Long-term repeated fed-batch ethanol fermentation in aerated condition

In this study, we attempted to assess the process stability of long-term fed-batch ethanol fermentation in the absence and presence of aeration (0.33 vvm). To examine the effect of aeration, a long-term repeated fed-batch operation was conducted for 396 h to mimic a long-term industrial bioethanol production process. In this long-term repeated fed-batch ethanol fermentation experiments, withdrawal-fill operation were conducted every 36 h for 10 repeat cycles. The whole operation was stably sustained in a quasi-steady state. The average maximal cell concentration and the average maximal ethanol production during operation were increased by 81.63 and 12.12%, respectively, when aeration was used. In addition, since aeration was carried out, the average ethanol yield slightly decreased by 4.03% and the average specific ethanol production rate decreased by 46.75% during operation. However, the average ethanol productivity increased by 17.53% when aeration was carried out. After 396 h of long-term repeated fed-batch ethanol fermentation, 1,908.9 g of ethanol was cumulatively produced when aeration was used, which was 12.47%, higher than when aeration was not used (1,697.2 g). Meanwhile, glycerol production was greatly decreased during long-term repeated fed-batch ethanol fermentation, in which the glycerol concentration in the culture broth decreased from about 34∼15 g/L. Thus, we can conclude that cell growth was greatly improved by overcoming ethanol inhibition and glycerol production was remarkably decreased when aeration was carried out, although aeration in ethanol fermentation decreased the specific ethanol production rate and ethanol yield.     

High-level production of ethanol during fed-batch ethanol fermentation with a controlled aeration rate and non-sterile glucose powder feeding of Saccharomyces cerevisiae

In this study, we utilized a unique strategy for fed-batch fermentation using ethanol-tolerant Saccharomyces cerevisiae to achieve a high-level of ethanol production that could be practically applied on an industrial scale. During this study, the aeration rate was controlled at 0.0, 0.13, 0.33, and 0.8 vvm to determine the optimal aeration conditions for the production of ethanol. Additionally, non-sterile glucose powder was fed during fed-batch ethanol fermentation and corn-steep liquor (CSL) in the medium was used as an organic N-source. When aeration was conducted, the ethanol production and productivity were superior to that when aeration was not conducted. Specifically, the maximum ethanol production reached approximately 160 g/L, when the fermentor was aerated at 0.13 vvm. These findings indicate that the use of a much less expensive C-source may enable the fermentation process to be directed towards the improvement of overall ethanol production and productivity in fermentors that are aerated at 0.13 vvm. Furthermore, if a repeated fed-batch process in which the withdrawal and fill is conducted prior to 36 h can be employed, aeration at a rate of 0.33 and/or 0.8 vvm may improve the overall ethanol productivity     

Aeration strategy: a need for very high ethanol performance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> fed-batch process

In order to identify an optimal aeration strategy for intensifying bio-fuel ethanol production in fermentation processes where growth and production have to be managed simultaneously, we quantified the effect of aeration conditions—oxygen limited vs non limited culture (micro-aerobic vs aerobic culture)—on the dynamic behaviour of Saccharomyces cerevisiae cultivated in very high ethanol performance fed-batch cultures. Fermentation parameters and kinetics were established within a range of ethanol concentrations (up to 147 g l^–1), which very few studies have addressed. Higher ethanol titres (147 vs 131 g l^–1 in 45 h) and average productivity (3.3 vs 2.6 g l^–1 h^–1) were obtained in cultures without oxygen limitation. Compared to micro-aerobic culture, full aeration led to a 23% increase in the viable cell mass as a result of the concomitant increase in growth rate and yield, with lower ethanol inhibition. The second beneficial effect of aeration was better management of by-product production, with production of glycerol, the main by-product, being strongly reduced from 12 to 4 g l^–1. We demonstrate that aeration strategy is as much a determining factor as vitamin feeding (Alfenore et al. 2002) in very high ethanol performance (147 g l^–1 in 45 h) in order to achieve a highly competitive dynamic process.     

