Effect of limonene on the heterotrophic growth and

polyhydroxybutyrate production by Cupriavidus necator

H16

Authors

Guzman Lagunesa, F., Winterburna*, J.B.

a School of Chemical Engineering and Analytical Science, The Mill, The University of

Manchester, Manchester, M13 9PL, UK

*Corresponding author: James.Winterburn@manchester.ac.uk

Tel: +44(0)161 306 4891

Abstract

The inhibitory effect of limonene on polyhydroxybutyrate (PHB) production in

Cupriavidus necator H16 was studied. Firstly, results demonstrate the feasibility of

using orange juicing waste (OJW) as a substrate for PHB production. An intracellular

PHB content of 81.4 % (w/w) was attained for a total dry matter concentration of 9.58 g

L−1, when the OJW medium was used. Later, a mineral medium designed to mimic the

nutrient levels found in the complex medium derived from OJW was used to study the

effect of limonene on the production of PHB. Results showed a drop in specific growth

rate (μ) of more than 50% when the initial limonene concentration was 2% (v/v)

compared to the limonene free medium. This work highlights the importance of a

1

limonene recovery stage prior to fermentation, to maintain levels below 1 % (v/v) in the

medium, adding value to the OJW and enhancing the fermentation process productivity.

Keywords:

Limonene; inhibition; Cupriavidus necator; PHB; orange peel.

1. Introduction

The wider uptake and utilisation of microbially produced biopolymers is dependent on

our ability to efficiently and economically produce these polymers, ultimately to either

give significant benefits in applications, as biomaterials, or to be available at a price

comparable to oil derived polymers. In order to achieve either of these aims a low cost,

a widely available source of carbon substrate is required, along with sufficient

biorefining and bioprocessing techniques. Approximately 1.5 million tonnes of orange

peel waste from the juicing industry are available every year as a source of fermentable

fructose for PHB production (USDA, 2016).

The production of polyhydroxyalkanoates (PHAs) has been studied during the last few

decades, as an alternative to petrochemical polymers. PHAs are a group of polyesters

that can be synthesised by a range of different microbial strains as carbon and energy

reservoir under stress conditions. Cupriavidus necator is recognised as the model

microorganism for PHA production due to its capacity of accumulate up to 80% of total

dry weight in biopolymer and the simplicity of the metabolic pathway that involves

three enzymatic reactions (Choi and Lee, 1999). PHAs not only have similar mechanical

properties to polyethylene terephthalate (PET) or polypropylene (PP) (Koller et al.,

2

2010; Lee, 1996), but they are also susceptible to biodegradation and can be produced

from renewable raw materials thus reducing environmental impact and lowering

petroleum dependency. However, production costs are still a major disadvantage to a

wider use of PHAs and implementation of production strategies on a large scale

(Braunegg et al., 2004; Lee and Choi, 1998; Sudesh et al., 2000).

Many studies have been performed in order to improve productivities and reducing

production costs involving different fermentation strategies, microbial strains, upstream

and downstream processing (Chanprateep, 2010; Koller and Braunegg, 2015; Wang et

al., 2014). Moreover, the use of purified carbon sources accounts around 40% of the

total cost of production leading to several investigation groups to find alternative culture

media that can provide the conditions for the production of PHAs (Lee, 1996; Urtuvia et

al., 2014). Numerous lignocellulosic materials have been studied as potential raw

materials for the production of a medium rich in the nutrients necessary for fermentation

processes (Jain and Tiwari, 2015). These materials are often obtained as by-products

from other processes and are generally used as fuel for heat generation (Castilho et al.,

2009). The approach of such studies is to recover the fermentable sugars, to be used for

biopolymer production, through a pre-treatment step and then the solid residue can still

be used as a heat source (Kawaguchi et al., 2016; Oh et al., 2015; Tripathi et al., 2011).

One of the main lignocellulosic materials worldwide is produced by the citrus

processing industry, with orange juice being the main product (Angel Siles López et al.,

2010; Boukroufa et al., 2014). Approximately 50 million tonnes of orange fruit are

produced annually, with 3 million tonnes being used to produce fruit juice of which

3

about 1.5 million tonnes of unwanted waste material (OJW) are produced every year.

