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CHINESE JOURNAL OF BIOTECHNOLOGY Volume 24, Issue 11, November 2008 Online English edition of the Chinese language journal RESEARCH PAPER

Cite this article as: Chin J Biotech, 2008, 24(11), 1943í1948.

Performance Optimization of Property-improved Biodiesel Manufacturing Process Coupled with Butanol Extractive Fermentation Longyun Zhang, Ying Yang, and Zhongping Shi Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China

Abstract:

In traditional acetone–butanol (AB) fermentation, the products concentrations are too low. Therefore, large amount of

energy is consumed in the distillation and product recovery process. Aiming at direct utilization of the fermentation products, in this study, the optimization of property-improved biodiesel manufacturing process coupled with AB extractive fermentation was conducted, under the condition of using the biodiesel that originated from waste cooking oil as the extractant and high concentrated corn flour as medium. The effect of biodiesel/broth volume ratio, waste supernatant recycle ratio, and electron carrier addition on major process performance index was carefully investigated. Under the optimized condition, the biodiesel quality was improved with cetane number (CN) increased from 51.4 to 54.4; “actual butanol yield” reached the level of 18%, and waste supernatant recycle ratio exceeded 50%. In this way, elimination of energy-consuming product recovery process and realization of “energy-saving & waste minimization” industrial production mode advocated by the state government could be potentially expected. Keywords: extractive fermentation; butanol; energy-saving & waste minimization; biodiesel

Introduction The industrially important solvents such as butanol, acetone, and so on, could be produced from corn flour by acetone–butanol (AB) fermentation with solventogenic Clostridium. One of the major problems in AB fermentation is the severe growth inhibition from the primary end-product, butanol[1]. To overcome the problem, in situ extractive fermentation was developed to relief butanol inhibitory effect and to enhance the fermentation productivity [2-3]. Among various fermentation extractants tested, methylated palm oil (a kind of biodiesel) was reported to be a considerably good extractant for butanol in AB fermentation[4]. However, even with extractive fermentation, butanol concentrations in both extractant and

aqueous phases are still very low because of which a large amount of energy has to be consumed in the subsequent products purification process. The huge energy cost is one of the significant factors that limits the recovery and development of fermentative AB industry. As a result, a new process or method that directly utilizes fermentative products and completely eliminates the energy consuming down-stream purification step, are of great interest. As one of the clean and renewable fuels, biodiesels have been well accepted by many countries particularly in the current era of high international crude oil price. Oil-seed crops-based biodiesels competitively use food oil resources, and cautious measures must be taken in development of related biodiesel manufacturing processes to always prioritize the worldwide food-oil safety. However, as the

Received: May 3, 2008; Accepted: September 3, 2008 Corresponding author: Zhongping Shi. Tel: +86-510-85918292; Fax: +86-510-85918296; E-mail: [email protected] Supported by: the Major State Basic Research Development Program of China (973 Program, No.2007CB714306). Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.

Longyun Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1943–1948

most populated country in the world, China is enriched with various biomass resources including waste restaurant and cooking oils. These waste biomass resources could be considered as wealthy and stable raw materials for manufacturing biodiesels in China. On the other hand, biodiesels, particularly the waste cooking oil-originated biodiesel, still suffer a couple of shortcomings, such as lower combustion power, cetane number (CN), and quality, which limit their future practical application and promotion worldwide[5,6]. Report on diesel properties showed that addition of butanol into diesel in a proper manner could improve ignition performance and combustion power of diesel engines. It has been recognized that the butanol mixed diesel could be used as a fuel with high combustion quality as its CN is generally higher than that of the ordinary diesel[5]. Theoretically, the above-mentioned methylated palm oil extracting fermentative butanol could be considered as a kind of high quality or properties-improved biodiesel ready for use directly. The properties-improved biodiesel manufacturing process coupled with AB extractive fermentation with biodiesel itself as the extractant has at least the following two advantages: 1) improvement in a couple of key properties of ordinary biodiesels is expected, raising the reality of replacing fossil diesel with biodiesels; 2) production of properties-improved biodiesel could be carried out in a most energy-saving manner. In previous studies by the same authors[7,8], the entire performance of properties-improved biodiesel manufacturing process coupled with AB extractive fermentation was not much improved, because starch was eventually consumed with 7% initial corn flour medium (prepared by gelatinization) as the fermentation material. To further increase initial corn flour content in the medium would cause very high viscosity so that the subsequent gelatinization and inoculation operation fails to continue. In practice, addition of tiny amount of D-amylase/ glucoamylase would first hydrolyze a portion of solid starch powders into glucose and soluble dextrin to reduce the viscosity of fermentation medium, and to meet the requirements of normal inoculation and fermentation operations. The major objectives of this study are: to enhance performance index of the property-improved biodiesel manufacturing process coupled with AB extractive fermentation, with initially high corn flour concentrated medium (15%) and under optimized environmental conditions. The index include: 1) butanol concentration in biodiesel or CN of the biodiesel; 2) biodiesel productivity; 3) “actual butanol yield”.

