Formate hydrogenlyase in the hyperthermophilic archaeon, <Emphasis Type="Italic">Thermococcus litoralis</Emphasis>

Background  

Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis.     

Critical factors in chitin production by fermentation of shrimp biowaste

Factors affecting Lactobacillus fermentation of shrimp waste for chitin and protein liquor production were determined. The objective of the fermentation is medium conditioning by Lactobacillus through production of proteases and lowering of the pH. The efficiency was tested by conducting fermentation of biowaste in 1-l beakers with or without pH adjustment using different acids. Addition of 5% glucose to the biowaste supported the growth of lactic acid bacteria and led to better fermentation. Among four acids tested to control pH at the start and during fermentation, acetic acid and citric acid proved to be the most effective. In biowaste fermented with 6.7% L. plantarum inoculum, 5% glucose, and pH 6.0 adjusted with acetic acid, 75% deproteination and 86% demineralization was achieved. Replacement of acetic acid by citric acid gave 88% deproteination and 90% demineralization. The fermentation carried out in the presence of acetic acid resulted in a protein fraction that smelled good and a clean chitin fraction. Received: 4 April 2000 / Received revision: 9 June 2000 / Accepted: 9 June 2000     

Isolation and Characterization of Antifungal Substances from Burkholderia sp. Culture Broth

A new antagonistic Burkholderia strain, designated MP-1 and producing antifungal activities against various filamentous plant pathogenic fungi, was isolated from the rhizoshere in the Naju area. Cultural characteristic studies strongly suggested that this strain belongs to the genus Burkholderia. The nucleotide sequence of the 16S rRNA gene (1491 pb) of strain MP-1 exhibited close similarity (99% to 100%) with other Burkholderia 16S rRNA genes. Extraction of fermentation broth of Burkholderia sp. MP-1 and various separations and purification steps led to isolation of four pure active molecules. The chemical structure of these four compounds—named phenylacetic acid, hydrocinnamic acid, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetate methyl ester—was established on the basis on their gas chromatography–electron impact–mass spectrometry (GC-EI-MS) and trimethylsilation GC-EI-MS data. The four isolated compounds inhibited filamentous fungal growth on potato dextrose agar medium supplemented with 100 mg/L of phenylacetic acid, hydrocinnamic acid, 4-hydroxyphenylacetic acid and 4-hydroxyphenylacetate methyl ester individually.     

Ethanol fermentation from lignocellulosic hydrolysate by a recombinant xylose- and cellooligosaccharide-assimilating yeast strain

The sulfuric acid hydrolysate of lignocellulosic biomass, such as wood chips, from the forest industry is an important material for fuel bioethanol production. In this study, we constructed a recombinant yeast strain that can ferment xylose and cellooligosaccharides by integrating genes for the intercellular expressions of xylose reductase and xylitol dehydrogenase from Pichia stipitis, and xylulokinase from Saccharomyces cerevisiae and a gene for displaying β-glucosidase from Aspergillus acleatus on the cell surface. In the fermentation of the sulfuric acid hydrolysate of wood chips, xylose and cellooligosaccharides were completely fermented after 36 h by the recombinant strain, and then about 30 g/l ethanol was produced from 73 g/l total sugar added at the beginning. In this case, the ethanol yield of this recombinant yeast was much higher than that of the control yeast. These results demonstrate that the fermentation of the lignocellulose hydrolysate is performed efficiently by the recombinant Saccharomyces strain with abilities for xylose assimilation and cellooligosaccharide degradation.     

NADH- vs NADPH-coupled reduction of 5-hydroxymethyl furfural (HMF) and its implications on product distribution in Saccharomyces cerevisiae

Saccharomyces cerevisiae alcohol dehydrogenases responsible for NADH-, and NADPH-specific reduction of the furaldehydes 5-hydroxymethyl-furfural (HMF) and furfural have previously been identified. In the present study, strains overexpressing the corresponding genes (mut-ADH1 and ADH6), together with a control strain, were compared in defined medium for anaerobic fermentation of glucose in the presence and absence of HMF. All strains showed a similar fermentation pattern in the absence of HMF. In the presence of HMF, the strain overexpressing ADH6 showed the highest HMF reduction rate and the highest specific ethanol productivity, followed by the strain overexpressing mut-ADH1. This correlated with in vitro HMF reduction capacity observed in the ADH6 overexpressing strain. Acetate and glycerol yields per biomass increased considerably in the ADH6 strain. In the other two strains, only the overall acetate yield per biomass was affected. When compared in batch fermentation of spruce hydrolysate, strains overexpressing ADH6 and mut-ADH1 had five times higher HMF uptake rate than the control strain and improved specific ethanol productivity. Overall, our results demonstrate that (1) the cofactor usage in the HMF reduction affects the product distribution, and (2) increased HMF reduction activity results in increased specific ethanol productivity in defined mineral medium and in spruce hydrolysate.     