This waste includes the skin, pulp and seeds and accounts for about 50% (w/w) of the

total amount of oranges processed for juice, making it a very wasteful process and

representing a disposal challenge for the industries involved (USDA, 2016; Boukroufa

et al., 2014; Choi et al., 2013). Different strategies for the valorisation this waste as a

whole have been tested, from burning it as heat and power source to the use as hard

metals sieve in water treatment (Balu et al., 2012; Bampidis and Robinson, 2006;

Santos et al., 2015). The use of OJW as starting material for different biotechnological

processes is currently being assessed; however, the complexity of the composition, and

the presence of toxic substances, the process has not been successfully established

(Pourbafrani et al., 2007). The use of an autohydrolysate of orange peel (Rivas et al.,

2008) as complex medium rich in fructose for the production of PHAs by C. necator

H16 represents a new approach for the use of this carbon-rich material that has obtained

promising results. However, our preliminary studies revealed that even when fructose

concentration was close to the optimum found for a mineral medium (Aramvash et al.,

2015), a significant drop in the specific growth rate is triggered by increasing the

concentration of OJW solids at the beginning of the media preparation process. This

suggested that some inhibitory substance was accumulating in the medium.

OJW can contain up to 1.6 % (w/w) of orange essential oil (OEO), with important

applications in several industries, including food, cosmetics and pharmaceutical. This

essential oil accumulates in small oil sacs of 0.4 to 0.6 mm in diameter and is located at

irregular depths in the flavedo at the outer peel of the fruit (Angel Siles López et al.,

4

2010) and in addition to its characteristic smell it also has shown inhibitory effects on

the growth of several pathogenic strains (Zahi et al., 2015) (Muthaiyan et al., 2012;

Subramenium et al., 2015). Approximately 90% of the OEO consists of limonene, a

naturally occurring monoterpene; consequently, studies on the antimicrobial effect of

orange essential oil have focused on the limonene titration. According to literature,

concentrations as low as 0.05% can inhibit cell growth for bioethanol production (Choi

et al., 2013; Joshi et al., 2015). Furthermore, different approaches focused on the

holistic implementation of citrus wastes have highlighted the importance of recovery of

the OEO prior its biotechnological processing, enhancing the productivities of the

microbiological stage, and adding value to the starting material (Lohrasbi et al., 2010;

Ruiz and Flotats, 2014).

In this work, the biomass and PHB accumulation by C. necator H16 using an OJW

autohydrolysed medium were evaluated. The effect of limonene on the strain’s growth

kinetics was as well assessed with the objective to determine its tolerance to the main

component of OEO.

2. Materials and Methods

2.1.Microbial strain

Freeze dried C. necator H16, from the DSMZ-German Collection of Microorganisms

and Cell Cultures, DSM No. 428, was purchased and activated according to supplier

instructions. Master and working stock were created using MicroBankTM cryovial

system (Pro-Lab Diagnostics, UK) and kept at the −80 °C. Short term storage plates 5

consisting of nutrient agar (Sigma-Aldrich, UK) were prepared every time a batch of

experiments was started.

2.2. Media preparation

2.2.1. Orange juicing waste medium

The feasibility of using OJW as starting material for the production of PHAs was

assessed. OJW was obtained from a local juicing bar. Material was stored as received at

−20° C until used. The process proposed by Rivas et al. (2008) (Rivas et al., 2008) to

produce sugar rich medium from OJW was followed. After defrosting, the OJW was

submitted to a drying stage at 60° C for 48h and then ground using a standard food

processor. An extra run was performed using fresh material, whole and ground, to

measure the effect of the drying stage over the carbohydrates extraction process. Two

initial ratios of OJW solids to distilled water were used, 1:8 and 1:12 (w:w). A

hydrolysis stage was then performed using an autoclave where the temperature was

maintained at 121° C for 20 minutes. In order to determine the effect of the grinding

stage on the sugar concentration in the medium, both options, ground OJW and whole

OJW were tested during the extraction step. Solids were spun down using a centrifuge

Sigma 6-16S (Sigma, Germany) at 7000 rpm and supernatant was separated by

decantation. A 10M NaOH solution was used to adjust the initial pH of the media to a

value of 7.0 ± 0.2. Finally, solutions were sterilised by filtration using 0.2 μm

polyethersulfone (PES) membrane filtration units (Thermo Fisher Scientific Inc., UK)

and transferred to shake flasks for the fermentation experiments.

