Enhancement of acetate production by a novel coupled syntrophic acetogenesis with homoacetogenesis process

A novel process was developed and demonstrated that a coupled syntrophic acetogenesis with homoacetogenesis reaction was able to enhance acetate production from high strength synthetic wastewater containing glucose by mixed cultures. A coupling system was constructed with two bioreactors which were connected via a silicon rubber pipe. The first reactor (bioreactor A) was for syntrophic acetogenesis, in which glucose was converted to volatile fatty acids consisting primarily of acetate. The second (bioreactor H) was for homoacetogenesis in which CO₂ and H₂ from bioreactor A were converted to acetate. Acetate yield in the coupling system was 87% higher than that in control 1, in which the homoacetogenesis did not occur. Also, acetate yield in the coupling system was 52% higher than that in control 2, which consisted of only bioreactor A and the gas in the headspace was released manually once a day. Enhancement of acetate production was contributed principally to relieve of the products (H₂ and CO₂) inhibition to syntrophic acetogenesis in bioreactor A, in which the degradation of glucose and the conversion of ethanol were enhanced. This coupling process provides a strategy for increasing acetate production and the degradation rate of the substrate.     

pH值和产甲烷抑制剂对两相耦合系统污泥发酵定向产乙酸的影响

采用产氢产乙酸/同型产乙酸两相耦合工艺对剩余污泥进行了半连续式厌氧发酵,主要研究了pH值和产甲烷抑制剂2-bromoethanesulphonate(BES)对耦合系统定向产乙酸的影响.结果表明:碱性pH(pH=10.0)和添加BES都能促进A相乙酸的积累,提高乙酸的产率,同时碱性pH比添加BES更有利于污泥的水解.当...     

Acetate oxidation by syntrophic association between Geobacter sulfurreducens and a hydrogen-utilizing exoelectrogen

Anodic microbial communities in acetate-fed microbial fuel cells (MFCs) were analyzed using stable-isotope probing of 16S rRNA genes followed by denaturing gradient gel electrophoresis. The results revealed that Geobacter sulfurreducens and Hydrogenophaga sp. predominated in the anodic biofilm. Although the predominance of Geobacter sp. as acetoclastic exoelectrogens in acetate-fed MFC systems has been often reported, the ecophysiological role of Hydrogenophaga sp. is unknown. Therefore, we isolated and characterized a bacterium closely related to Hydrogenophaga sp. (designated strain AR20). The newly isolated strain AR20 could use molecular hydrogen (H₂), but not acetate, with carbon electrode as the electron acceptor, indicating that the strain AR20 was a hydrogenotrophic exoelectrogen. This evidence raises a hypothesis that acetate was oxidized by G. sulfurreducens in syntrophic cooperation with the strain AR20 as a hydrogen-consuming partner in the acetate-fed MFC. To prove this hypothesis, G. sulfurreducens strain PCA was cocultivated with the strain AR20 in the acetate-fed MFC without any dissolved electron acceptors. In the coculture MFC of G. sulfurreducens and strain AR20, current generation and acetate degradation were the highest, and the growth of strain AR20 was observed. No current generation, acetate degradation and cell growth occurred in the strain AR20 pure culture MFC. These results show for the first time that G. sulfurreducens can oxidize acetate in syntrophic cooperation with the isolated Hydrogenophaga sp. strain AR20, with electrode as the electron acceptor.     

High-titer n-butanol production by clostridium acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping

