EUREKA 2016 conference

Bioethanol Production Via Simultaneous Saccharification and Fermentation Using Unwashed Pretreated CorncobComment by Yoon Li Wan: Title might need to change a bit as this paper is published as Eureca paper before. Can consider changing it to:Potential of bioethanol production from unwashed alkali-pretreated corncob via simultaneous saccharification and fermentation

Assessment of bioethanol production from unwashed pretreated corncob via simultaneous saccharification and fermentation

Eureca 2016 – Conference Paper Paper Number 2CE12

3

AbstractComment by Yoon Li Wan: Abstract flow:Introduction (1 to 2 sentence) + problem statementEg:The conversion of lignocellulosic biomass to bioethanol could be realized by applying pretreatment, enzymatic hydrolysis and fermentation. One of the major concern on choosing the effective pretreatment strategy is the huge amount of wash water produced after pretreating the biomass.Objectives: (1 sentence)This study aims to investigate the potential of applying unwashed pretreated corncob for ethanol production via SSF.Brief methodology (2 to 3 sentence) Mentioned about the parameters studied and the test involved.Results and discussionOnly focus on significant resultsConclusionAnswer whether the objectives has been met and name 1 recommendation for future work. Emphasis on what is the significance for this study.

Maize is one of the main food sources throughout major parts of the world and this leads to large scale of corn cobs being disposed as agrowastes without being utilised for its high cellulosic composition. Large scale bioethanol production utilizing corn cob is a great idea to overcome this issue and in the present study, enzymatic hydrolysis and simultaneous saccharification and fermentation is investigated for feasible bioethanol yield. Choice of pretreatment is a contributing factor in bioethanol production to allow better sugar digestibility in hydrolysis and fermentation thus, this study involves utilisation of 2% NaOH (alkaline) of 1:7 solid to liquid ratio onto corncob. The treated corncobs are prepared as washed and unwashed substrate which undergoes enzymatic hydrolysis with the aid of cellulase from Trichoderma Longibrachiatum of concentration 30 FPU/g-corncob and citrate buffer (0.1M citric acid and 0.1M sodium citrate). The washed pretreated corncob is prepared using 1:7.5 ratio of raw corn cob to rinsing water. The parameters varied in this study are the pH (ranging from pH 4 to 6), temperature (40°C, 50°C and 60°C) and, duration. This is done to attain an optimum condition for enzymatic saccharification. The expected outcome convinces that unwashed NaOH pretreated corncob gives good rate of fermentable sugar conversion (hydrolysis yield of 36.97%) with not much difference compared to the washed substrate (glucose yield of 37.69%).

Keywords: Corncob, SSF, unwashed pretreated biomass, NaOH pretreatment, bioethanol.Comment by Yoon Li Wan: Keyword is good

1. IntroductionComment by Yoon Li Wan: Introduction: Need to restructure. The first 2 paragraphs can be combined. Inhibitors production can be eliminated because we are not focusing on inhibitors in the study.FORMAT: (This is part of your literature review also, do more on compiling the review on others’ study)Start with a general statement:Eg: Lignocellulosic biomass which is being viewed as waste can be converted to multiple value-added productsThe first 2 paragraphs can be combined.In the second and third paragraph, talked about the usual route for ethanol production. Pretreatment, enzymatic hydrolysis and fermentation. Talk also about the current development of the route in bioethanol production. In the following part, it is basically your problem statement. Talk about washed and unwashed. What are the works being done on unwashed/In the final section, bring out the benefits of unwashed pretreated biomass. Talk about the objectives of the study.After introduction, reader should be able to get an idea on:Why are we producing bioethanol from biomassWhy are we using alkaline pretreatmentWhy SSFWhy unwashed

In the recent years, biofuel such as bioethanol has become one of the most sought after resources to replace the depleting fossil fuel. The production of bioethanol appeared to be more cost effective when the low cost agricultural waste such as sugarcane bagasse, wheat straw and corn corb was proven as promosing feedstock (Cardona et al. 2010; Talebnia et al. 2010; Sumphanwanich et al. 2008). These agricultural waste which is lignocellulosic in nature serve as potential feedstocks for a variety of other chemicals such as fermentable sugars, organic acid and xylitol (Pandey et al. 2000).