6

2.2.2. Mineral medium

The effect of limonene over the cell growth of C.necator H16 was studied adding

different concentrations of the terpene to the mineral media developed by Aramvash et

al. (2015) for the production of PHB. A basal mineral salt medium was prepared with

the following composition: KH2PO4 1.75 g L−1; MgSO4.7H2O 1.2 g L−1; NH4CL 2 g

L−1; citric acid 1.7 g L−1; trace elements solution 10 ml L−1. The trace elements

solution was composed of ZnSO4.7H2O 2.25 mg L−1; FeSO4.7H2O 10 mg L−1;

CaCl.2H2O 2 mg L−1; Na2B4O7.7H2O 0.23 mg L−1; (NH4)6Mo7O24 0.1 mg L−1;

CuSO4.5H2O 1 mg L−1; MnSO4.5H2O 0.6 mg L−1; HCl (35%) 10 mL L−1. Fructose was

used as the carbon source at a concentration of 25 g L−1. Salts, trace elements and

fructose solutions were prepared separately. All solutions were autoclaved at 121 °C

during 20 mins; once they reached room temperature, the tree solutions were mixed.

The initial pH of the medium was adjusted to 6.8. Limonene (Thermo Fisher Scientific,

UK) was filtered using a 0.2 μm PET membrane filter to assure sterility; the

corresponding quantity was then added to the mineral media to reach the concentration

required. Concentrations of 0, 0.5, 1, 1.5 and 2 % (v/v) of limonene were tested for this

work.

2.3.Cultivation conditions and inoculum preparation

For every experiment, a single colony from the short term storage stock was taken,

keeping aseptic conditions, and inoculated into 10 mL of nutrient broth No. 2 (Sigma-

Aldrich, UK) contained in 50 mL falcon tubes. Tubes were placed on an orbital shaker

where conditions were maintained at 30° C and 200 rpm. After 24 h of cultivation, an

7

adaptation stage was performed taking 2 mL of the broth to inoculate 20 ml of, either,

OJW-based medium or limonene-free mineral media contained in 50 ml falcon tubes

and cultivated for 48 h under the same conditions. Finally, for the limonene effect

experiments, 10 ml of the limonene-free medium were used to inoculate 100 ml of the

mineral media with limonene added, using 500 ml Erlenmeyer flasks. When working

with the OJW-based medium 10 ml of the adaptation stage broth were used to inoculate

100 ml of identical medium in 500 ml shake flasks. All experiments were run in

triplicate; results are presented as the mean, with error bars showing ± 1 standard

deviation.

2.4.Analytical methods

2.4.1. Partial OJW characterization

Total carbohydrates, crude protein, crude fibre and water content measurements were

carried out to the OJW in order to characterise the material. The phenol-sulphuric acid

method described by Nielsen was used for total carbohydrate determination (Nielsen,

2010). Standard procedures 954.01, 962.09 described in the Official Methods of

Analysis for the AOAC (AOAC, 1990) were followed for the determination of crude

protein and fibre. A protein factor of 6.25 was used to calculate the protein content.

Water content was determined by measuring the weight difference between fresh

material and the material after dried. Samples of fresh OJW were located into a drying

oven at 60° C during a period of 48 h.