1

Materials and methods

1.1 Microorganism Clostridium acetobutylicum ATCC824 was maintained at

spore suspension in 5% corn flour medium at 4qC. The methods for inoculation and preculture followed those described in literature[9]. 1.2 Pretreatment of medium and extractant (biodiesel) The corn flour (raw starch content about 60% W/W) obtained at local market was sieved with mesh size 40. When corn flour content in medium was below 7%, the medium was prepared by gelatinization in boiling water bath for 60 min, otherwise medium was pretreated by adding a tiny amount of D-amylase (8 u/g-corn, heated in boiling water bath for 45 min) and then glucoamylase (120 u/g-corn, heated at 62qC for 60 min). Subsequently, the viscosity-reduced medium was autoclaved at 121qC and natural pH for 15 min. The DE (Dextrose equivalent) values for corn flour mediums of 7% and 15% were 0 and 73, respectively, in terms of absolute glucose concentration (g/L). Biodiesel was autoclaved at 121qC for 15 min and then added into the fermentation bottle, or was directly poured into the bottle without sterilization (experimental result elsewhere indicated that contamination did not occur during fermentation if biodiesel was directly added ). 1.3 Major reagents The waste cooking oil-originated biodiesel, the extractant for butanol, was kindly provided by Huahong Biofuel Co., Wuxi, China. D-amylase (20 000 u/mL) and glucoamylase (100 000 u/mL) were purchased from Genencor Biotech Co., Wuxi, China. 1.4 Major instruments The fermentation products were measured by a GC112A gas chromatograph (Shanghai Precision Science Instrument Co., Shanghai, China) equipped with a flame ionization detector (FID) and ALPHA-Col PEG column (SGE Int’l Pty. Ltd., Australia), and analyzed with a N-2000 GC data processing workstation (Zhida Information Co. of Zhejiang Univ., China). All of the inoculation and biodiesel addition operations were performed anaerobically in an ACII-2E anaerobic chamber (Sheldon Manufacturing Inc., USA). 1.5 Experimental methods 1.5.1 Fermentation method and condition: Based on the required ratio, the biodiesel was first added into 100 mL anaerobic fermentation bottle containing 40 mL corn flour medium, followed by inoculation with an inoculum size of 10% (V/V). The bottles were kept at vacuum condition for 2 min to remove oxygen possibly dissolved in the medium, and then placed in a water bath for static fermentation at 37qC. Figure 1 is the schematic diagram of property-improved biodiesel manufacturing process coupled with AB fermentation. Under static condition, the fermentative solvents diffused from broth phase into extractant phase (biodiesel) by the self-generated gas bubbles during fermentation. After each fermentation run, the extractant extracting butanol (also small amount of acetone) entered

Longyun Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1943–1948

Fig. 1 Diagram of property-improved biodiesel manufacturing process coupled with butanol fermentation

