-stage anaerobic digestion for the treatment of thin stillage. Bioresource Technology, 2012. 111: p. 122-126.

27. Moestedt, J., et al., Biogas Production from Thin Stillage on an Industrial Scale—Experience and Optimisation. Energies, 2013. 6(11): p. 5642.

28. Liu, K. and K.A. Rosentrater, Distillers Grains: Production, Properties, and Utilization, ed. C. Press. 2016: CRC Press. 564.

29. Djukić-Vuković, A.P., et al., Lactic acid production on liquid distillery stillage by Lactobacillus rhamnosus immobilized onto zeolite. Bioresource Technology, 2013. 135: p. 454-458.

30. Bento, J.M.A. and H.L. Fleming, Membrane-based process for the recovery of lactic acid and glycerol from a "corn thin stillage" stream. 1993, Google Patents.

31. Yen, H.-W., Y.-C. Yang, and Y.-H. Yu, Using crude glycerol and thin stillage for the production of microbial lipids through the cultivation of Rhodotorula glutinis. Journal of Bioscience and Bioengineering, 2012. 114(4): p. 453-456.

32. Ahn, J.-H., B.-I. Sang, and Y. Um, Butanol production from thin stillage using Clostridium pasteurianum. Bioresource Technology, 2011. 102(7): p. 4934-4937.

33. Biebl, H., Fermentation of glycerol by Clostridium pasteurianum — batch and continuous culture studies. Journal of Industrial Microbiology and Biotechnology, 2001. 27(1): p. 18-26.

34. Gonzalez, R., P. Campbell, and M. Wong, Production of ethanol from thin stillage by metabolically engineered Escherichia coli. Biotechnology Letters, 2010. 32(3): p. 405-411.

35. Hsieh, C., T.-H. Hsu, and F.-C. Yang, Production of polysaccharides of Ganoderma lucidum (CCRC36021) by reusing thin stillage. Process Biochemistry, 2005. 40(2): p. 909-916.

36. Agler, M.T., et al., Waste to bioproduct conversion with undefined mixed cultures: the carboxylate platform. Trends in Biotechnology, 2011. 29(2): p. 70-78.

37. Cope, J.L., et al., Evaluating the performance of carboxylate platform fermentations across diverse inocula originating as sediments from extreme environments. Bioresource Technology, 2014. 155: p. 388-394.

38. Wu, G.H., et al., Stepwise engineering to produce high yields of very long-chain polyunsaturated fatty acids in plants. Nature Biotechnology, 2005. 23(8): p. 1013-1017.

39. Holtzapple, M.T. and C.B. Granda, Carboxylate Platform: The MixAlco Process Part 1: Comparison of Three Biomass Conversion Platforms. Applied Biochemistry and Biotechnology, 2009. 156(1-3): p. 525-536.

40. Varrone, C., et al., Comparison of Different Strategies for Selection/Adaptation of Mixed Microbial Cultures Able to Ferment Crude Glycerol Derived from Second-Generation Biodiesel. BioMed Research International, 2017. 2017: p. 1.

41. Dengate, S. and A. Ruben, Controlled trial of cumulative behavioural effects of a common bread preservative*. Journal of Paediatrics and Child Health, 2002. 38(4): p. 373-376.

42. Finch, C.A., Polyvinyl alcohol; properties and applications. 1973: John Wiley & Sons. 43. Serop, M., Cellulose acetate butyrate semipermeable membranes and their production. 1971,

Google Patents. 44. Weiss, S.C., et al., A randomized controlled clinical trial assessing the effect of betamethasone

valerate 0.12% foam on the short-term treatment of stasis dermatitis. Journal of drugs in dermatology : JDD, 2005. 4(3): p. 339-345.

45. Wang, J., et al., Anaerobic Fermentation for Production of Carboxylic Acids as Bulk Chemicals from Renewable Biomass, in Anaerobes in Biotechnology, R. Hatti-Kaul, G. Mamo, and B. Mattiasson, Editors. 2016, Springer International Publishing: Cham. p. 323-361.

46. Schäfer, T. and P. Schönheit, Maltose fermentation to acetate, CO2 and H2 in the anaerobic hyperthermophilic archaeon Pyrococcus furiosus: evidence for the operation of a novel sugar fermentation pathway. Archives of Microbiology, 1992. 158(3): p. 188-202.

47. Chen, Y., et al., High-purity propionate production from glycerol in mixed culture fermentation. Bioresource Technology, 2016. 219: p. 659-667.

48. Berlett, B.S. and E.R. Stadtman, Protein Oxidation in Aging, Disease, and Oxidative Stress. Journal of Biological Chemistry, 1997. 272(33): p. 20313-20316.

49. Liu, K., et al., Continuous syngas fermentation for the production of ethanol, n-propanol and n-butanol. Bioresource Technology, 2014. 151: p. 69-77.