8

2.4.2. Biomass measurements

Samples were taken periodically throughout the experiments and the cell density was

evaluated by optical density measurements at a wavelength (λ) of 600 nm (OD600),

using a spectrophotometer UVmini-1240 (Shimadzu, USA). The dry matter content of

fermentation media was measured by transferring approximately 2 mL of cell

containing broth into a pre-weighed 2 mL micro test tube (Eppendorf, DE), cells were

then spun down at 13,000 rpm for 10 minutes using an Eppendorf MiniSpin centrifuge

(Fisher Scientific, UK). The resulting supernatant was decanted and frozen to be used in

residual nutrient determinations. The remaining cell pellet was washed twice using

distilled water and then dried at 60C until constant weight was reached, 48 hours after.

Residual biomass concentration was calculated by the subtraction of the PHB

concentration from the total dry matter.

2.4.3. PHB determination

Gas chromatography (GC) was employed for PHB quantification according to the

method developed by Riis and Mai (1988) (Riis and Mai, 1988). A gas chromatography

system model 7820A (Agilent Technologies, USA) coupled with an autosampler

Combi/Pal from Varian was used for this study. A Poraplot Q-HT 10×32 mm column

was used and the detection system selected was a flame ionization detector (FID) set at

200° C. The injection volume and temperature were 1 μL and 230° C respectively.

Temperature program started at 120°C to be gradually increased during 3 minutes until

230° C, temperature was then held until finish the analysis. Helium was used as the

carrier gas. A calibration curve was prepared using purified PHB as a standard (Sigma-

9

Aldrich, UK) at different known concentrations. Peak areas of the samples were then

correlated to concentration using the calibration curve obtained.

2.4.4. Carbohydrate measurement

The concentration of fructose, glucose and sucrose, in the supernatant collected from

TDM samples, was determined using a Dionex Ultimate 3000 HPLC equipment. The

refractive index intensity of the samples was measured using a RefractoMax 521

(ThermoFisher Scientific, UK) detector, set at 50 C, peak area and concentration were

correlated using a calibration curve constructed by running standards of known

concentration. An Aminex HPX-87C Column was used to achieve the separation at a

temperature of 50 C. The mobile phase used was 5 mM sulphuric acid at a flow rate of

0.6 mL min−1. Samples were diluted 10 times to assure a good column performance

using HPLC grade water and filtered using nylon syringe filters 0.45 μm pore size prior

analysis.

2.4.5. Total nitrogen measurement

Total nitrogen quantification was performed using a Shimadzu TOC-VC equipment

coupled with both an ASI-V autosampler unit and a TNM-1 total nitrogen detector. A

calibration curve was created by the equipment, from a master solution of NH4Cl at a

concentration of 50 mg L−1. An aliquot of 750 μL of free solids supernatant was taken

to a final volume of 15 mL, required for the machine, and filtered through nylon syringe

filters 0.45 μm pore before injection.

10

3. Results and discussion

3.1.OJW as starting material for PHA production

Results for the water content of the material show a solids content around 20 ± 0.6 %

(w/w) for the OJW tested, this is similar to that observed by Pourbafrani et al.(2010),

20± 0.8 % of solids content, when working with citrus waste coming from a juice

factory (Pourbafrani et al., 2010). The difference in the water content removed from the

ground OJW and the whole “as juiced” material was around only 6.5 % (w/w) after 48 h

of drying, leading to the decision of grinding the material after the drying stage. The

composition of the OJW material was found to be similar to those reported in other

studies on the valorisation of this by-product. Results for the total carbohydrates and

crude fibre assays were 18.4 and 66. 31 %, respectively. This corresponds to those

reported by Rivas et al. [29] a total soluble sugars content of 16.9 % and a crude protein

of 63.05 %. The protein determination by Kjendhal digestion yielded a content of 7.22

%, slightly above the 6.50 % reported by Rivas et al.; this variation can be expected

when analysing natural materials of different origin.

