Preparation of immobilized whole cell biocatalyst and biodiesel production using a packed-bed bioreactor

Rhizopus oryzae NBRC 4697 was selected from among promising candidates as a biocatalyst for biodiesel production. This microorganism was immobilized on to polyurethane foam coated with activated carbon for reuse, and, for biodiesel production. Vacuum drying of the immobilized cells was found to be more efficient than natural or freeze-drying processes. Although the immobilized cells were severely inhibited by a molar ratio of methanol to soybean oil in excess of 2.0, stepwise methanol addition (3 aliquots at 24-h feeding intervals) significantly prevented methanol inhibition. A packed-bed bioreactor (PBB) containing the immobilized whole cell biocatalyst was then operated under circulating batch mode. Stepwise methanol feeding was used to mitigate methanol inhibition of the immobilized cells in the PBB. An increase in the feeding rate (circulating rate) of the reaction mixture barely affected biodiesel production, while an increase in the packing volume of the immobilized cells enhanced biodiesel production noticeably. Finally, repeated circulating batch operation of the PBB was carried out for five consecutive rounds without a noticeable decrease in the performance of the PBB for the three rounds.     

Application of a Burkholderia cepacia lipase-immobilized silica monolith to batch and continuous biodiesel production with a stoichiometric mixture of methanol and crude Jatropha oil

Background

The enzymatic production of biodiesel through alcoholysis of triglycerides has become more attractive because it shows potential in overcoming the drawbacks of chemical processes. In this study, we investigate the production of biodiesel from crude, non-edible Jatropha oil and methanol to characterize Burkholderia cepacia lipase immobilized in an n-butyl-substituted hydrophobic silica monolith. We also evaluate the performance of a lipase-immobilized silica monolith bioreactor in the continuous production of biodiesel.

Results

The Jatropha oil used contained 18% free fatty acids, which is problematic in a base-catalyzed process. In the lipase-catalyzed reaction, the presence of free fatty acids made the reaction mixture homogeneous and allowed bioconversion to proceed to 90% biodiesel yield after a 12 hour reaction time. The optimal molar ratio of methanol to oil was 3.3 to 3.5 parts methanol to one part oil, with water content of 0.6% (w/w). Further experiments revealed that B. cepacia lipase immobilized in hydrophobic silicates was sufficiently tolerant to methanol, and glycerol adsorbed on the support disturbed the reaction to some extent in the present reaction system. The continuous production of biodiesel was performed at steady state using a lipase-immobilized silica monolith bioreactor loaded with 1.67 g of lipase. The yield of 95% was reached at a flow rate of 0.6 mL/h, although the performance of the continuous bioreactor was somewhat below that predicted from the batch reactor. The bioreactor was operated successfully for almost 50 days with 80% retention of the initial yield.

Conclusions

The presence of free fatty acids originally contained in Jatropha oil improved the reaction efficiency of the biodiesel production. A combination of B. cepacia lipase and its immobilization support, n-butyl-substituted silica monolith, was effective in the production of biodiesel. This procedure is easily applicable to the design of a continuous flow-through bioreactor system.     

Determination of kinetic parameters of 1,3-propanediol fermentation by Clostridium diolis using statistically optimized medium

1,3-propanediol (1,3-PD) is a chemical compound of immense importance primarily used as a raw material for fiber and textile industry. It can be produced by the fermentation of glycerol available abundantly as a by-product from the biodiesel plant. The present study was aimed at determination of key kinetic parameters of 1,3-PD fermentation by Clostridium diolis. Initial experiments on microbial growth inhibition were followed by optimization of nutrient medium recipe by statistical means. Batch kinetic data from studies in bioreactor using optimum concentration of variables obtained from statistical medium design was used for estimation of kinetic parameters of 1,3-PD production. Direct use of raw glycerol from biodiesel plant without any pre-treatment for 1,3-PD production using this strain investigated for the first time in this work gave results comparable to commercial glycerol. The parameter values obtained in this study would be used to develop a mathematical model for 1,3-PD to be used as a guide for designing various reactor operating strategies for further improving 1,3-PD production. An outline of protocol for model development has been discussed in the present work.     