Fed-batch cultivation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on lignocellulosic hydrolyzate

Saccharomyces cerevisiae grows very poorly in dilute acid lignocellulosic hydrolyzate during the anaerobic fermentation for fuel ethanol production. However, yeast cells grown aerobically on the hydrolyzate have increased tolerance for the hydrolyzate. Cultivation of yeast on part of the hydrolyzate has therefore the potential of enabling increased ethanol productivity in the fermentation of the hydrolyzate. To evaluate the ability of the yeast to grow in the hydrolyzate, fed-batch cultivations were run using the ethanol concentration as input variable to control the feed-rate. The yeast then grew in an undetoxified hydrolyzate with a specific growth rate of 0.19 h⁻¹ by controlling the ethanol concentration at a low level during the cultivation. However, the biomass yield was lower for the cultivation on hydrolyzate compared to synthetic media: with an ethanol set-point of 0.25 g/l the yield was 0.46 g/g on the hydrolyzate, compared to 0.52 g/g for synthetic media. The main reason for the difference was not the ethanol production per se, but a significant production of glycerol at a high specific growth rate. The glycerol production may be attributed to an insufficient respiratory capacity.     

Fed-batch xylitol production with recombinantXYL-1-expressingSaccharomyees cerevisiae using ethanol as a co-substrate

The bioconversion of xylose into xylitol in fed-batch fermentation with a recombinantSaccharomyces cerevisiae strain, transformed with the xylose-reductase gene ofPichia stipitis, was studied. When only xylose was fed into the fermentor, the production of xylitol continued until the ethanol that had been produced during an initial growth phase on glucose, was depleted. It was concluded that ethanol acted as a redox-balance-retaining co-substrate. The conversion of high amounts of xylose into xylitol required the addition of ethanol to the feed solution. Under O₂-limited conditions, acetic acid accumulated in the fermentation broth, causing poisoning of the yeast at low extracellular pH. Acetic acid toxicity could be avoided by either increasing the pH from 4.5 to 6.5 or by more effective aeration, leading to the further metabolism of acetic acid into cell mass. The best xylitol/ethanol yield, 2.4 gg^–1 was achieved under O₂-limited conditions. Under anaerobic conditions ethanol could not be used as a co-substrate, because the cell cannot produce ATP for maintenance requirements from ethanol anaerobically. The specific rate of xylitol production decreased with increasing aeration. The initial volumetric productivity increased when xylose was added in portions rather than by continuous feeding, due to a more complete saturation of the transport system and the xylose reductase enzyme.     

The effect of aeration on xylose fermentation by Candida shehatae and Pachysolen tannophilus

Summary The ability of a Candida shehatae and a Pachysolen tannophilus strain to ferment D-xylose to ethanol was evaluated in defined and complex media under different levels of aeration. Aeration enhanced the ethanol productivity of both yeasts considerably. C. shehatae maintained a higher fermentation rate and ethanol yield than P. tannophilus over a wide range of aeration levels. Ethanol production by C. shehatae commenced during the early stage of the fermentation, whereas with P. tannophilus there was a considerable lag between the initiation of growth and ethanol production. Both yeasts produced appreciable quantities of xylitol late in the fermentation. P. tannophilus failed to grow under anoxic conditions, producing a maximum of only 0.5 g · l^-1 ethanol. In comparison, C. shehatae exhibited limited growth in anoxic cultures, and produced ethanol much more rapidly. Under the condition of aeration where C. shehatae exhibited the highest ethanol productivity, the fermentation parameters were: maximum specific growth rate, 0.15 h^-1; maximum volumetric and specific rates of ethanol production, 0.7 g (l · h)^-1 and 0.34 g ethanol (g cells · h)^-1 respectively; ethanol yield, 0.36 g (g xylose)^-1. The best values obtained with P. tannophilus were: maximum specific growth rate, 0.14 h^-1; maximum volumetric and specific rates of ethanol production, 0.22 g (l · h)^-1 and 0.07 h^-1 respectively; ethanol yield coefficient, 0.28. Because of its higher ethanol productivity at various levels of aeration, C. shehatae has a greater potential for ethanol production from xylose than P. tannophilus.     