There are different routes to convert these lignocellulosic waste into value-added bioethanol. In general, the lignocellulosic waste needs to be first pretreated in order to alter its recalcitrant nature for further processing. Some of the commonly applied and effective pretreatment method include acid pretreatment, alkali pretreatment and steam explosion method. After pretreatment, lignin content and cellulose crystallinity could be reduced, depending on the pretreatment method applied (Banerjee et al. 2009; Kumar et al. 2009). The structure of the pretreated biomass would therefore become more porous and easily accessible by enzymes (Perez-Pimienta et al. 2013).

The pretreated waste is then subjected to enzymatic saccharification, whereby cellulase enzymes would depolymerized cellulose in pretreated biomass into glucose monomers. These sugars are further fermented into bioethanol by yeast. Instead of performing enzymatic saccharification and fermentation as two separate steps, both processes can be combined in a single reactor by adopting simultaneous saccharification and fermentation route. In simultaneous saccharification and fermentation, cellulase enzymes and yeast were added into the reactor simultaneously in order to convert the glucose produced from enzymatic saccharification into bioethanol continuously (Dwivedi et al. 2009). This will help to prevent the accumulation of glucose which causes end-product inhibition to cellulase enzymes. As a result, the saccharification rate increases (Menon & Rao 2012) with higher ethanol yield in shorter processing duration (Sun & Cheng 2002).

In the recent years, improving the cost effectiveness of production route and the yield of bioethanol are the two main focuses in the research field. While some of the ongoing researches are developing the potential genetically engineered ethanolic microorganisms (Matsushika et al. 2009; Olson et al. 2015; Dien et al. 2003), others are working on enhancing the production scheme of bioethanol. For instance, consolidated bioprocess that involves the usage of a single microorganism or microbial community for cellulase production, enzymatic saccharification and fermentation in a single reactor was investigated (Dashtban et al. 2009; Dwivedi et al. 2009; La Grange et al. 2010). Besides that, application of high solid loading in saccharification and fermentation could improve the bioethanol yield as well as reduce the cost of distillation (Wang et al. 2014; Liu et al. 2016; López-Linares et al. 2014).

Generally, a huge amount of wastewater is generated from washing of pretreated substrate before the saccharification process in order to neutralize the pretreatment agent used. Therefore, the cost of bioethanol production could be greatly reduced when the amount of wastewater generation is minimized. Studies have shown that fermentable sugars production from unwashed alkali-pretreated lignocellulosic waste is feasible (Yu et al. 2016). To the best of our knowledge, unwashed pretreated lignocellulosic waste for bioethanol production have not been examined. In this study, the effect of pH and temperature on sugar production via enzymatic saccharification of washed and unwashed pretreated substrates was investigated. Ethanol production from both unwashed and washed pretreated substrate via simultaneous saccharification and fermentation was then examined.

2. Materials and Methods

2.1 Lignocellulosic Material

Corncob was obtained from a local market (Perak, Malaysia) and were dried in an oven at 60°C for 48 hours. The corncob was subjected to pre-milling until reaching particle size less than 1 mm. It is sieved to obtain fine powdered corncob with particle size of 500µm or less. Comment by Yoon Li Wan: Put reference if you have.

