Butanol production by Clostridium acetobutylicum in a continuous packed bed reactor

In this study, we report on a butanol production process by immobilized Clostridium acetobutylicum in a continuous packed bed reactor (PBR) using Tygon^® rings as a carrier. The medium was a solution of lactose (15–30 g/L) and yeast extract (3 g/L) to emulate the cheese whey, an abundant lactose-rich wastewater. The reactor was operated under controlled conditions with respect to the pH and to the dilution rate. The pH and the dilution rate ranged between 4 and 5, the dilution rate between 0.54 and 2.4 h^?1 (2.5 times the maximum specific growth rate assessed for suspended cells). The optimal performance of the reactor was recorded at a dilution rate of 0.97 h^?1: the butanol productivity was 4.4 g/Lh and the selectivity of solvent in butanol was 88%_w.     

2,3-Butanediol production by Enterobacter aerogenes in continuous culture: role of oxygen supply

Summary The influence of oxygen on growth and production of 2,3-butanediol and acetoin by Enterobacter aerogenes was studied in continuous culture. At all dilution rates (D) studied cell mass increased steadily with increasing oxygen uptake rate (OUR), hence the micro-aerobic cultivation was energy-limited. The biomass yield on oxygen increased with D which suggests that cells need more energy for maintenance functions at lower D. At each D an optimal OUR giving highest volumetric productivity for the sum of butanediol and acetoin was found. The optimal OUR increased with D. The occurrence of optimal OURs results from the various effects of O₂ on growth and specific productivity. The latter was found to be a linear function of the specific OUR irrespective of D. At optimal OUR the cells proved to have equal specific OURs and equal specific productivities representing a fixed relationship between fermentative and respiratory metabolism. The product yield based on glucose and corrected for biomass formation was 80%. A product concentration as high as 43 g/l was obtained at D =0.1 h^–1 while the volumetric productivity was the highest at D =0.28 h^–1 (5.6 g/l and hour). The findings further indicate that growth and product generation are obviously non-associated phenomena. Hence, high productivities may be achievable by cell recycling and cell immobilisation systems. Offprint requests to: W.-D. Deckwer     

Improved hydrogen production under microaerophilic conditions by overexpression of polyphosphate kinase in Enterobacter aerogenes

Effects of different microaerophilic conditions on cell growth, glucose consumption, hydrogen production and cellular metabolism of wild Enterobacter aerogenes strain and polyphosphate kinase (PPK) overexpressing strain were systematically studied in this paper, using NaH(2)PO(4) as the phosphate sources. Under different microaerophilic conditions, PPK-overexpressing strain showed better cell growth, glucose consumption and hydrogen production than the wild strain. In the presence of limited oxygen (2.1%) and by PPK overexpression, the hydrogen production per liter of culture, the hydrogen production per cell and the hydrogen yield per mol of glucose increased by 20.1%, 12.3% and 10.8%, respectively, compared with the wild strain under strict anaerobic conditions. Metabolic analysis showed that the increase of the total hydrogen yield was attributed to the improvement of NADH pathway. The result of more reductive cellular oxidation state balance also further demonstrated that, under proper initial microaerophilic conditions and by PPK overexpression, the cell could adjust the cellular redox states and make more energy flow into hydrogen production pathways.     

产气肠杆菌fhlA基因克隆、表达及重组菌株产氢量

【目的】本研究以产氢细菌产气肠杆菌Enterobacter aerogenes ATCC13408为研究对象,克隆甲酸-氢裂解酶(formate hydrogen lyase,FHL)系统的转录激活蛋白FHL activator(fhlA)基因,构建过表达重组菌株,以提高菌株产氢效率。【方法】利用简并引物和Genome walking技术,克隆fhlA的全长基因,将该基因连接到改造质粒pGEX-4T-2-Cat中,电击转化得到重组菌株,用厌氧发酵方法测定重组细菌的产氢量。【结果】E.aerogenes ATCC13408fhlA ORF全长2073bp,编码一个含690个氨基酸残基的蛋白(GenBank accessionGU188474)。SDS-PAGE和Western blot分析证明fhlA基因在重组菌中得到了融合表达。对重组后菌株的产氢量进行了测定,结果表明:底物产氢潜力由原来的1.23±0.08mol H2/mol葡萄糖提高到了1.48±0.04mol H2/mol葡萄糖,提高了20.36%。【结论】本研究首次克隆了E.aerogenes ATCC13408的fhlA基因,并将该基因在原菌中过量表达。重组后菌株的产氢量得到显著提高,为进一步研究和开发利用E.aerogenes ATCC13408的fhlA基因提供了基础。     