The concentrations of carbohydrates measured in the supernatants obtained for the

different conditions tested are showed in table 1.The fructose concentration in the

aqueous extracts was improved by almost 15 % comparing the whole material to the

ground OJW, after the drying step. The maximum concentration of fructose obtained

was 24.74 g L−1 when ground OJW was used. The drying strategy simplifies the

handling of OJW, reducing the risk of microbial growth and, as the results show,

11

concentrating the target compounds in the solid fraction. The results also confirmed the

observations made in previous reports that have studied the effect of the particle size on

hydrolysis and extraction processes for citrus by-products, namely that the grinding

stage enhances carbohydrate recovery by increasing the surface area in contact with the

aqueous fraction (Agbor et al., 2011; Choi et al., 2013; Lopresto et al., 2014). The

process proposed in this contribution only focused on the effect of the grinding stage,

not taking into account the resulting particle size. Nevertheless, previous studies

focused on lignocellulosic materials show that reduction of particle size below 0.400

mm has little impact on the rates and yields of hydrolysis process (Agbor et al., 2011).

Three carbohydrate peaks were identified by HPLC analysis, glucose, fructose and

sucrose; consumption over time was determined from the difference in the peak areas.

Initial solids loading during media preparation led to a corresponding difference in the

fructose extracted from the peels. The autohydrolysis treatment proved effective for

fructose recovery, where Rivas et al. (2008) (Rivas et al., 2008) reported maximum

concentrations of 16 g L−1 of fructose, this study obtained 23 g L−1. Treatment with an

initial ratio of orange peel of 1:12 (w) lead to an initial fructose concentration of 14 g

L−1 and complete consumption was achieved after 72 h of fermentation. The depletion

of fructose for treatments with ratio 1:8, initial fructose concentration of 23 g L−1, was

not achieved for the frame time of the study, indicating that other nutrients were

limiting.

Figure 1 shows the cell growth curves as well as the PHB concentration time course for

the extraction treatments studied. The specific growth rate value for the media with an

12

initial solids load of 1:12 (w:v), (figure 1.b) reached the highest value for the different

treatments studied, 0.18 h−1, with an intracellular PHB percentage above 80%. These

values are similar to those obtained for C. necator H16 when grown in a phosphate

buffered medium, using organic acids as carbon source reaching a maximum PHA

content of 83.7 % (Yang et al., 2010). Other efforts have focused on the implementation

of glycerol as carbon source for PHA production, as this by-product of the biodiesel

process is available in great quantities. In 2012, Tanadchangsaeng and Yu growing C.

necator H16 in a mineral media added with 20 g L−1 of glycerol achieving a μmax of

0.11h−1 and 70% of PHB accumulation (Tanadchangsaeng and Yu, 2012). The

treatment with an initial ratio of solids of 1:8 (figure 1.a) exhibited slower growth for a

higher concentration of fructose, this can be related to some inhibitors present in the

broth as result of the extraction process conditions (Mohan et al., 2015; Talebnia et al.,

2007). A recent study showed that methanol contained in the crude glycerol can inhibit

the cell growth of C. necator DSM4058, while a μmax of 0.47 h−1 was obtained when 50

g L−1 of glycerol were used as carbon source in inhibition free conditions (Salakkam

and Webb, 2015).

A maximum dry matter concentration of 9.58 g L−1 with a percentage of PHB of 76 %

was achieved with a medium prepared from whole (non-ground) orange peel. However,

an increased intracellular PHB content, 80%, was reached for the same concentration of

initial solids, 1:8 (w:w), but adding the grinding stage. With yields on consumed

fructose (YX/S) of 0.41 and 0.39, whole and ground peel respectively. These results

indicate that the medium prepared with whole OJW generates provides slightly better

13

conditions for cell propagation, whereas ground OJW gives an improved PHB

production. Final biomass and intracellular polymer concentrations are comparable to

those attained by Aramvash et al. (2015), using a mineral medium with an initial

fructose concentration of 35 g L−1, reaching 7.48 g L−1 of PHB with a maximum of 90

% of PHB accumulation. Other strains have been tested on different wastes, Halomonas

campisialis is capable of using 84 % of the sugars in an orange peel based medium,

leading to a final PHB content of 42 % (w/w) corresponding to 0.33 g L−1 after 48 h of

fermentation, lower than those reported here (Kulkarni et al., 2015).

Residual biomass results show that after the first 30 hours the total matter reaches a

plateau stage and stays constant for a period of 40 h when it starts to increase again.