Enhancing biodiesel production by immobilized whole cells by optimizing reaction conditions and adding glycerol and water

In this study, several methods were devised and evaluated to enhance biodiesel production by whole cells immobilized onto the polyurethane foam coated with activated carbon. Biodiesel conversion was increased to 76.4% with the increase in the number of polyurethane foam until it occupied 18.0 or 2.4% of reaction mixture based on apparent or actual volume of supports, respectively. Stepwise methanol addition to prevent methanol inhibition on the immobilized whole cells was optimized in terms of number of aliquot and feeding interval. When 4.5 molar ratio of methanol to soybean oil was divided into 4 equal aliquots and each aliquot was fed to the reaction mixture every 24 h, the highest final biodiesel conversion of 82.4% was achieved. Chemical treatment of the immobilized cells with 0.1% of chloroform for 2 h enhanced biodiesel conversion to 90.5%. The initial addition of 5% glycerol in the fresh reaction mixture increased biodiesel conversion to 90.3% while the removal of glycerol during biodiesel production barely increased biodiesel conversion. The biodiesel conversion was increased with the increase of initial water content in the fresh reaction mixture and the highest value was 92.7% at 3.0% of water content, but decreased thereafter. The effects of co-addition of glycerol and water on biodiesel production were also investigated, and the co-addition of 3.125% of glycerol and 1.875% of water relative to soybean oil substantially increased biodiesel conversion to 95.0%. By these optimization of reaction conditions and co-adding glycerol and water, initial biodiesel production rate and final biodiesel conversion were remarkably enhanced by 26.8 and 24.1%, respectively.     

Process design and evaluation of value-added chemicals production from biomass

Three different biodiesel production processes were simulated using the SuperPro Designer program. The process for producing biodiesel from soybean oil and methanol was designed using commercial chemical catalysts. This chemical process was compared with the biological process catalyzed by immobilized enzymes. In addition, a hybrid process consisting of catalytic biodiesel production and enzymatic glycerol carbonate production was designed and simulated for the conversion of waste glycerol to value-added chemical. Finally, the economics and productivity of these processes were evaluated to determine economic feasibility.     

酶法合成生物柴油工业化研究进展

介绍了北京化工大学近年来酶法合成生物柴油工业化研究的结果。主要内容包括以下几个方面:高产脂肪酶菌株的选育、脂肪酶发酵工艺优化及放大、脂肪酶固定化方法、酶反应器放大、生物柴油分离精制及副产物甘油综合利用。该脂肪酶假丝酵母Candida sp.99-125在5 m3罐发酵活力不低于8 000 IU/mL,然后将该脂肪酶吸附固定在织物膜上并进行表面改性,用于搅拌罐式反应器生产每吨甲酯的需酶量仅为4.2 kg,产品经分离精制调质后,其各项指标完全符合德国生物柴油生产标准。副产物甘油可用于1,3-丙二醇发酵,30 L发酵罐中1,3-丙二醇的产量可达到76.1 g/L。     

One-step enzymatic production of fatty acid ethyl ester from high-acidity waste feedstocks in solvent-free media

This work aims to demonstrate the enzymatic production of fatty acid ethyl ester biodiesel from highly acidic feedstock in a single-step reaction, without co-solvents and avoiding the inhibition of the enzyme by ethanol and glycerol. Additionally, an empirical equation is proposed to predict the kinetics of the production reaction as a function of the used feedstock and catalyst concentration. Biodiesel production from highly acidic feedstock perform via simultaneous esterification of free fatty acids and transesterification of triacylglycerols. Enzymatic catalysis is one of the most promising alternative technologies for the biodiesel production. Increasing of the enzymatic bioactivity is crucial for the success of such process in industrial scale. Currently, stepwise addition of the alcohol or the use of co-solvents have been proposed to avoid enzyme inhibition, such strategies add downstream processes to the production. These results can be applied to the development economical-viable enzymatic production of biodiesel in industrial scale.     