Heterologous expression of a hydrogenase gene in Enterobacter aerogenes to enhance hydrogen gas production

The hydrogenase gene from Enterobacter cloacae (IIT-BT 08) was amplified and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-4T-2-Cat/hydA). The recombinant plasmid was transformed into a hydrogen-producing strain of Enterobacter aerogenes (ATCC13408). SDS–PAGE and western blot analysis confirmed the successful expression of the GST-tagged hydA protein. Anaerobic fermentation for the production of hydrogen from glucose was investigated using E. aerogenes ATCC13408 and the recombinant strain. The results showed that the hydrogen yield markedly increased, from 442.82 ± 22.61 ml/g glucose in the ATCC13408 strain to 864.02 ± 36.8 ml/g glucose in the recombinant. The maximum rate of hydrogen production was found to be 53.49 ± 3.34 ml l⁻¹ h⁻¹ using 1% (w/v) glucose as the substrate at pH 6.0 and a reaction temperature of 37°C.     

Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor

Continuous hydrogen gas evolution by self-flocculated cells of Enterobacter aerogenes, a natural isolate HU-101 and its mutant AY-2, was performed in a packed-bed reactor under glucose-limiting conditions in a minimal medium. The flocs that formed during the continuous culture were retained even when the dilution rate was increased to 0.9 h⁻¹. The H₂ production rate increased linearly with increases in the dilution rate up to 0.67 h⁻¹, giving maximum H₂ production rates of 31 and 58 mmol l⁻¹ h⁻¹ in HU-101 and AY-2 respectively, at a dilution rate of more than 0.67 h⁻¹. The molar H₂ yield from glucose in AY-2 was maintained at about 1.1 at dilution rates between 0.08 h⁻¹ and 0.67 h⁻¹, but it decreased rapidly at dilution rates more than 0.8 h⁻¹. Received: 27 August 1997 / Received revision: 11 November 1997 / Accepted: 14 December 1997     

Optimization of key process variables for enhanced hydrogen production by Enterobacter aerogenes using statistical methods

The individual and mutual effects of glucose concentration, temperature and pH on the hydrogen production by Enterobacter aerogenes were investigated in a batch system. A Box-Behnken design and response surface methodology (RSM) were employed to determine the optimum condition for enhanced hydrogen production. The hydrogen production rate was investigated by simultaneously changing the three independent variables, which all had significant influences on the hydrogen production rate. The maximum hydrogen production rate of 425.8 ml H(2)(g dry cell h)(-1) was obtained under the optimum condition of glucose concentration 118.06 mM, temperature 38 degrees C and pH 6.13. The experimental results showed that the RSM with the Box-Behnken design was a useful tool for achieving high rate of hydrogen production by E. aerogenes.     

Hydrogen production from biodiesel byproduct by immobilized Enterobacter aerogenes

The recent rapid growth of the biodiesel industry has generated a significant amount of glycerol as a byproduct. As a result, the price of glycerol is currently relatively low, making it an attractive starting material for the production of chemicals with higher values. Crude glycerol can be directly converted through microbial fermentation into various chemicals such as hydrogen. In this study, we optimized immobilization of a facultative hydrogen producing microorganism, Enterobacter aerogenes, with the goal of developing biocatalysts that was appropriate for the continuous hydrogen production from glycerol. Several carriers were tested and agar was found to be the most effective. In addition, it was clearly shown that variables such as the carrier content and cell loading should be controlled for the immobilization of biocatalysts with high hydrogen productivity, stability, and reusability. After optimization of these variables, we were able to obtain reusable biocatalysts that could directly convert the byproduct stream from biodiesel processes into hydrogen in continuous processes.     

Alkaloid formation by Catharanthus roseus cells in a packed column biofilm reactor

Summary Catharanthus roseus cells producing indole alkaloids were grown on surfaces of Ca-alginate beads within the interspacial volume of a packed column. Production media was circulated through the packed column in an upflow mode. Growth and indole alkaloid formation were quantified and compared with suspension culture of cells. Final alkaloid concentration and alkaloid yield obtained in the packed bed was superior to those obtained in suspension culture. This is thought to be due to improved cell-cell contact and interaction in the packed column.     