Acetone–butanol–ethanol (ABE) fermentation with a hyper‐butanol producing Clostridium acetobutylicum JB200 was studied for its potential to produce a high titer of butanol that can be readily recovered with gas stripping. In batch fermentation without gas stripping, a final butanol concentration of 19.1 g/L was produced from 86.4 g/L glucose consumed in 78 h, and butanol productivity and yield were 0.24 g/L h and 0.21 g/g, respectively. In contrast, when gas stripping was applied intermittently in fed‐batch fermentation, 172 g/L ABE (113.3 g/L butanol, 49.2 g/L acetone, 9.7 g/L ethanol) were produced from 474.9 g/L glucose in six feeding cycles over 326 h. The overall productivity and yield were 0.53 g/L h and 0.36 g/g for ABE and 0.35 g/L h and 0.24 g/g for butanol, respectively. The higher productivity was attributed to the reduced butanol concentration in the fermentation broth by gas stripping that alleviated butanol inhibition, whereas the increased butanol yield could be attributed to the reduced acids accumulation as most acids produced in acidogenesis were reassimilated by cells for ABE production. The intermittent gas stripping produced a highly concentrated condensate containing 195.9 g/L ABE or 150.5 g/L butanol that far exceeded butanol solubility in water. After liquid–liquid demixing or phase separation, a final product containing ～610 g/L butanol, ～40 g/L acetone, ～10 g/L ethanol, and no acids was obtained. Compared to conventional ABE fermentation, the fed‐batch fermentation with intermittent gas stripping has the potential to reduce at least 90% of energy consumption and water usage in n‐butanol production from glucose. Biotechnol. Bioeng. 2012; 109: 2746–2756. © 2012 Wiley Periodicals, Inc.     

A novel feeding strategy for enhanced protein production by fed-batch fermentation in recombinant Pichia pastoris

A novel feeding strategy for enhanced protein production of hepatitis B virus surface antigen (HBsAg) in fed-batch fermentation, recombinant Pichia pastoris, has been developed. A minimal salt medium was used to grow cells in the initial batch fermentation, followed by a glycerol+salts fed-batch phase. At the end of the fed-batch phase a dry cell weight of 130 g l⁻¹ was achieved. In the absence of basal salts, the same amount of glycerol feed resulted in only 90 g l⁻¹ cell dry weight. When a limited amount of casamino acids were also included every 24 h during methanol induction, there was a two-fold increase in expression levels of HBsAg. After 192 h of induction, the expression levels of HBsAg (soluble and insoluble) reached >1 g l⁻¹ using the Mut⁻ strain. Thus, the use of basal salts in the glycerol feed, along with the addition of limited amounts of casamino acids with the methanol feed, resulted in an increased expression of total HBsAg.     

Ethanol production from paper material using a simultaneous saccharification and fermentation system in a fed-batch basis

In this work, a recycled paper-derived feedstock was used to produce ethanol by the simultaneous saccharification and fermentation (SSF) process using the thermotolerant yeast Kluyveromyces marxianus CECT 10875. At standard SSF conditions, the highest yield (about 80% of theoretical) was obtained at low substrate concentration and high enzyme loading. With increasing substrate concentration, mixing difficulties appeared which prevented an adequate SSF process performance and limited ethanol production. An SSF fed-batch procedure was then used which permitted an increase in substrate concentrations while maintaining SSF yields similar to that obtained at standard SSF, thus allowing an increased final ethanol production (about 18 g/l).     

Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44 h, glycerol (20 g L⁻¹) and corn steep liquor (CSL) (30 g L⁻¹) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2 g L⁻¹ h⁻¹) and continuous feeding of CSL (1 g L⁻¹ h⁻¹) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5±0.8 g L⁻¹; maximum EH production was 351.2±13.1 U L⁻¹ with a specific activity of 14.3±0.5 U g⁻¹. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.     

Use of a novel air separation system in a fed-batch fermentative culture of Escherichia coli

A novel air separation system based on permeable membrane gas separation technology was used to cultivate Escherichia coli. The system fulfilled the dissolved oxygen requirements of a culture of E. coli grown on a glucose synthetic medium at a high and constant growth rate of 0.55 h-1. A biomass yield of 45 g (dry weight) per liter was achieved, and no by-product inhibition by acetate or CO2 was observed.     

Ethanol fermentation of raw cassava starch with Rhizopus koji in a gas circulation type fermentor

Studies have been conducted in a gas circulation type fermentor in order to characterize the ethanol fermentation of uncooked cassava starch with Rhizopus koji. Results showed that ethanol concentration reached 13-14% (v/v) in 4-day broth, and the maximum productivity of ethanol was 2.3 g ethanol/L broth h. This productivity was about 50% compared to the productivity of a glucose-yeast system. Ethanol yield reached 83.5-72.3% of the theoretical yield for the cassava starch used. The fermentor used in the present work has been proven by experiment to be suitable for ethanol fermentation of the broth with solid substrate.     