Kinetics and mechanism of the cutinase-catalyzed transesterification of oils in AOT reversed micellar system

The kinetics of the enzymatic transesterification between a mixture of triglycerides (oils) and methanol for biodiesel production in a bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane reversed micellar system, using recombinant cutinase from Fusarium solani pisi as a catalyst, was investigated. In order to describe the results that were obtained, a mechanistic scheme was proposed, based on the literature and on the experimental data. This scheme includes the following reaction steps: the formation of the active enzyme–substrate complex, the addition of an alcohol molecule to the complex followed by the separation of a molecule of the fatty acid alkyl ester and a glycerol moiety, and release of the active enzyme. Enzyme inhibition and deactivation effects due to methanol and glycerol were incorporated in the model. This kinetic model was fitted to the concentration profiles of the fatty acid methyl esters (the components of biodiesel), tri-, di- and monoglycerides, obtained for a 24 h transesterification reaction performed in a stirred batch reactor under different reaction conditions of enzyme and initial substrates concentration.     

Soluble expression of Candida antarctica lipase B in Escherichia coli by fusion with Skp chaperone

Candida antarctica lipase B (CalB) is an industrially versatile enzyme, especially for biodiesel production and organic synthesis. Recombinant expression using the E. coli system has advantages, such as lower costs, easier handling, and higher number of clones that can be screened daily compared to expression using higher organism. But the expression of CalB in E. coli is not feasible because insoluble aggregates are formed and proteolytic degradation is known to occur during expression. In this study, fusion proteins were designed to express soluble CalB in E. coli. The periplasmic chaperone of E. coli, Skp was fused with CalB and this fusion protein showed a high solubility (yielding 82.5 ??g/mL). The fusion protein system can be applied to the rapid expression and evaluation of CalB variants for functional improvement.     

Improvement of enzymatic biodiesel production by controlled substrate feeding using silica gel in solvent free system

A silica gel-based substrate feeding system was developed to prevent methanol inhibiting the catalyst during enzymatic biodiesel synthesis. In the system, silica gel swelled upon methanol addition and subsequently released it in a controlled manner to prevent excess methanol affecting the enzyme. Biodiesel was synthesized by the enzymatic transesterification of canola oil with methanol. For this reaction, enzyme loading, methanol/oil molar ratio, silica gel dosage, glycerol content, and methanol feeding method were tested using commercial immobilized enzymes (Novozym 435 and Lipozyme RM IM from Novozymes). The results showed that conversion was highest with controlled substrate feeding rather than direct methanol addition, suggesting that the method developed here can easily prevent enzyme inhibition by limiting methanol concentration to an acceptable level.     

Microbial utilization of crude glycerol for the production of value-added products

Energy fuels for transportation and electricity generation are mainly derived from finite and declining reserves of fossil hydrocarbons. Fossil hydrocarbons are also used to produce a wide range of organic carbon-based chemical products. The current global dependency on fossil hydrocarbons will not be environmentally or economically sustainable in the long term. Given the future pessimistic prospects regarding the complete dependency on fossil fuels, political and economic incentives to develop carbon neutral and sustainable alternatives to fossil fuels have been increasing throughout the world. For example, interest in biodiesel has undergone a revival in recent times. However, the disposal of crude glycerol contaminated with methanol, salts, and free fatty acids as a by-product of biodiesel production presents an environmental and economic challenge. Although pure glycerol can be utilized in the cosmetics, tobacco, pharmaceutical, and food industries (among others), the industrial purification of crude glycerol is not economically viable. However, crude glycerol could be used as an organic carbon substrate for the production of high-value chemicals such as 1,3-propanediol, organic acids, or polyols. Microorganisms have been employed to produce such high-value chemicals and the objective of this article is to provide an overview of studies on the utilization of crude glycerol by microorganisms for the production of economically valuable products. Glycerol as a by-product of biodiesel production could be used a feedstock for the manufacture of many products that are currently produced by the petroleum-based chemical industry.     