Effect of crude glycerol-derived inhibitors on ethanol production by Enterobacter aerogenes

In this study, ethanol production from pure and crude glycerol using Enterobacter aerogenes ATCC 29007 was evaluated under anaerobic culture conditions. Inhibitory effects of substrate concentrations, pH, and salt concentrations were investigated based on crude glycerol components. Ethanol production was performed with pure glycerol concentrations ranging from 5 to 30 g/L to evaluate the effects of substrate concentration and osmotic pressure. The consumed glycerol was 5-14.33 g/L, and the yield of ethanol was higher than 0.75 mol ethanol/mol glycerol after 24 h of cultivation. To evaluate the inhibitory effects of salts (NaCl and KCl), experiments were performed with 0-20 g/L of each salt. Inhibitory effects of salts were strongest at high salt concentrations. The inhibitory effect of pH was performed in the pH range 4-10, and cell growth and ethanol production were highest at pH 5-6. Also, ethanol production was slightly inhibited at low concentration of crude glycerol comparison with pure glycerol. However, significant inhibitory effects were not observed at 1.5 and 2% crude glycerol which showed higher ethanol production compared to pure glycerol.     

Study of the role of anaerobic metabolism in succinate production by Enterobacter aerogenes

Succinate is a core biochemical building block; optimizing succinate production from biomass by microbial fermentation is a focus of basic and applied biotechnology research. Lowering pH in anaerobic succinate fermentation culture is a cost-effective and environmentally friendly approach to reducing the use of sub-raw materials such as alkali, which are needed for neutralization. To evaluate the potential of bacteria-based succinate fermentation under weak acidic (pH <6.2) and anaerobic conditions, we characterized the anaerobic metabolism of Enterobacter aerogenes AJ110637, which rapidly assimilates glucose at pH 5.0. Based on the profile of anaerobic products, we constructed single-gene knockout mutants to eliminate the main anaerobic metabolic pathways involved in NADH re-oxidation. These single-gene knockout studies showed that the ethanol synthesis pathway serves as the dominant NADH re-oxidation pathway in this organism. To generate a metabolically engineered strain for succinate production, we eliminated ethanol formation and introduced a heterogeneous carboxylation enzyme, yielding E. aerogenes strain ΔadhE/PCK. The strain produced succinate from glucose with a 60.5 % yield (grams of succinate produced per gram of glucose consumed) at pH <6.2 and anaerobic conditions. Thus, we showed the potential of bacteria-based succinate fermentation under weak acidic conditions.     

H2 production from starch by a mixed culture of Clostridium butyricum and Enterobacter aerogenes

A mixed continuous culture of Clostridium butyricum and Enterobacter aerogenes removed O2 in a reactor and produced H2 from starch with yield of more than 2 mol H2/mol glucose without any reducing agents in the medium. Co-immobilized cells of the bacteria on porous glass beads evolved H2 from starch at 1.3 l/l.h, with H2 yield of 2.6 mol H2/ mol glucose at dilution rate of 1.0 h–1 in a continuous culture.     

Effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes in continuous culture

Summary The effect of pH and acetic acid on growth and 2,3-butanediol production of Enterobacter aerogenes from glucose was investigated in a microaerobic continuous culture. At a dilution rate of 0.20 h^–1 and a fixed oxygen uptake rate (OUR) of 31.5 mmol l^–1 h^–1 the biomass concentration increased with pH ranging from 5.0 to 7.0, while the specific ATP requirement of the cells decreased. In the pH range 5.5–6.5 the product concentration (butanediol + acetoin) was maximal and nearly constant. However, the specific production continuously declined with increasing pH. Experiments with addition of acetic acid showed that the various effects of pH are due to inhibition of the by-product acetic acid on cell growth. The strength of the acetic and inhibition depended only on the concentration of its undissociated form [HAc]. The biomass concentration and the specific OUR were also only functions of [HAc], irrespective of the pH. Although the specific ATP requirement (q ATP) strongly depended on the pH, [HAc] at constant pH. Offprint requests to: W.-D. Deckwer     

Studies on nutritional and oxygen requirements for production of L-asparaginase by Enterobacter aerogenes

The carbon and nitrogen sources most suitable for L-asparaginase production by Enterobacter aerogenes were selected and their concentrations optimized in shake-flask cultures. Sodium citrate (1.0%) and diammonium hydrogen phosphate (0.16%) proved to be the best sources of carbon and nitrogen, respectively. Nitrogen catabolite repression of enzyme formation was absent in this bacterium. Cultivation in a reactor showed that the dissolved oxygen level is the limiting factor for L-asparaginase production by E. aerogenes. Glucose was found to be a repressor of enzyme synthesis. Asparagine was absent intracellularly when the L-asparaginase level was high. An increase in the extracellular alanine level when the dissolved oxygen remained low indicated a shift from aerobic to fermentative metabolism. Received: 20 July 1999 / Accepted: 2 October 1999     