the “properties-improved biodiesel tank”, which was then was utilized directly as the biodiesel with high quality. The supernatant of waste broth should be reutilized as much as possible for medium preparation for the next run to save the valuable fresh water resource, and the unusable broth residuals was disposed to environment after subject to waste water treatment. Here, a new performance index was designed and defined: “actual butanol yield” was defined as the ratio of butanol accumulation amount in biodiesel to starch consumption amount. This is because only the butanol extracted into the biodiesel is the real target product for the property-improved biodiesel manufacturing process coupled with AB fermentation and without fermentative products recovery. 1.5.2 Measurements of solvents: The concentrations of fermentative solvents, butanol, acetone, and ethanol were determined using the chromatograph mentioned above, with iso-butanol as the internal standard. 1.5.3 Measurements of initial and residual concentrations of starch and reductive sugar: Starch could be consecutively hydrolyzed into dextrin, maltose, and eventually glucose under acidic condition. The starch content of the broth or fresh medium was determined in the following way: 2 mL sample was taken, which was then accurately diluted to 25 mL with 2% HCl. The diluted solution was placed in boiling water bath reacting for 3 h. The supernatant obtained after centrifugation was further diluted for suitable folds to measure glucose concentration using a biosensor detection unit (SBA-40, Shandong Science Academy, China). Starch concentration was calculated as 0.9 u glucose concentration. 1.5.4 Measurement of fermentative gas production: AB fermentation is a typical growth-associated fermentation, and the gas production could be simply used as an indicator for cell growth and solvents production. After fermentation

was initiated for several hours, the evolved gas amounts were determined by collecting in a graduated cylinder filled with water in every 2–3 h interval. 1.5.5 Preparation of recycled waste supernatant At the end of each fermentation run, the waste supernatant for reutilization was obtained by centrifugation and then stocked at 4qC for future use.

2

Results and discussion

2.1 Possible substrate inhibition when using medium with high corn flour concentration (15%) Acetone–butanol fermentation is typically anaerobic and growth associated, with possible substrate inhibition. Figure 2 shows the gas production curves in batch fermentation when using the two corn flour mediums with different initial concentrations (7% and 15%). The gas production curves in the two cases basically overlapped in the first 30 h and no gas production lag was observed. This suggested that substrate inhibition did not occur even when the medium of very high corn flour concentration (15%) was used. Gas production completely ceased at 43 h because of starch exhaustion when using 7% initial corn flour medium, whereas gas production continued until 65 h when 15% initial corn flour medium was used. The total solvents (butanol plus acetone, whereas ethanol was ignored for its small production amount) and butanol concentrations reached levels of 20.9 g/L and 13.6 g/L for the latter case, but the residual starch concentration still remained at a high level of 43.7 g/L. The facts clearly indicated that the fermentation stoppage was not due to the starch exhaustion but because of butanol inhibition. As a result, the medium of 15% initial corn flour content was used as the standard medium in the subsequent experiments unless indicated otherwise.

Longyun Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1943–1948

Fig. 2 Gas production under different initial corn flour concentrations

2.2 Effect of amount of biodiesel addition on performance of AB extractive fermentation The biodiesel addition ratio (volume ratio of extractant phase to broth phase) or amount directly related with performance index such as butanol concentration in biodiesel (biodiesel quality), properties-improved biodiesel productivity, total fermentative solvents concentration, and so on. Fig. 3 shows the effect of different biodiesel addition ratio on gas production. The initial gas production rates of extractive fermentation were slower than that of control (traditional batch fermentation), and the bigger the biodiesel addition ratio, the slower the initial gas production rate. This fact suggested that the waste cooking oil-originated biodiesel is somewhat inhibitory to cell growth. However, due to the extractive effect of the biodiesel for the primary inhibitory metabolite-butanol, all of the gas production amounts in extractive fermentations eventually exceeded that of control in the end. Figure 4 indicates the effect of biodiesel addition ratio on the performance of AB extractive fermentation. The bigger the ratio, much more fermentative butanol are extracted into biodiesel, in other words, higher properties-improved biodiesel productivity. On the other hand, the bigger the ratio, the lower the butanol concentration in biodiesel, and butanol concentration in biodiesel is a direct indicator of biodiesel quality. When the extractant/ broth ratio increased from 0.4:1 to 1.2: 1, butanol concentration in biodiesel decreased from 13.6 g/L to 7.8 g/L. Comprehensively considering both the biodiesel productivity and quality, extractant/broth volume ratio of 1:1 was set as the standard ratio for the subsequent experiments unless indicated otherwise. 2.3 Reutilization of waste supernatant of AB extractive fermentation There were still certain amounts of solvent products (butanol and acetone about 10 g/L and 5–6 g/L, respectively) in the broth when each operation run finished. Firstly, the focus was only on direct utilization of waste supernatant for medium preparation for next fermentation run, aiming at saving valuable fresh water resources and eliminating recovery of the residual solvents in the waste broth for