Continuous production of 1,3-propanediol using raw glycerol with immobilized Clostridium beijerinckii NRRL B-593 in comparison to suspended culture

The continuous production of 1,3-propanediol (1,3-PDO) was investigated with Clostridium beijerinckii NRRL B-593 using raw glycerol without purification obtained from a biodiesel production process. Ceramic rings and pumice stones were used for cell immobilization in a packed-bed bioreactor. For comparison purpose, a control bioreactor with suspended culture was also run. The effect of hydraulic retention time (HRT) on the production of 1,3-PDO in both immobilized and suspended bioreactors were also investigated. The study revealed that HRT is an important factor for both immobilized and suspended systems and a HRT of 2 h is the best one in terms of volumetric production rate (g 1,3-PDO/L/h). Furthermore, cell immobilization had also obvious benefits especially for the robustness and the reliability of the production. The results indicated that cell immobilization achieved a 2.5-fold higher productivity in comparison to suspended cell system. Based on our results, continuous production of 1,3-PDO with immobilized cells is an efficient method, and raw glycerol can be utilized without any pretreatment.     

An overview of enzymatic production of biodiesel

Biodiesel production has received considerable attention in the recent past as a biodegradable and nonpolluting fuel. The production of biodiesel by transesterification process employing alkali catalyst has been industrially accepted for its high conversion and reaction rates. Recently, enzymatic transesterification has attracted much attention for biodiesel production as it produces high purity product and enables easy separation from the byproduct, glycerol. But the cost of enzyme remains a barrier for its industrial implementation. In order to increase the cost effectiveness of the process, the enzyme (both intracellular and extracellular) is reused by immobilizing in a suitable biomass support particle and that has resulted in considerable increase in efficiency. But the activity of immobilized enzyme is inhibited by methanol and glycerol which are present in the reacting mixture. The use of tert-butanol as solvent, continuous removal of glycerol, stepwise addition of methanol are found to reduce the inhibitory effects thereby increasing the cost effectiveness of the process. The present review analyzes these methods reported in literature and also suggests a suitable method for commercialization of the enzymatic process.     

Metabolic modelling of syntrophic-like growth of a 1,3-propanediol producer, Clostridium butyricum, and a methanogenic archeon, Methanosarcina mazei, under anaerobic conditions

Clostridium butyricum can convert glycerol into 1,3-propanediol, thereby generating unfortunately a high amount of acetate, formate and butyrate as inhibiting by-products. We have proposed a novel mixed culture comprising C. butyricum and a methane bacterium, Methanosarcina mazei, to relieve the inhibition and to utilise the by-products for energy production. In order to examine the efficiency of such a mixed culture, metabolic modelling of the culture system was performed in this work. The metabolic networks for the organisms were reconstructed from genomic and physiological data. Several scenarios were analysed to examine the preference of M. mazei in scavenging acetate and formate under conditions of different substrate availability, including methanol as a co-substrate, since it may exist in glycerol solution from biodiesel production. The calculations revealed that if methanol is present, the methane production can increase by 130%. M. mazei can scavenge over 70% of the acetate secreted by C. butyricum.     

Lipase-catalyzed biodiesel production from soybean oil deodorizer distillate with absorbent present in tert-butanol system

Lipase-catalyzed alcoholysis of soybean oil deodorizer distillate (SODD) for biodiesel production was studied. During this system both free fatty acids and glycerides could be converted to biodiesel simultaneously. tert-Butanol has been adopted as the reaction medium, in which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated completely. There was no obvious loss in lipase activity even after being repeatedly used for 120 cycles. Fine-pored silica gel and 3 Å molecular were found to be effective to control by-product water concentration and much higher biodiesel yield could be achieved with those adsorbents present in the reaction system. The highest biodiesel yield of 97% could be achieved with 3 Å molecular sieve as the adsorbent.     

Effect of impurities in biodiesel-derived waste glycerol on the performance and feasibility of biotechnological processes