Lactic acid production by Lactobacillus delbrueckii in a dual reactor system using packed bed biofilm reactor

Aims:  An integrated dual reactor system for continuous production of lactic acid by Lactobacillus delbrueckii using biofilms developed on reticulated polyurethane foam (PUF) is demonstrated.
Methods and Results:  Lactobacillus delbrueckii was immobilized on PUF, packed in a bioreactor and used in lactic acid fermentation. The rate of lactic acid production was significantly high with a volumetric productivity of 5 g l⁻¹ h⁻¹ over extended period of time. When coupled to a bioreactor, the system could be operated as dual reactor for over 1000 h continuously without augmentation of inoculum and no compromise on productivity.
Conclusions:  Polyurethane foams offer an excellent support for biofilm formation.
Significance and Impact of the Study:  The system was very robust and could be operated for prolonged period at a volumetric productivity of 4–6 g l⁻¹ h⁻¹.     

Exopolysaccharide and storage polymer production in Enterobacter aerogenes type 8 strains

Many exopolysaccharide (EPS)-producing bacterial strains also synthesize storage polymers. The production of slime EPS and of the storage polymer glycogen was compared in batch cultures of EPS⁺ and EPS^- isogenic strains of Enterobacter aerogenes type 8. Conditions of nutrient imbalance with high C: N ratios favoured both EPS and storage polymer synthesis and resulted in little subsequent degradation of glycogen. In the EPS⁺ strain, glycogen synthesis was consistently lower than in the EPS^- strain, indicating that substrate was preferentially used for EPS production. Reduced levels of carbon substrate in the growth medium resulted in lower storage polymer synthesis and in the degradation of the glycogen formed in EPS-producing bacteria. Considerable differences in the synthesis and breakdown of intracellular carbohydrate were observed between bacteria grown in synthetic media with ammonium salts and the same bacteria grown in medium with casein hydrolysate as the nitrogen source. Growth in media depleted in magnesium was slower than in complete media but high yields of glycogen were obtained in both the EPS⁺ and EPS^- strains.     

Deletion of lactate dehydrogenase in Enterobacter aerogenes to enhance 2,3-butanediol production

2,3-Butanediol is an important bio-based chemical product, because it can be converted into several C4 industrial chemicals. In this study, a lactate dehydrogenase-deleted mutant was constructed to improve 2,3-butanediol productivity in Enterobacter aerogenes. To delete the gene encoding lactate dehydrogenase, λ Red recombination method was successfully adapted for E. aerogenes. The resulting strain produced a very small amount of lactate and 16.7% more 2,3-butanediol than that of the wild-type strain in batch fermentation. The mutant and its parental strain were then cultured with six different carbon sources, and the mutant showed higher carbon source consumption and microbial growth rates in all media. The 2,3-butanediol titer reached 69.5 g/l in 54 h during fed-batch fermentation with the mutant,which was 27.4% higher than that with the parental strain.With further optimization of the medium and aeration conditions,118.05 g/l 2,3-butanediol was produced in 54 h during fed-batch fermentation with the mutant. This is by far the highest titer of 2,3-butanediol with E. aerogenes achieved by metabolic pathway engineering.     

A continuous process for biodiesel production in a fixed bed reactor packed with cation-exchange resin as heterogeneous catalyst

Continuous esterification of free fatty acids (FFA) from acidified oil with methanol was carried out with NKC-9 cation-exchange resin in a fixed bed reactor with an internal diameter of 25 mm and a height of 450 mm to produce biodiesel. The results showed that the FFA conversion increased with increases in methanol/oil mass ratio, reaction temperature and catalyst bed height, whereas decreased with increases in initial water content in feedstock and feed flow rate. The FFA conversion kept over 98.0% during 500 h of continuous esterification processes under 2.8:1 methanol to oleic acid mass ratio, 44.0 cm catalyst bed height, 0.62 ml/min feed flow rate and 65°C reaction temperature, showing a much high conversion and operational stability. Furthermore, the loss of sulfonic acid groups from NKC-9 resin into the production was not found during continuous esterification. In sum, NKC-9 resin shows the potential commercial applications to esterification of FFA.