Fig. 3 Effect of biodiesel/broth ratio on gas production

saving energy. The medium with 15% initial corn flour concentration was prepared by mixing waste supernatant with fresh water based on the required recycle ratio. The residual solvents particularly the volatile acetone, would be lost in large amounts under high temperature during operations of gelatinization, hydrolyzation, and sterilization. However, in this case, the loss of solvent products was not a key problem subject to consideration as the aim was to recycle waste supernatant to effectively use the limited fresh water resources. The experimental results (data not shown) showed that gas production lag did not occur during traditional batch fermentation when recycling 25% and 50% waste supernatant (initial butanol concentration about 2–4 g/L), and total gas production amounts were even higher than that of 0% recycle ratio (when 100% fresh water was used for medium preparation). This fact suggested that the nonvolatile and soluble nutrients and proteins remained in the waste supernatant could even somewhat promote the next fermentation run. However, when the recycle ratio exceeded 75%, the next fermentation failed. There might be two reasons for the fermentation stoppage: 1) initial butanol concentration in the medium was too high (about 4–6 g/L) as only a small portion of the nonvolatile butanol (boiling point 119qC) was distilled out or escaped during medium preparation at high temperature; 2) some unknown substances inhibitory to cell growth likely formed when treating the soluble proteins-enriched waste supernatant at high temperature. Final butanol and total solvents concentrations achieved in 50% recycle ratio case were highest among all the cases. The results for the extractive fermentation using recycled waste supernatant were also very similar to those of batch fermentation. Fig. 5 depicts the relationship between the performance indexes of butanol concentration in extractant, total solvent concentration as well as “actual butanol yield” and supernatant recycle ratio in extractive fermentation cases. With recycle ratio of 50%,

Longyun Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1943–1948

Fig. 4 Effect of biodiesel/broth ratio on butanol extractive fermentation performance

Fig. 5 Effect of fermented supernatant recycle ratio on butanol extractive fermentation

butanol concentration in extractant, total solvent concentration, and “actual butanol yield” each reached maximum level of 11.0 g/L, 32.0 g/L and 14%, respectively. When recycle ratio exceeded 50% and reached 75%, all performance indexes decreased significantly. 2.4 Cetane number (CN) of biodiesel extracting fermentative butanol Cetane number (CN) is one of the most important indexes in evaluating diesel or biodiesel quality. The bigger the CN, the shorter the ignition time delay, and the higher the combustion efficiency, the less the black smoke exhaustion. The Nanjing Jingling Branch of China PetroChem Co. was consigned to measure CN of several samples of the waste cooking oil-originated biodiesel extracting fermentative butanol of more than 10 g/L, and then compared with that of the original biodiesel without butanol extraction, to actually testify the enhancement of their combustion properties. The measurement results were as follows: Here, A, B, and C referred to the samples of original biodiesel, biodiesel extracting 10.8 g/L butanol, and biodiesel extracting 12.0 g/L butanol combined with simple

after-dehydration treatment with 1.6% (W/V) Na2SO4 for 1 h, respectively. The CN of sample B was not enhanced as compared with that of sample A. This could be ascribed to the small amount of miscellaneous water added into biodiesel during fermentation offsetting the positive effect of butanol extraction. On the other hand, CN of the biodiesel extracting 12.0 g/L butanol combining with simple dehydration (sample C) increased CN up to 3 units compared with that of sample A; the positive effect of butanol extraction began to appear and the CN enhancement magnitude were within the acceptable range of the relevant combustion standard/literature on diesels[4, 10]. In addition, CNs of the original biodiesel with/without dehydration treatment were compared, and no difference was found. 2.5 Increase of “actual butanol yield” by adding tiny amount of electron carrier The liquid-liquid extraction coefficient of the waste cooking oil-originated biodiesel for butanol is not very high[7], therefore, “actual butanol yield” could only stay at a lower level of 13%–14% generally. Direct utilization of partial waste supernatant for the next fermentation run and elimination of energy-consuming product recovery process move toward the ideal “energy-saving & waste minimization” industrial production mode advocated by the state government. However, they actually conflict with the principle of effective raw materials utilization as the waste supernatant still contains certain amounts of solvent products. As a result, a new method that could simultaneously satisfy the two targets of “energy-saving & waste minimization” and “effective raw materials utilization” at maximum extent should be considered, to increase the “actual butanol yield” at the same reducing solvent contents in the waste supernatant. A previous report[11] showed that adding tiny amount of electron carrier such as neutral red in AB fermentation could change metabolic flow and enhance nicotinamide adenine dinucleotide hydrate (NADH) availability. Final concentration of butanol could be increased more than 30% and acetone be decreased about 50%. The central carbon flow branches at node of acetoacetyl-CoA in AB metabolic pathway: acetone forms in one flow under the regulation of CoA transferase and acetoacetate decarboxylase; butanol forms in another flow by the catalysis of NADH, H2, and multiple NADH-dependent coenzyme. Butanol fermentation has the following characteristics from the branch node: acetone formation requires or consumes neither NADH nor H2 evolved in the EMP (Embden-Meyerhof-Parnas) route; but butanol formation must rely on or consume a large amount of NADH and H2. The intercellular NADH comes from two routes, EMP and the electron transport shuttle system composed of ferredoxin (reductive/oxidative) and ferredoxin reductase (reductive/oxidative). Addition of electron carrier causes excess of proton and force the proton