(a) (b)

(c)

Figure 2.1 Comparison view of (a) unprepared raw corncob, (b) grinded raw corncob and (c) sieved raw corncob.Comment by Yoon Li Wan: Don’t have to put this figure in

2.2 Sodium Hydroxide Pretreatment and Solids Washing

60 g of corncob was treated with 2% (w/v) NaOH using autoclave (Model no. WAC-60) at 121°C for 30 minutes with a ratio of 1g of raw sieved corncob to 7 mL of NaOH solution. Upon completion of NaOH pretreatment, the pretreated slurry was collected and squeezed with a strainer manually to remove moisture. After the first straining of the substrate, the product is considered to be the unwashed pretreated corncob. For solids washing, the product obtained is mixed with 600 mL pure ultrapure water (UPW) with 1:7.5 ratio of raw corn cob to water and stirred thoroughly for 5 minutes, followed by straining method. The washing sequence is repeated according to desired number of washing cycle. Comment by Yoon Li Wan: PretreatedDon’t start the sentence with numberSentence is too long. Break down to 2 partsChange to:Alkali pretreatment was conducted with 60 g of corncob by using an autoclave at 121 deg C for 30 min. The ratio of….Comment by Yoon Li Wan: 30 minComment by Yoon Li Wan: Eliminate Comment by Yoon Li Wan: Remove excess pretreatment reagent. Comment by Yoon Li Wan: As a comparison, washing of pretreated corncob was conducted by mixing the solid ….Comment by Yoon Li Wan: Mention specifically 2 cycles

2.3 Enzymatic Hydrolysis

10 g of unwashed pretreated corncob was weighed and placed in three different 250 mL conical flasks. Same steps are repeated for corncob that is washed once. The solids are then buffered with 80 mL of citrate buffer (0.1 M of citric acid and 0.1 M of sodium citrate, pH 4) [7]. Two repetitions were done for pH 5 and 6. 10 mL of cellulase from Trichoderma Longibrachiatum (Sigma-Aldrich, C9748-100G) with an activity of 30 FPU/g substrate was added into each flask and the enzymatic hydrolysis was carried out for 48 hours using orbital incubator shaker (Model no. TS-520D) at 50°C (120 rpm) and these steps were repeated using another two-temperature variation of 40°C and 60°C. The flasks were sealed with rubber stopper and a layer of aluminium foil to prevent air contamination.Comment by Yoon Li Wan: eliminateComment by Yoon Li Wan: 10 g of unwashed and washed alkali-pretreated corncob was subjected to enzymatic saccharification….The pretreated corncob was buffered with….Comment by Yoon Li Wan: The same enzymatic saccharification steps was conducted for pH 5 and pH 6Comment by Yoon Li Wan: Brand of the equipment, model of the equipment.Comment by Yoon Li Wan: Don’t have to bracket…just put at 50 deg and 120 rpmComment by Yoon Li Wan: Don’t have to mention

(a) (b)

Figure 2.2 Comparison view of unwashed (dark) and washed pretreated corncob (light) (a) before adding buffer solution and (b) after adding buffer solution.

Table 2.1 Levels of independent variables applied in the enzymatic saccharification comparison test between washed and unwashed pretreated corn cob.

Variable

Coding

Units

Levels

-1

0

1

pH

A

-

4

5

6

Temperature

B

°C

40

50

60

Duration

C

hours

3 pre-determined intervals (till 48 hours)

Total of 5 runs of enzymatic hydrolysis were carried out with the different combinations of the independent variable values stated in Table 2.1. At each interval, the flasks were placed in the freezer at -20°C for 20 minutes to deactivate the enzymes.

2.4 Analytical MethodsComment by Yoon Li Wan: Add the writeup for SSF

HPLC equipment is used for quantitative analysis to determine the concentration of glucose in HPLC standard samples and experimental samples. The mobile phase applied was pure ultrapure water (UPW) with flow rate 0.6 mL/min [7]. Before the sample analysis via HPLC, High Performance Liquid Chromatography with refractive index detector (Agilent, USA Model no. 1220-G1362A) was carried out, samples are centrifuged (Multipurpose Centrifuge Scanspeed Model no. 1236MG) for 15 minutes at 4,500 rpm. Supernatant is filtered through nylon syringe filter (0.2 µm pore size) and subjected for analysis and 65°C was the set column temperature and column used was Hi-Plex H (7.7mm x 300, Agilent, USA).Comment by Yoon Li Wan: High performance liquid chromatography (HPLC) equipped with refractive index detector was used in….Comment by Yoon Li Wan: Glucose and ethanolComment by Yoon Li Wan: Mentioned in previous sentence.Comment by Yoon Li Wan: Mentioned that the samples are centrifuged, supernatant part is collected and filtered with nylon syringe filter before subjecting it to HPLC analysis.