The inhibitory effect of ethanol on ethanol production by Zymomonas mobilis

Summary In an effort to establish the reasons for the limitations in the final ethanol concentration of Zymomonas mobilis fermentation, the effects of CO₂ and ethanol on the fermentation were investigated using continuous and fed-batch cultivation systems. The nucleation and stripping out of CO₂ from the fermenter using diatomaceous earth or nitrogen gas or both exhibited a profound effect on the glucose uptake rate during the early stages of fed-batch fermentation, but did not improve final ethanol yields. The addition of ethanol together with above mentioned experiments confirmed conclusively that ethanol inhibition is responsible for the final ethanol concentration obtainable during Zymomonas mobilis fermentation. The final concentration lies between 90 and 110 gl⁻¹ or approximately 12–15% (v/v) ethanol.     

补料分批发酵过程中两阶段发酵生产1,3-丙二醇

在补料分批发酵过程中提高比生长速率不仅减少乙醇、甲酸的生成,而且提高1,3-丙二醇的得率和比生产速率.发酵后期甘油的浓度在15～26 g/L时有利于提高1,3-丙二醇的生产.采取在发酵前期控制菌体较高比生长速率和发酵后期控制适宜甘油浓度相结合的策略,有效地提高了1,3-丙二醇的生产,降低副产物乳酸和乙醇的生成.     

Performance of batch, fed-batch, and continuous A-B-E fermentation with pH-control

Batch, fed-batch, and continuous A-B-E fermentations were conducted and compared with pH controlled at 4.5, the optimal range for solvent production. While the batch mode provides the highest solvent yield, the continuous mode was preferred in terms of butanol yield and productivity. The highest butanol yield and productivity found in the continuous fermentation at dilution rate of 0.1 h⁻¹ were 0.21 g-butanol/g-glucose and 0.81 g/L/h, respectively. In the continuous and fed-batch fermentation, the time needed for passing acidogenesis to solventogenesis was an intrinsic hindrance to higher butanol productivity. Therefore, a low dilution rate is suggested for the continuous A-B-E fermentation, while the fed-batch mode is not suggested for solvent production. While 3:6:1 ratio of acetone, butanol, and ethanol is commonly observed from A-B-E batch fermentation by Clostridium acetobutylicum when the pH is uncontrolled, up to 94% of the produced solvent was butanol in the chemostat with pH controlled at 4.5.     

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l⁻¹) and less than 200 g l⁻¹ total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l⁻¹ xylitol concentration, 0.75 g xylitol g xylose⁻¹ xylitol yield and 3.9 g xylitol l⁻¹ h⁻¹ volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257 Received 15 October 2001/ Accepted in revised form 30 March 2002     

Sugar fermentation by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> to produce ethanol

As a first step in the research on ethanol production from lignocellulose residues, sugar fermentation by Fusarium oxysporum in oxygen-limited conditions is studied in this work. As a substrate, solutions of arabinose, glucose, xylose and glucose/xylose mixtures are employed. The main kinetic and yield parameters of the process are determined according to a time-dependent model. The microorganism growth is characterized by the maximum specific growth rate and biomass productivity, the substrate consumption is studied through the specific consumption rate and biomass yield, and the product formation via the specific production rate and product yields. In conclusion, F. oxysporum can convert glucose and xylose into ethanol with product yields of 0.38 and 0.25, respectively; when using a glucose/xylose mixture as carbon source, the sugars are utilized sequentially and a maximum value of 0.28 g/g ethanol yield is determined from a 50% glucose/50% xylose mixture. Although fermentation performance by F.␣oxysporum is somewhat lower than that of other fermenting microorganisms, its ability for simultaneous lignocellulose-residue saccharification and fermentation is considered as a potential advantage.     

Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp.