This growth coincides with a loss of intracellular PHB. This cessation of cell growth is

usually related to the nitrogen source reaching the limiting concentration, triggering

PHA accumulation at the same time (Koller et al., 2010; Rodríguez-Contreras et al.,

2015). An initial reduction of the total nitrogen concentration was observed during the

first stages of the fermentation with a corresponding accumulation of biomass, which

stayed constant after 40 % of the initial total nitrogen had been consumed, for all the

conditions tested. This indicates that only a part of the total nitrogen is accessible and

available to the microorganism (Haas et. al., 2015). This could be caused by the

Maillard reaction between sugars, proteins and peptides during autoclaving, or protein

conversion into a protease-resistant form. Bioavailable nitrogen was nonetheless the

limiting nutrient and the triggering factor for bacteria to switch its metabolism to PHB

14

production, as the synthesis of the polymer started when the nitrogen consumption, and

hence cellular growth, stopped. Table 2 presents a summary of all results obtained.

3.2.Effect of limonene on C. necator growth and PHB production

The variation of cell growth, fructose uptake, residual nitrogen and polymer production

over the course of the experiments is shown in figure 2 for the different limonene

concentrations tested. The experiments were carried as previously described and

proceeded as expected, with biomass and PHA production occurring within the first

72h. Higher initial levels of limonene led to lower titre readings for biomass and

polymer accumulation.

The biomass concentrations were followed through time for all limonene conditions

tested. For each limonene concentration different kinetic behaviour was exhibited by C.

necator H16. As the initial limonene concentration was increased, stronger growth

inhibition effect can be observed. The lag phase observed during the first hours of

fermentation increased considerably with the concentration of limonene, lasting around

40 h when the limonene concentration was 2 % (v/v) (figure 2.e). The final

concentrations of biomass and PHB were strongly affected by the presence of the

terpene, losing about 30% of intracellular PHB content when the initial concentration of

limonene rose from 1 to 1.5%.

Many studies on the antimicrobial effect of orange essential oil have centred on the

effect of limonene, as this terpene represents 90% (w/w) of OES (Di Pasqua et al.,

2006; Vuuren and Viljoen, 2007). In the present study, results for the cell growth and

15

PHB production of C. necator H16, indicate that the addition of limonene at

concentrations as low as 0.5% (v/v) has a negative effect, leading to a value in specific

growth rate 9% lower than those observed for a limonene free medium, dropping from

0.19 to 0.17 h−1. This agrees with previous studies testing the inhibitory effect of orange

essential oils and limonene against several organisms, which showed that the minimal

inhibitory concentration of limonene was in the range of 1-4% for Gram-negative

strains, while for the Gram-positive Staphylococcus aureus and Brochotrix

thermospacta a maximum sublethal concentration of 1.68 mg L−1 was determined (Ruiz

and Flotats, 2014).

The final concentrations of dry matter, as well as PHB titre, were affected by the

presence of limonene. Experiments run in the absence of limonene, figure 2.a, reached a

maximum dry matter concentration of 10.18 g L−1 after 60 hours of fermentation while

adding 0.5% (v/v) of limonene, figure 2.b, caused a drop in the dry matter concentration

to 8.28 g L−1 after 72 h. It is interesting to notice that higher limonene concentrations

shortened the time taken to reach the trigger point for the synthesis of the biopolymer

when compared to lower concentrations of the terpene, whilst it was possible to detect a

slight amount of PHB earlier on in the fermentation for the higher concentrations tested

indicating that the presence of limonene acts as an external stress on the cell population.

It was also possible to observe for all the limonene concentrations a decrease in residual

biomass during the second half of the fermentation. These results are in agreement with

those reported by Aramvash et al. (2015) using 35 g L−1 of fructose leading to 7.48 g

L−1 of PHB at maximum concentration, for a medium optimised for PHB production.

16

The lag phase present during the first hours of fermentation increased considerably with

the concentration of limonene, lasting around 40 h for the maximum concentration

tested. The final concentration of biomass, as reported for other strains (Marques et al.,

2014; Mohammad Pourbafrani, 2007), and PHB was strongly affected by the presence

of limonene.