Comparative mycelial and spore yield by Trichoderma viride in batch and fed-batch cultures

The effects of cultural parameters such as carbon and nitrogen source and environmental factors including temperature and pH were investigated on spore and mycelial yield of Trichoderma viride, which has potential as a biocontrol agent against species of Fusarium in batch culture and fed-batch culture where there was limiting nutrient. The results obtained indicated that growth and sporulation of T. viride were greatly influenced by various carbon and nitrogen sources, and by environmental factors such as pH and temperature. Mannitol, wheat bran and rice bran as sole carbon sources appear to stimulate high mycelial growth and spore yield in fed-batch culture. Growth and sporulation were also favoured by NaNO₃, peptone and NH₄SO₄ as the nitrogen sources in fed-batch and batch cultures_. Maximum growth and sporulation was between pH 4.5 and 6.0. Temperatures between 30 and 37 °C were good for mycelium growth of T. viride while temperatures between 30 to 45 °C were good for sporulation. The amount of spore and mycelium produced and the time required for attainment of maximum spore yield increased with increasing carbon and nitrogen source in batch culture. The final spore yield obtained in fed-batch culture was two times higher than the apparent spore-carrying capacity of batch culture. These results show that T. viride is capable of growing and sporulating with varied nutritional and environmental conditions, and, therefore, this strain of T. viride may be useful as a biocontrol agent under diverse physiological and environmental conditions.     

Use of a novel air separation system in a fed-batch fermentative culture of Escherichia coli.

A novel air separation system based on permeable membrane gas separation technology was used to cultivate Escherichia coli. The system fulfilled the dissolved oxygen requirements of a culture of E. coli grown on a glucose synthetic medium at a high and constant growth rate of 0.55 h-1. A biomass yield of 45 g (dry weight) per liter was achieved, and no by-product inhibition by acetate or CO2 was observed.     

Operation of a fixed-bed bioreactor in batch and fed-batch modes for production of inulinase by solid-state fermentation

This work is focused on the inulinase production by solid-state fermentation (SSF) in a fixed-bed reactor (34 cm diameter and 50 cm height) with working capacity of 2-kg of dry substrate operated in batch and fed-batch modes. It was investigated different strategies for feeding the inlet air in the bioreactor (saturated and unsaturated air) as alternative to remove the metabolic heat generated during the microbial growth by evaporative cooling. The kinetic evaluation of the process carried out in batch mode using unsaturated air showed that the evaporative cooling decreasing the mean temperature of the solid-bed, although the enzyme production was lower than that obtained using saturated air. Results showed that maximum enzyme activity (586 ± 63 U gds⁻¹) was obtained in the fed-batch mode using saturated air after 24 h of fermentation. The enzymatic extract obtained by fed-batch mode was characterized and presented optimum temperature and pH in the range of 52–57 °C and 4.8–5.2, respectively. For a temperature range from 40 to 70 °C the enzyme presented decimal reduction time, D-value, ranging from 5748 to 47 h, respectively. For a pH range from 3.5 to 5.5 the enzyme showed good stability, presenting D-values higher than 2622 h. In terms of Michaelis–Mentem parameters were demonstrated that the crude inulinase activity presented higher affinity for substrate sucrose compared to inulin.     

Growth monitoring and control through computer-aided on-line mass balancing in a fed-batch penicillin fermentation

A computer-aided methodology is developed for on-line monitoring and control of cell growth in fed-batch penicillin fermentation using a semidefined medium containing low corn steep liquor concentration (5.7 g/L). Cell growth is monitored and controlled with the use of experimental correlation and carbon-balancing equatiions on a real-time basis throughout the fermentation. Through a combination of feed-forward and feedback control of sugar addition, residual glucose concentration in the broth was maintained below 1 g/L and cell-growth rate was kept at constant at preset vaiues. The accuracy and reproducibility of this technique are demonstrated. The use of real-time control of cell growth is expected to aid future investigations of this antibiotic fermentation.     