Figure 2.3 Supernatant collected after enzymatic hydrolysis of unwashed (dark) and washed pretreated corncob (light)Comment by Yoon Li Wan: eliminate

The results obtained from the HPLC readings were compared using the results of HPLC standard calibration pure glucose samples (calibration curve) The area under peak given by HPLC was converted to amount of glucose (g/L). The glucose (hydrolysis) yield percentages are calculated manually using related formula Eq. (2.1) and (2.2) below:Comment by Yoon Li Wan: can put the ref on where did you obtain the formula from.

(2.1)

(2.2)

3. Results and DiscussionComment by Yoon Li Wan: R&DSectioned it into 3.1 enzymatic hydrolysis3.2 bioethanol production from unwashed

Comment by Yoon Li Wan: This looks like literature review where you can placed in introduction

Alkaline pretreatment assists in eliminating most of the lignin composition of the biomass used and it improves the reactivity of the cellulose and hemicellulose [3]. Low chemical loadings of NaOH during pretreatment allows better alteration of the crystallinity structure of the cellulose, thus giving a better hydrolysis yield compared to other alkaline pretreatment NH3 and KOH even with higher concentrations [3]. In this study, the enzymatic hydrolysis is considered the vital process in simultaneous saccharification and fermentation (SSF) to produce bioethanol from lignocellulosic biomass, in this case which is corncob. Since glucose is converted to ethanol during fermentation, the glucose yield from saccharification is to be the contributing factor for a better ethanol yield. However, selection between washed or unwashed pretreated biomass is said to have an effect onto the sugar yield based on other research done on rice straws (Yu-Shen Cheng et.al.,2010). Hence, this study determines the productivity comparison of washed and unwashed pretreated corn cob in hydrolysis yield as it gives better understanding of the economic feasibility of unwashed pretreated lignocellulosic biomass. The main reason the unwashed factor is being focused is due to the effect of reduced water consumption for the process. Bioethanol production requires large amount of water throughout and by implementing this, the production cost can be reduced as less wastewater needs to be treated.

3.1 HPLC Analysis Comment by Yoon Li Wan: Enzymatic hydrolysisAs opening remark, you can mention

3.1.1 Effect of pH on Enzymatic HydrolysisComment by Yoon Li Wan: Focus on discussion of the results, instead of merely reporting the results.Instead of putting the info in Table, keep the graphs only. Compare your result with other studies (eg Yu et al., 2016) Reduce the decimal points of glucose concentration to 1. Instead of 32.172 g/L, change to 32.2 g/L.

The optimal pH value for saccharification using cellulase from Trichoderma Longibrachiatum is investigated and a set point temperature is selected to carry out this part. The optimum temperature for saccharification by cellulase is researched to be at 50°C and the growth tolerant pH for Trichoderma species is between 3 to 6 [8, 9]. So, this experiment was done using three different pH variables which is 4, 5 and 6 at a constant incubation temperature of 50°C. For the result, the highest final concentrations of glucose yield using unwashed and washed pretreated corn cob are obtained at pH 5 which are 32.172 g/L and 34.259 g/L respectively. The percentage difference calculated is 6.09 % and a small concentration difference between washed and unwashed substrate is observed through this experiment. Besides that, the concentration values obtained for the saccharification between pH 4 till 6 gradually increases from time to time and stops increasing when the duration is at 24~25 hours.

Table 3.1 HPLC and calculation analysis value of enzymatic hydrolysis at pH 4, 50°C.

Area

Concentration (g/L)

Time (hours)

Unwashed

Washed

Unwashed

Washed

Unwashed

Washed

40676.92

47264.17

25.81

29.99

19.58

19.58

43182.84

51888.30

27.40

32.93

39.53

39.53

44396.61

52371.54

28.17

33.24

46.08

46.08

Table 3.2 HPLC and calculation analysis of enzymatic saccharification at pH 5, 50°C.