The rapid development of biodiesel production technology has led to the generation of tremendous quantities of glycerol wastes, as the main by-product of the process. Stoichiometrically, it has been calculated that for every 100 kg of biodiesel, 10 kg of glycerol are produced. Based on the technology imposed by various biodiesel plants, glycerol wastes may contain numerous kinds of impurities such as methanol, salts, soaps, heavy metals, and residual fatty acids. This fact often renders biodiesel-derived glycerol unprofitable for further purification. Therefore, the utilization of crude glycerol though biotechnological means represents a promising alternative for the effective management of this industrial waste. This review summarizes the effect of various impurities-contaminants that are found in biodiesel-derived crude glycerol upon its conversion by microbial strains in biotechnological processes. Insights are given concerning the technologies that are currently applied in biodiesel production, with emphasis to the impurities that are added in the composition of crude glycerol, through each step of the production process. Moreover, extensive discussion is made in relation with the impact of the nature of impurities upon the performances of prokaryotic and eukaryotic microorganisms, during crude glycerol bioconversions into a variety of high added-value metabolic products. Finally, aspects concerning ways of crude glycerol treatment for the removal of inhibitory contaminants as reported in the literature are given and comprehensively discussed.     

Lipase-catalyzed transesterification of rapeseed oils for biodiesel production with a novel organic solvent as the reaction medium

tert-Butanol, as a novel reaction medium, has been adopted for lipase-catalyzed transesterification of rapeseed oil for biodiesel production, with which both the negative effects caused by excessive methanol and by-product glycerol could be eliminated. Combined use of Lipozyme TL IM and Novozym 435 was proposed further to catalyze the methanolysis and the highest biodiesel yield of 95% could be achieved under the optimum conditions (tert-butanol/oil volume ratio 1:1; methanol/oil molar ratio 4:1; 3% Lipozyme TL IM and 1% Novozym 435 based on the oil weight; temperature 35 °C; 130 rpm, 12 h). There was no obvious loss in lipase activity even after being repeatedly used for 200 cycles with tert-butanol as the reaction medium. Furthermore, waste oil was also explored for biodiesel production and it has been found that lipase also showed good stability in this novel system.     

Biodiesel production using a membrane reactor

The immiscibility of canola oil in methanol provides a mass-transfer challenge in the early stages of the transesterification of canola oil in the production of fatty acid methyl esters (FAME or biodiesel). To overcome or rather, exploit this situation, a two-phase membrane reactor was developed to produce FAME from canola oil and methanol. The transesterification of canola oil was performed via both acid- or base-catalysis. Runs were performed in the membrane reactor in semi-batch mode at 60, 65 and 70 degrees C and at different catalyst concentrations and feed flow rates. Increases in temperature, catalyst concentration and feedstock (methanol/oil) flow rate significantly increased the conversion of oil to biodiesel. The novel reactor enabled the separation of reaction products (FAME/glycerol in methanol) from the original canola oil feed. The two-phase membrane reactor was particularly useful in removing unreacted canola oil from the FAME product yielding high purity biodiesel and shifting the reaction equilibrium to the product side.     

Glycerol extracting dealcoholization for the biodiesel separation process

By means of utilizing sunflower oil and Jatropha oil as raw oil respectively, the biodiesel transesterification production and the multi-stage extracting separation were carried out experimentally. Results indicate that dealcoholized crude glycerol can be utilized as the extracting agent to achieve effective separation of methanol from the methyl ester phase, and the glycerol content in the dealcoholized methyl esters is as low as 0.02 wt.%. For the biodiesel separation process utilizing glycerol extracting dealcoholization, its technical and equipment information were acquired through the rigorous process simulation in contrast to the traditional biodiesel distillation separation process, and results show that its energy consumption decrease about 35% in contrast to that of the distillation separation process. The glycerol extracting dealcoholization has sufficient feasibility and superiority for the biodiesel separation process.     

毕赤酵母发酵产S-腺苷蛋氨酸的甘油-甲醇混合流加策略

在毕赤酵母发酵生产S-腺苷蛋氨酸(SAM)的诱导阶段,以不同甘油-甲醇比例的甘油-甲醇混合培养基进行诱导培养,结果表明以10%(w/v)甘油含量的甘油-甲醇混合培养基进行诱导培养时最有利于SAM的表达,SAM产量达6.09 g/L,比0%甘油含量条件下的SAM产量提高了20.4%。对诱导方式进行优化,先以100%甲醇诱导24 h,然后再连续流加10%(w/v)甘油含量的甘油-甲醇混合培养基,SAM产量可达7.94 g/L,在此基础上,进一步改进诱导方式,SAM产量得到进一步的提高,达到9.80 g/L。