PHB content inside the cells dropped from 68 % to 59 % (w/w) when 0.5% (v/v) of

limonene was added to the mineral media. Some growth was still observed, however,

with cell growth in the presence of 2 % (v/v) of terpene, reaching 2.6 g L−1 of dry mass

with a content of 22 % of PHB after 98h of fermentation. This differs to previous

reports on the ethanol producing yeast Saccharomyces cerevisiae, where no cell growth

was observed in the presence of 0.5 % of limonene and cell viability reached 0 after 3

hours of cultivation (Pourbafrani et al., 2007). More recently, Tao et al. (2014)

described the antifungal effect of limonene over a pathogenic strain that infects citrus

plants, Penicililum digitatum, at concentrations around 0.24 %, reporting a mycelial

growth inhibition of 43 %. C. necator showed stronger resistance when compared with

above mentioned strains losing 25% of its growth with concentrations up to 1% (v/v) of

limonene. A greater effect was triggered when the concentration of limonene was

increased to 1.5% (v/v) with C. necator H16 losing 50% of its specific growth rate.

Total nitrogen measurements never reached complete depletion. However, a constant

residual concentration of 220 mg L−1 of nitrogen was reached for the limonene free

control experiment after 40 hours, and similar behaviour can be observed for limonene

concentrations of 0.5 and 1 % (v/v). The nitrogen uptake was significantly reduced

17

when the concentration of limonene was increased from 1% to 1.5% (v/v), the residual

total nitrogen being 60% higher when compared to lower concentrations. Figure 3

shows the percentage of total nitrogen uptake for the different concentrations of

limonene.

Biomass and product yields, as well as specific growth rate and maximum PHB content,

were calculated for the different limonene concentrations tested and the results are

shown in figure 4. The trend for specific growth rate and PHB content shows sigmoidal

behaviour with a major drop occurring between 1 and 1.5 % (v/v) of initial limonene.

The effect was less pronounced for the consumed substrate yields on dry matter and

product; however total consumptions were considerably lower for concentrations of

limonene above 1%, as previous data showed.

The values of μ showed a decrease of more than 50% when the concentration of

limonene was 2 % (v/v); however, compared with the reports for some pathogenic

bacterial strains, C. necator was able to show some growth in such conditions (Espina et

al., 2011; Marques et al., 2014). Results indicate that C. necator is able to grow in

media prepared from orange peel media if the concentration if limonene is as low as

1%, with a 10% reduction in specific growth rate. When Salakkam and Webb (2015)

studied the effect of methanol on a glycerol consuming strain of C. necator, results

showed that, similar to limonene, the presence of the alcohol inhibited growth at all of

the concentrations tested. Even when both compounds exhibit similar sigmoidal profile

as inhibitors of cellular growth, limonene triggered a stronger inhibition at lower

concentrations. This similar behaviour could be due to the mechanism by which

18

methanol and limonene inhibit cell growth. Alcohol and terpenes are molecules that

affect the structure of the plasmatic membrane of the cell and its functions. Lipid

composition and protein conformation is changed, leading to leakage of intracellular

material in the case of limonene, and lowering the proton motive force in case of

methanol (Mohammad Pourbafrani, 2007; Zahi et al., 2015; Salakkam and Webb,

2015). However, direct comparison between different systems needs to be considered

carefully as the affinity of the different compounds for the microorganism’s membrane

may play a role.

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4. Conclusions

The technical feasibility of value added biopolymer production using OJW as a starting

material has been demonstrated and a process established to recover fructose from

OJW, to a maximum concentration of 24.74 g L−1. From this OJW medium a maximum

PHB concentration of 7.34 g L−1 was obtained with no nutriment supplementation.

The effect of the initial limonene concentration on PHB productivity and cell growth

was assessed, using a mineral media to replicate the OJW medium, with the addition of

different limonene concentrations. The investigation into limonene tolerance indicates

the existence of a threshold inhibitory concentration of 1 %(v/v).

Acknowledgements

We thank Dr. Saul Alonso Tuero for his insightful advice during the writing process.