Area

Concentration (g/L)

Time (hours)

Unwashed

Washed

Unwashed

Washed

Unwashed

Washed

45923.45

48557.45

29.14

30.82

16.50

16.50

48793.52

50417.32

30.97

32.00

22.97

22.97

50694.78

53982.03

32.17

34.26

47.00

47.00

Table 3.3 HPLC and calculation analysis of enzymatic hydrolysis at pH 6, 50°C.

Area

Concentration (g/L)

Time (hours)

Unwashed

Washed

Unwashed

Washed

Unwashed

Washed

18887.96

27801.40

11.98

17.64

16.77

16.77

22630.84

29054.39

14.36

18.44

23.00

23.00

24444.92

40845.01

15.51

25.92

46.97

46.97

(c)

(b)

(a)

Figure 3.1 Glucose concentration-time curve for (a) pH 4, (b) pH 5 and (c) pH 6 at 50 °C with different sample: unwashed pretreated corn cob. washed pretreated corncob.

Comment by Yoon Li Wan: In enzymatic hydrolysis section, focus on:What is the impact of washing and not washing steps on glucose productionWhat is the relationship between temp/pH/ time with washed and unwashed substrate for glucose production?

3.1.2 Effect of Temperature on Enzymatic Hydrolysis

The hydrolysis of cellulose is also affected by the incubation temperature. Hence, a balanced temperature is needed to be confirmed at the optimal pH obtained previously and this experiment allows the study of sugar concentration produced from hydrolyzed corn cob with constant pH value of 5 but, varying incubation temperature such as 40 °C and 60 °C since the results for 50 °C has been noted. Based on results obtained at the end of experiment, the final highest concentration is noted at 40 °C for unwashed pretreated corn cob which is 33.611 g/L whereas for washed substrate, it is 34.259 g/L at 50 °C. The difference in concentration between both substrates is calculated to be 1.89 % which can be negligible.Comment by Yoon Li Wan: Comment by Yoon Li Wan: Can keep the writeup but add in more discussion and comparison. For eg, Regardless of washed and unwashed substrate, temperature effect on glucose production is the same. This phenomenon is different from pH (there is a significant increase on glucose production for pH 6)

Table 3.4 HPLC and calculation analysis value of enzymatic hydrolysis at 40°C, pH 5.Comment by Yoon Li Wan: Eliminate Table. Keep only graph

Area

Concentration (g/L)

Time (hours)

Unwashed

Washed

Unwashed

Washed

Unwashed

Washed

47704.49

49243.67

30.27

31.25

15.95

15.95

51600.41

48880.51

32.75

31.02

21.97

21.97

52961.15

45147.37

33.61

28.65

46.92

46.92

Table 3.5 HPLC and calculation analysis of enzymatic saccharification at 50°C, pH 5.

Area

Concentration (g/L)

Time (hours)

Unwashed

Washed

Unwashed

Washed

Unwashed

Washed

45923.45

48557.45

29.14

30.82

16.50

16.50

48793.52

50417.32

30.97

32.00

22.97

22.97

50694.78

53982.03

32.17

34.26

47.00

47.00

Table 3.6 HPLC and calculation analysis of enzymatic hydrolysis at 60°C, pH 5.

Area

Concentration (g/L)

Time (hours)

Unwashed

Washed

Unwashed

Washed

Unwashed

Washed

40720.42

41887.33

25.84

26.58

17.75

17.75

44757.55

45805.26

28.40

29.07

23.00

23.00

47093.26

50785.11

29.89

32.23

47.77

47.77

(c)

(b)

(a)

Figure 3.2 Glucose concentration-time curve for (a) 40 °C, (b) 50 °C and, (c) 60 °C at pH 5 with different sample: unwashed pretreated corn cob. washed pretreated corncob.