Enhancement of biomass and fermentation activity of surplus brewers' yeast in a fed-batch process

The growth of surplus brewers' yeast in a fed-batch process was studied with the aim of increasing the fermentation activity of the yeast cells and of optimizing the growth conditions: 20 h cultivation at 30° C and pH 5.0–5.5 using beet molasses as substrate, with a regulated feeding rate, showed satisfactory results. Under the chosen conditions, the final amount of biomass increased more than fivefold, achieving a specific growth rate of 0.1 h^–1 and substrate yield coefficient of 0.54 g·g^–1. The increase in fermentation activity of yeast cells during cultivation correlated very well with the concentration of reduced glutathione, which increased from 1.2 to 2.7 mg·g^–1 (dry matter). At the same time the fermentation activity increased fivefold, which related to the nitrogen content of the yeast cells. Ethanol formation throughout the cultivation did not exceed 0.5 g·l^–1. Correspondence to: B. Strel     

Focusing transgene expression in Drosophila by coupling Gal4 with a novel split-LexA expression system

Here we report the development of a ternary version of the LexA::VP16/LexAop system in which the DNA-binding and trans-activating moieties are independently targeted using distinct promoters to achieve highly restricted, intersectional expression patterns. This Split LexA system can be concatenated with the Gal4/upstream activating sequence system to refine the expression patterns of existing Gal4 lines with minimal genetic manipulations.     

Simultaneous saccharification and fermentation of sweet sorghum carbohydrates to ethanol in a fed-batch process

Summary The simultaneous saccharification and fermentation (SSF) of sweet sorghum carbohydrates to ethanol by Fusarium oxysporum F3 alone or in mixed culture with Saccharomyces cerevisiae 2541 or Zymomonas mobilis CP4 in a fed-batch fermentation process was studied. While SSF was adequately carried out by the first microorganism the process achieved its maximum value by the mixed culture of the fungus and yeast. Under optimum conditions, ethanol yields and concentrations as high as 29.7 g of ethanol per 100 g of dry sorghum stalk and 7.5 % (w/v) respectively were obtained. These values together with the high yield of sorghum crop in Greece make this process promising and worthy of further investigation for the production of fuel bioethanol.     

Fungal morphology and fragmentation behavior in a fed-batch Aspergillus oryzae fermentation at the production scale

It is well known that high-viscosity fermentation broth can lead to mixing and oxygen mass transfer limitations. The seemingly obvious solution for this problem is to increase agitation intensity. In some processes, this has been shown to damage mycelia, affect morphology, and decrease product expression. However, in other processes increased agitation shows no effect on productivity. While a number of studies discuss morphology and fragmentation at the laboratory and pilot scale, there are relatively few publications available for production-scale fungal fermentations. The goal of this study was to assess morphology and fragmentation behavior in large-scale, fed-batch, fungal fermentations used for the production of protein. To accomplish this, a recombinant strain of Aspergillus oryzae was grown in 80 m(3) fermentors at two different gassed, impeller power-levels (one 50% greater than the other). Impeller power is reported as energy dissipation/circulation function (EDCF) and was found to have average values of 29.3 +/- 1.0 and 22.0 +/- 0.3 kW m(-3) s(-1) at high and low power levels, respectively. In all batches, biomass concentration profiles were similar and specific growth rate was < 0.03 h(-1). Morphological data show hyphal fragmentation occurred by both shaving-off of external clump hyphae and breakage of free hyphae. The fragmentation rate constant (k(frag)), determined using a first-order model, was 5.90 and 5.80 h(-1) for high and low power batches, respectively. At the end of each batch, clumps accounted for only 25% of fungal biomass, most of which existed as small, sparsely branched, free hyphal elements. In all batches, fragmentation was found to dominate fungal growth and branching. We speculate that this behavior was due to slow growth of the culture during this fed-batch process.     

A novel fed-batch based cultivation method provides high cell-density and improves yield of soluble recombinant proteins in shaken cultures

Background  

Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.