Longyun Zhang et al. / Chinese Journal of Biotechnology, 2008, 24(11): 1943–1948

Table 1 Improvement of butanol content in biodiesel and “actual butanol yield” by adding neutral red Operation mode Biodiesel extraction (1:1) Biodiesel extraction (1:1) + 0.1% neutral red addition

Acetone conc. (g/L)

Butanol conc. (g/L)

Total solvent

Butanol/Acetone

Actual butanol yield (%)

Oil

Broth

Oil

Broth

conc. (g/L)

yield

2.87

7.78

11.29

8.29

30.23

1.84

13.83

2.25

5.64

13.41

10.04

31.34

2.97

18.13

Note: fermentation time 81 h

to act with ferredoxin-NAD reductase to overproduce NADH, resulting in an enhanced butanol formation but further strengthening butanol inhibition in turn. If an effective coupling of the selective butanol extractive fermentation with NADH overproduction is obtained, a more efficient property-improved biodiesel manufacturing process coupled with extractive fermentation could be expected. Addition of an electron carrier such as neutral red would reduce fermentative gas production, indicating the enhancement of fermentative reductive power. In addition, the more amount of neutral red added, the more H2 was utilized and more gas production reduction was observed. Based on the relationship between neutral red addition amount and reduction in total gas production determined experimentally (data not shown), 0.1% (W/V) was selected as the standard addition amount of neutral red. Table 1 shows performance of the “combinational AB extractive fermentation” and the relevant comparison results, where addition/use of neutral red enhancing NADH availability and biodiesel selectively extracting butanol was carried out simultaneously. Compared with the control (extractive fermentation without neutral red addition), butanol concentration in biodiesel and “actual butanol yield” increased from 11.3 g/L and 13.8% to 13.4% and 18.1%, the relative enhancement magnitude reached 19% and 31% respectively. Acetone concentration in broth phase also decreased 28%. However, the magnitudes of “actual butanol yield” increase and acetone concentration reduction in waste supernatant, in other words, the “high butanol/low acetone” fermentative characteristics still did not reach either the expected levels or the pursued levels. In addition, as a kind of dye, color change of biodiesel occurs even when very tiny amount of neutral red penetrates into the extractant phase. Therefore, the problem of final product depigmentation remains to be solved As for future prospects, progress has been achieved in enhancing the entire process performance compared with previous studies by the authors[7,8]. However, the results are still not very satisfactory and continuous efforts have to be made to further improve the process performance in the following aspects: 1) productivity of the properties-improved biodiesel; 2) quality of the properties-improved biodiesel, in other words, increasing butanol concentration in the biodiesel or CN of the biodiesel; 3) “actual butanol yield; 4) recycle ratio of waste supernatant, and it is desirable that more than 70%

waste fermentation broth could be recycled to further reduce waste water amount and environmental pollution.

3

Conclusions

1) Substrate inhibition does not occur when using initial medium with high corn flour concentration of 15%. 2) Under the optimized conditions, butanol concentration in biodiesel and “actual butanol yield” could reach the levels of 14.0 g/L and 18%. 3) CN of biodiesel extracting more than 10 g/L fermentative butanol increased 3 units compared with that of original biodiesel, and its high quality nature was verified through combustion testing. 4) Recycling 50% waste supernatant to next fermentation run could not only improve fermentation performance but also save valuable fresh water resources.

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