50. Knifton, J.F., Syngas reactions. Journal of Catalysis, 1985. 96(2): p. 439-453.

54

51. Ding, Y.-H., W. Huang, and Y.-G. Wang, Direct synthesis of acetic acid from CH4 and CO2 by a step-wise route over Pd/SiO2 and Rh/SiO2 catalysts. Fuel Processing Technology, 2007. 88(4): p. 319-324.

52. Ganigue, R., et al., Microbial electrosynthesis of butyrate from carbon dioxide. Chemical Communications, 2015. 51(15): p. 3235-3238.

53. Buasri, A., et al., Biodiesel production from crude palm oil with a high content of free fatty acids and fuel properties. Vol. 8. 2009. 115-124.

54. Angenent, L.T., et al., Chain Elongation with Reactor Microbiomes: Open-Culture Biotechnology To Produce Biochemicals. Environmental Science & Technology, 2016. 50(6): p. 2796-2810.

55. Mancini, A., et al., Biological and Nutritional Properties of Palm Oil and Palmitic Acid: Effects on Health. Molecules, 2015. 20(9): p. 17339-17361.

56. Wasewar, K.L. and D.Z. Shende, Extraction of Caproic Acid Using Tri-n-butyl Phosphate in Benzene and Toluene at 301 K. Journal of Chemical and Engineering Data, 2010. 55(9): p. 4121-4125.

57. Adam, G.A. and S.A. Needham, Antimicrobial polymers and methods for their production. 2015, Google Patents.

58. Sun, C.Q., C.J. O'Connor, and A.M. Roberton, The antimicrobial properties of milkfat after partial hydrolysis by calf pregastric lipase. Chemico-Biological Interactions, 2002. 140(2): p. 185-198.

59. Gabbe, S., Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate (vol 348, pg 2379, 2003). New England Journal of Medicine, 2003. 349(13): p. 1299-1299.

60. Yalkowsky, S.H., Y. He, and P. Jain, Handbook of: Aqueous Solubility Data. Handbook of, ed. C. Press. 2010.

61. Marounek, M., K. Fliegrova, and S. Bartos, Metabolism and Some Characteristics of Ruminal Strains of Megasphaera-Elsdenii. Applied and Environmental Microbiology, 1989. 55(6): p. 1570-1573.

62. Lundquist, F., et al., Ethanol metabolism and production of free acetate in the human liver. J Clin Invest, 1962. 41: p. 955-61.

63. Steinbusch, K.J.J., et al., Biological formation of caproate and caprylate from acetate: fuel and chemical production from low grade biomass. Energy & Environmental Science, 2011. 4(1): p. 216-224.

64. Van Eerten-Jansen, M.C.A.A., et al., Bioelectrochemical Production of Caproate and Caprylate from Acetate by Mixed Cultures (vol 1, pg 513, 2013). Acs Sustainable Chemistry & Engineering, 2013. 1(8): p. 1069-1069.

65. Cavalcante, W.d.A., et al., Anaerobic fermentation for n-caproic acid production: A review. Process Biochemistry, 2017. 54: p. 106-119.

66. Kucek, L.A., M. Nguyen, and L.T. Angenent, Conversion of L-lactate into n-caproate by a continuously fed reactor microbiome. Water Research, 2016. 93: p. 163-171.

67. Zhu, X.Y., et al., The synthesis of n-caproate from lactate: a new efficient process for medium-chain carboxylates production. Scientific Reports, 2015. 5.

68. Gonzalez-Cabaleiro, R., et al., Linking thermodynamics and kinetics to assess pathway reversibility in anaerobic bioprocesses. Energy & Environmental Science, 2013. 6(12): p. 3780-3789.

69. Spirito, C.M., et al., Chain elongation in anaerobic reactor microbiomes to recover resources from waste. Current Opinion in Biotechnology, 2014. 27: p. 115-122.

70. Zhou, Z., Q. Meng, and Z. Yu, Effects of Methanogenic Inhibitors on Methane Production and Abundances of Methanogens and Cellulolytic Bacteria in In Vitro Ruminal Cultures. Applied and Environmental Microbiology, 2011. 77(8): p. 2634-2639.

71. Zhuang, L., et al., Methanogenesis Control using 2-Bromoethanesulfonate for Enhanced Power Recovery from Sewage Sludge in Air-cathode Microbial Fuel Cells. International Journal of Electrochemical Science, 2012. 7.

72. Van Kessel, J.A.S. and J.B. Russell, The effect of pH on ruminal methanogenesis. FEMS Microbiology Ecology, 1996. 20(4): p. 205-210.

73. Ge, S., et al., Long-Term n-Caproic Acid Production from Yeast-Fermentation Beer in an Anaerobic Bioreactor with Continuous Product Extraction. Environ Sci Technol, 2015. 49(13): p. 8012-21.

74. Vasudevan, D., H. Richter, and L.T. Angenent, Upgrading dilute ethanol from syngas fermentation to n-caproate with reactor microbiomes. Bioresource Technology, 2014. 151: p. 378-382.