The effect of aeration on the performance of docosahexaenoic acid (DHA) production by Schizochytrium sp. was investigated in a 1,500-L bioreactor using fed-batch fermentation. Six parameters, including specific growth rate, specific glucose consumption rate, specific lipid accumulation rate, cell yield coefficient, lipid yield coefficient, and DHA yield coefficient, were used to understand the relationship between aeration and the fermentation characteristics. Based on the information obtained from the parameters, a stepwise aeration control strategy was proposed. The aeration rate was controlled at 0.4 volume of air per volume of liquid per minute (vvm) for the first 24 h, then shifted to 0.6 vvm until 96 h, and then switched back to 0.4 vvm until the end of the fermentation. High cell density (71 g/L), high lipid content (35.75 g/L), and high DHA percentage (48.95%) were achieved by using this strategy, and DHA productivity reached 119 mg/L h, which was 11.21% over the best results obtained by constant aeration rate.     

Co-fermentation of a mixture of glucose and xylose to ethanol by Zymomonas mobilis and Pachysolen tannophilus

This research was designed to maximize ethanol production from a glucose-xylose sugar mixture (simulating a sugar cane bagasse hydrolysate) by co-fermentation with Zymomonas mobilis and Pachysolen tannophilus. The volumetric ethanol productivity of Z. mobilis with 50 g glucose/l was 2.87 g/l/h, giving an ethanol yield of 0.50 g/g glucose, which is 98% of the theoretical. P. tannophilus when cultured on 50 g xylose/l gave a volumetric ethanol productivity of 0.10 g/l/h with an ethanol yield of 0.15 g/g xylose, which is 29% of the theoretical. On optimization of the co-fermentation with the sugar mixture (60 g glucose/l and 40 g xylose/l) a total ethanol yield of 0.33 g/g sugar mixture, which is 65% of the theoretical yield, was obtained. The co-fermentation increased the ethanol yield from xylose to 0.17 g/g. Glucose and xylose were completely utilized and no residual sugar was detected in the medium at the end of the fermentation. The pH of the medium was found to be a good indicator of the fermentation status. The optimum conditions were a temperature of 30°C, initial inoculation with Z. mobilis and incubation with no aeration, inactivation of bacterium after the utilization of glucose, followed by inoculation with P. tannophilus and incubation with limited aeration.     

Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review

One of the major challenges faced in commercial production of lignocellulosic bioethanol is the inhibitory compounds generated during the thermo-chemical pre-treatment step of biomass. These inhibitory compounds are toxic to fermenting micro-organisms. The ethanol yield and productivity obtained during fermentation of lignocellulosic hydrolysates is decreased due to the presence of inhibiting compounds, such as weak acids, furans and phenolic compounds formed or released during thermo-chemical pre-treatment step such as acid and steam explosion. This review describes the application and/or effect of biological detoxification (removal of inhibitors before fermentation) or use of bioreduction capability of fermenting yeasts on the fermentability of the hydrolysates. Inhibition of yeast fermentation by the inhibitor compounds in the lignocellulosic hydrolysates can be reduced by treatment with enzymes such as the lignolytic enzymes, for example, laccase and micro-organisms such as Trichoderma reesei, Coniochaeta ligniaria NRRL30616, Trametes versicolor, Pseudomonas putida Fu1, Candida guilliermondii, and Ureibacillus thermosphaericus. Microbial and enzymatic detoxifications of lignocellulosic hydrolysate are mild and more specific in their action. The efficiency of enzymatic process is quite comparable to other physical and chemical methods. Adaptation of the fermentation yeasts to the lignocellulosic hydrolysate prior to fermentation is suggested as an alternative approach to detoxification. Increases in fermentation rate and ethanol yield by adapted micro-organisms to acid pre-treated lignocellulosic hydrolysates have been reported in some studies. Another approach to alleviate the inhibition problem is to use genetic engineering to introduce increased tolerance by Saccharomyces cerevisiae, for example, by overexpressing genes encoding enzymes for resistance against specific inhibitors and altering co-factor balance. Cloning of the laccase gene followed by heterologous expression in yeasts was shown to provide higher enzyme yields and permit production of laccases with desired properties for detoxification of lignocellulose hydrolysates. A combination of more inhibitor-tolerant yeast strains with efficient feed strategies such as fed-batch will likely improve lignocellulose-to-ethanol process robustness.     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Microbial fed-batch production of 1,3-propanediol by Klebsiella pneumoniae under micro-aerobic conditions