3.1.3 Glucose Yield at Optimum Conditions

(a) (b)

Figure 3.3 Glucose yield (%) from enzymatic hydrolysis for (a) varying pH and, (b) varying temperature using different samples: unwashed pretreated corn cob. washed pretreated corncob.

According to the results shown in the Figure 3.3, the optimum condition for unwashed pretreated corncob is at pH 5 and 40 °C as the highest hydrolysis yield recorded shows 36.97 % is obtained during the 48 hours period. The highest sugar yield achievable using washed pretreated corn cob is 37.69 % at conditions of pH 5 and 50 °C. Hence, the difference in hydrolysis yield between the two substrates is calculated to be 0.72 %, which is < 1 %.Comment by Yoon Li Wan: What can you conclude when the difference is less than 1%?Mention it

4. ConclusionComment by Yoon Li Wan: Make conclusion simpler.Format:State significant resultsMention whether objectives are metFuture work that you can proceed. Eg: test with different pretreatment method, study inhibitors, scale up the production and etc.

Corn cob was used as lignocellulosic feedstock in the enzymatic hydrolysis carried out in order to determine the optimal conditions. The conditions recorded are necessary to be applied for simultaneous saccharification and fermentation using unwashed pretreated corn cob where hydrolysis yield plays a major role in bioethanol production (ethanol yield). In comparison between the glucose yield analysed, unwashed pretreated corncob has a slightly lower rate of sugar yield due to the presence of inhibitors such as hydroxymethylfurfural (HMF) and furfural as no rinsing is done. However, the concentration difference between both substrates are very small and this proves that it is feasible to utilize unwashed pretreated lignocellulosic biomass in large scale bioethanol production. Moreover, based on the analysis graphs, the graph line increases gradually from time to time at early stages referring to active hydrolysis rate however, the concentration value stays at a constant upon reaching a certain period. This is due to factors such as inaccessible surface area of enzyme and depletion of cellulose content affecting glucose conversion rate. This also proves that the enzymatic hydrolysis by cellulase Trichoderma Longibrachiatum requires not more than 30 hours according to parameters set in this study.

Acknowledgment (optional)

Sincere gratitude and thanks are to Dr. Yoon Li Wan for all the guidance and supervisions throughout the entire project. I would also like to thank the Faculty of Bioprocess of University Malaya for allowing me to use their equipment in order to complete this research.Comment by Yoon Li Wan: Comment by Yoon Li Wan: No need to thank one of the author..:)

ReferencesComment by Yoon Li Wan: Comment by Yoon Li Wan: Check ref format

[1] D. Lee, V. Owens, A. Boe, and P. Jeranyama. Composition of Herbaceous Biomass Feedstocks.

[2] R. Benson, C. Bradt, R.O. Benech, R.R. Lehoux, and G.E. Inc (2012). Patent US8287651 - cellulose pretreatment process.

[3] B. Marshall (2007). Gas From The Grass, Field & Stream (pp.40).

[4] Harmsen, P.F.H. (2010). Literature Review Of Physical And Chemical Pretreatment Processes For Lignocellulosic Biomass. Wageningen UR, Food & Biobased Research.

[5] K. Otulugbu (2012). Production of Ethanol from Cellulose (Sawdust).

[6] A. Raspolli Galletti and C. Antonetti (2011). Biomass pre-treatment: separation of cellulose, hemicellulose and lignin. Existing technologies and perspectives. University of Pisa.

[7] H. Zakpaa, E. Mak-Mensah and F. Johnson (2009). Production of bio-ethanol from corncobs using Aspergillus niger and Saccharomyces cerevisae in simultaneous saccharification and fermentation. African Journal of Biotechnology, vol. 8, no. 13.

[8] Zhang YHP, Lynd LR (2004). Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng (88: pp.797–824)

[9] Tao F, Miao JY, Shi GY, Zhang KC (2005). Ethanol fermentation by an acid-tolerant Zymomonas mobilis under non-sterilized condition. Process Biochem (40: pp.183–187)