55

75. Huang, C.B. and J.L. Ebersole, A novel bioactivity of omega-3 polyunsaturated fatty acids and their ester derivatives. Molecular Oral Microbiology, 2010. 25(1): p. 75-80.

76. van den Heuvel, J.C., et al., Determination of the Critical Concentration of Inhibitory Products in a Repeated Fed-Batch Culture. Biotechnology Techniques, 1992. 6(1): p. 33-38.

77. Kucek, L.A., et al., Waste Conversion into n-Caprylate and n-Caproate: Resource Recovery from Wine Lees Using Anaerobic Reactor Microbiomes and In-line Extraction. Frontiers in Microbiology, 2016. 7.

78. Fasahati, P. and J. Liu, Techno-economic analysis of production and recovery of volatile fatty acids from brown algae using membrane distillation. Proceedings of the 8th International Conference on Foundations of Computer-Aided Process Design, 2014. 34: p. 303-308.

79. Reyhanitash, E., et al., Extraction of volatile fatty acids from fermented wastewater. Separation and Purification Technology, 2016. 161: p. 61-68.

80. Xu, J., et al., In-line and selective phase separation of medium-chain carboxylic acids using membrane electrolysis. Chemical Communications, 2015. 51(31): p. 6847-6850.

81. Andersen, S.J., et al., Electrolytic extraction drives volatile fatty acid chain elongation through lactic acid and replaces chemical pH control in thin stillage fermentation. Biotechnology for Biofuels, 2015. 8.

82. Andersen, S.J., et al., Electrolytic Membrane Extraction Enables Production of Fine Chemicals from Biorefinery Sidestreams. Environmental Science & Technology, 2014. 48(12): p. 7135-7142.

83. Bermeo, M.A., et al., Use of electrodialysis as a vfa recovery process From acidogenic of msw synthetic leachates. 2014.

84. Zhang, J., et al., Simultaneous Saccharification and Ethanol Fermentation at High Corn Stover Solids Loading in a Helical Stirring Bioreactor. Biotechnology and Bioengineering, 2010. 105(4): p. 718-728.

85. Chen, W.S., et al., Production of Caproic Acid from Mixed Organic Waste: An Environmental Life Cycle Perspective. Environmental Science & Technology, 2017. 51(12): p. 7159-7168.

86. Scalbert, A., Antimicrobial properties of tannins. Phytochemistry, 1991. 30(12): p. 3875-3883. 87. Junicke, H., et al., Impact of the hydrogen partial pressure on lactate degradation in a coculture of

Desulfovibrio sp. G11 and Methanobrevibacter arboriphilus DH1. Applied Microbiology and Biotechnology, 2015. 99(8): p. 3599-3608.

88. Collet, C., et al., Acetate production from lactose by Clostridium thermolacticum and hydrogen-scavenging microorganisms in continuous culture—Effect of hydrogen partial pressure. Journal of Biotechnology, 2005. 118(3): p. 328-338.

89. Yerushalmi, L., B. Volesky, and T. Szczesny, Effect of increased hydrogen partial pressure on the acetone-butanol fermentation by Clostridium acetobutylicum. Applied Microbiology and Biotechnology, 1985. 22(2): p. 103-107.

90. Grootscholten, T.I.M., et al., Two-stage medium chain fatty acid (MCFA) production from municipal solid waste and ethanol. Applied Energy, 2014. 116: p. 223-229.

91. van Andel, J.G., et al., Glucose fermentation byClostridium butyricum grown under a self generated gas atmosphere in chemostat culture. Applied Microbiology and Biotechnology, 1985. 23(1): p. 21-26.

92. de Kok, S., et al., Impact of dissolved hydrogen partial pressure on mixed culture fermentations. Applied Microbiology and Biotechnology, 2013. 97(6): p. 2617-2625.

93. Zhang, F., et al., Fatty acids production from hydrogen and carbon dioxide by mixed culture in the membrane biofilm reactor. Water Research, 2013. 47(16): p. 6122-6129.

94. Mustafa, A.F., J.J. McKinnon, and D.A. Christensen, The nutritive value of thin stillage and wet distillers' grains for ruminants - Review. Asian-Australasian Journal of Animal Sciences, 2000. 13(11): p. 1609-1618.

95. Cazier, E.A., et al., Biomass hydrolysis inhibition at high hydrogen partial pressure in solid-state anaerobic digestion. Bioresource Technology, 2015. 190: p. 106-113.

96. Sun, Y. and J. Cheng, Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology, 2002. 83(1): p. 1-11.

97. Ganigue, R., et al., Low Fermentation pH Is a Trigger to Alcohol Production, but a Killer to Chain Elongation. Frontiers in Microbiology, 2016. 7.

production of caproate from undiluted thin stillage · [type here] - 3 - abstract the search for...

Documents