The microbial production of 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under micro-aerobic conditions was investigated in this study. The experimental results of batch fermentation showed that the final concentration and yield of 1,3-PD on glycerol under micro-aerobic conditions approached values achieved under anaerobic conditions. However, less ethanol was produced under microaerobic than anaerobic conditions at the end of fermentation. The batch micro-aerobic fermentation time was markedly shorter than that of anaerobic fermentation. This led to an increment of productivity of 1,3-PD. For instance, the concentration, molar yield, and productivity of 1,3-PD of batch micro-aerobic fermentation by K. pneumoniae DSM 2026 were 17.65 g/l, 56.13%, and 2.94 g l^–1 h^–1, respectively, with a fermentation time of 6 h and an initial glycerol concentration of 40 g/l. Compared with DSM 2026, the microbial growth of K. pneumoniae AS 1.1736 was slow and the concentration of 1,3-PD was low under the same conditions. Furthermore, the microbial growth in fed-batch fermentation by K. pneumoniae DSM 2026 was faster under micro-aerobic than anaerobic conditions. The concentration, molar yield, and productivity of 1,3-PD in fed-batch fermentation under micro-aerobic conditions were 59.50 g/l, 51.75%, and 1.57 g l^–1 h^–1, respectively. The volumetric productivity of 1,3-PD under microaerobic conditions was almost twice that of anaerobic fed-batch fermentation, at 1.57 and 0.80 g l^–1 h^–1, respectively.     

Enhanced production of 2,3-butanediol from glycerol by forced pH fluctuations

The glycerol fermentation by Klebsiella pneumoniae occurs by receiving more than five liquid products—organic acids, diols, and ethanol. Aiming to direct the glycerol conversion towards predominant production of 2,3-butanediol (2,3-BD), the main influencing parameters (the aeration and the pH) were investigated during fed-batch processes. The regime of intensive aeration (2.2 vvm air supply) was evaluated as most favorable for 2,3-BD synthesis and ensured the decrease of all other metabolites. Thus, without pH control, 52.5 g/l 2,3-BD were produced, as the carbon conversion of glycerol into 2,3-BD reached 60.6%. Additional enhancement in 2,3-BD production (by significant increase of glycerol utilization) was achieved by the development of a new method of “forced pH fluctuations”. It was realized by consecutive raisings of pH using definite ΔpH value, at exact time intervals, allowing multiple variations. Thus, the optimal conditions for maximal glycerol consumption were defined, and 70 g/l 2,3-BD were produced, which is the highest amount obtained from glycerol as a sole carbon source until now. The forced pH fluctuations emphasized pH as a governing factor in microbial conversion processes.     

Production of ethanol from mannitol by Zymobacter palmae

Extracts from brown seaweeds could possibly be fermented to ethanol, particularly seaweeds harvested in the autumn, which contain high levels of easily extractable laminaran and mannitol. Few microorganisms are able to utilise mannitol as a substrate for ethanol production and Zymobacter palmae was tested for this purpose. Bacterial growth as well as ethanol yield depended on the amount of oxygen present. Strictly anaerobic growth on mannitol was not observed. At excessive aeration, a change in the fermentation pattern was observed with high production of acetate and propionate. Under oxygen-limiting conditions, the bacteria grew and produced ethanol in a synthetic mannitol medium with a yield of 0.38 g ethanol (g mannitol)⁻¹. Z. palmae was also successfully applied for fermentation of mannitol from Laminaria hyperborea extracts. Journal of Industrial Microbiology & Biotechnology (2000) 24, 51–57. Received 27 June 1999/ Accepted in revised form 23 September 1999     

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.