This study was funded by a grant from the Mexican Council for Science and

Technology, CONACyT: 351189.

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List of Figures

Fig. 1. Fructose, dry matter, PHB and total nitrogen concentrations obtained for the

growth of C. necator H16 using different OJW media. Where: fructose, ; total

nitrogen, ; total dry matter, ; triangles are the concentration of PHB, ▲; and

residual biomass,▼. a) for an initial load of orange peel 1:8 (w:w); b) for the

1:12 (w:w) initial ratio OJW treatment.

Fig. 2. Variation cell growth, PHB production and, fructose and nitrogen uptakes, for

different initial concentrations of limonene. a) Limonene free; b) 0.5 % (v/v); c)

1 % (v/v) ; d) 1.5 % (v/v); e) 2 % (v/v). Where: fructose, ; total nitrogen, ; total

dry matter, ; concentration of PHB, ▲; and residual biomass, ▼.

Fig. 3. Percentage of total nitrogen uptake for the different initial percentages of

limonene in volume. Where: Limonene free (); 0.5 % (); 1 % (▲);1.5 % (▼); and for

2 % ().

Fig. 4. Variation of specific growth rate (), dry matter and PHB yields over substrate

( and ▲ respectively), and intracellular percentage of polymer (▼) as function

of the initial concentration of limonene.

List of Tables

Table 1 Carbohydrate concentrations obtained for different extraction treatments

studied

Table 2 Kinetic parameters obtained for the microbial growth of C. necator H16 using

the different OJW media obtained.

29

Table 1. Carbohydrate concentrations obtained for different extraction treatments studied.

%

WaterInitial solids ratio

[Initial fructose]

(g L−1)

[Initial glucose]

(g L−1)

[Initial sucrose]

(g L−1)

Ground OJW80

1:12 11.91 10.58

Ground OJW 1:8 24.74 13.62 9.90

Whole OJW73.5

1:12 12.09 10.97

Whole OJW 1:8 22.08 14.65 5.92

Manual peeling peels 60 1:12 14.13 10.74

Fresh OJW whole

N/D

1:8 4.51 4.78 3.06

Fresh OJW ground6.96 6.13 3.34

30

Table 2. Kinetic parameters obtained for the microbial growth of C. necator H16 using the different OJW media obtained.

Initial solids

Ratio

(w:v)

[Fructose]0

(g L−1)

*[Dry matter]f

(g L−1)

*[PHB]f

(g L−1)Yx/s Yp/s

μmax

(h−1)% PHB

Ground OJW 1:12 14.94 6.21 5.03 0.40 0.43 0.179 80.9

Ground OJW 1:8 23.14 9.01 7.34 0.46 0.39 0.122 81.5

Whole OJW 1:8 22.22 9.58 7.31 0.52 0.41 0.118 76.3

*Concentrations of dry matter and PHB were measured after 72 h. Yields were calculated against fructose consumed.

31

Fig. 1. Fructose, dry matter, PHB and total nitrogen concentrations obtained for the

growth of C. necator H16 using different OJW media. Where: fructose,; total nitrogen,

; total dry matter, ; triangles are the concentration of PHB, ▲; and residual

biomass,▼.

a) For an initial load of orange peel 1:8 (w:w).

b) For the 1:12 (w:w) initial ratio OJW treatment.

32

Fig. 2. Variation cell growth, PHB production and, fructose and nitrogen uptakes, for different initial concentrations of limonene . Where:

fructose, ; total nitrogen, ; total dry matter, ; concentration of PHB, ▲; and residual biomass, ▼.

a) Limonene free b) 0.5 % (v/v)

c) 1 % (v/v)

33

34

Fig. 3. Percentage of total nitrogen uptake for the different initial percentages of

limonene in volume. Limonene free (); 0.5 % (); 1 % (▲);1.5 % (▼); and for 2 %

().

34

Fig. 4. Variation of specific growth rate (), dry matter and PHB yields on substrate (

and ▲ respectively), and intracellular percentage of polymer (▼) as function of the

initial concentration of limonene.

35