Process optimization for large-scale production of TGF-α-PE40 in recombinant Escherichia coli: effect of medium composition and induction timing on protein expression

The effects of medium composition and induction timing on expression of a chimeric fusion protein TGF-α -PE40 (TP-40) in Escherichia coli strain RR1 were examined using a complex medium at several fermentor scales. Two distinctive phases in E. coli catabolism were identified during fermentation based on preferential utilization between protein hydrolysate and glycerol. Maximum specific and volumetric productivities were achieved by inducing the culture when the cells were switching substrate utilization from protein hydrolysate to glycerol. By increasing the yeast extract concentration in the production medium, initiation of the catabolic switch was delayed until high cell mass was achieved. The final titer of TP-40 at the 15-L fermentation scale was doubled from 400 mg L⁻¹ to 850 mg L⁻¹ by increasing the yeast extract concentration from 1% to 4% (w/v) and delaying the time of induction. This fermentation process was rapidly scaled up in 180-L and 800-L fermentors, achieving TP-40 titers of 740 and 950 mg L⁻¹, respectively. Received 26 August 1996/ Accepted in revised form 10 December 1996     

Bioethanol,biohydrogen and biogas production from wheat straw in a biorefinery concept

The production of bioethanol, biohydrogen and biogas from wheat straw was investigated within a biorefinery framework. Initially, wheat straw was hydrothermally liberated to a cellulose rich fiber fraction and a hemicellulose rich liquid fraction (hydrolysate). Enzymatic hydrolysis and subsequent fermentation of cellulose yielded 0.41 g-ethanol/g-glucose, while dark fermentation of hydrolysate produced 178.0 ml-H₂/g-sugars. The effluents from both bioethanol and biohydrogen processes were further used to produce methane with the yields of 0.324 and 0.381 m³/kg volatile solids (VS)_added, respectively. Additionally, evaluation of six different wheat straw-to-biofuel production scenaria showed that either use of wheat straw for biogas production or multi-fuel production were the energetically most efficient processes compared to production of mono-fuel such as bioethanol when fermenting C6 sugars alone. Thus, multiple biofuels production from wheat straw can increase the efficiency for material and energy and can presumably be more economical process for biomass utilization.     

Virgibacillus zhanjiangensis sp. nov., a marine bacterium isolated from sea water

A Gram-positive, endospore-forming, catalase- and oxidase-positive, motile, rod-shaped, aerobic bacterium, designated strain JSM 079157^T, was isolated from surface seawater off the coastline of Naozhou Island in South China Sea. The organism was able to grow with 1–15% (w/v) total salts (optimum, 4–7%), and at pH 6.0–10.0 (optimum, pH 7.5) and 10–45°C (optimum, 30°C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7, and the polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The major cellular fatty acids were anteiso-C_15:0 (45.1%) and anteiso-C_17:0 (16.2%), and the DNA G + C content was 39.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 079157^T should be assigned to the genus Virgibacillus, being related most closely to the type strains of Virgibacillus litoralis (97.4% sequence similarity), Virgibacillus necropolis (97.3%) and Virgibacillus carmonensis (97.1%). These four strains formed a distinct subcluster in the phylogenetic tree. The levels of DNA–DNA relatedness between the new isolate and the type strains of V. litoralis, V. necropolis and V. carmonensis were 30.4, 19.3 and 12.6%, respectively. The results of the phylogenetic analysis, combined with DNA–DNA relatedness data, phenotypic characteristics and chemotaxonomic information, support the suggestion that strain JSM 079157^T represents a new species of the genus Virgibacillus, for which the name Virgibacillus zhanjiangensis sp. nov. is proposed. The type strain is JSM 079157^T (=DSM 21084^T = KCTC 13227^T).     

Evaluation of cell recycling in continuous fermentation of enzymatic hydrolysates of spruce with Saccharomyces cerevisiae and on-line monitoring of glucose and ethanol

The maximum growth rate of Saccharomyces cerevisiae ATCC 96581, adapted to fermentation of spent sulphite liquor (SSL), was 7 times higher in SSL of hardwood than the maximum growth rate of bakers' yeast. ATCC 96581 was studied in the continuous fermentation of spruce hydrolysate without and with cell recycling. Ethanol productivity by ATCC 96581 in continuous fermentation of an enzymatic hydrolysate of spruce was increased 4.6 times by employing cell recycling. On-line analysis of CO₂, glucose and ethanol (using a microdialysis probe) was used to investigate the effect of fermentation pH on cell growth and ethanol production, and to set the dilution rate. Cell growth in the spruce hydrolysates was strongly influenced by fermentation pH. The fermentation was operated in continuous mode for 210 h and a theoretical ethanol yield on fermentable sugars was obtained. Received: 25 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998     

Harvesting biohydrogen from cellobiose from sulfide or nitrite-containing wastewaters using Clostridium sp. R1

Harvesting biohydrogen from inhibiting wastewaters is of practical interest since the toxicity of compounds in a wastewater stream commonly prevents the bioenergy content being recovered. The isolated Clostridium sp. R1 is utilized to degrade cellobiose in sulfide or nitrite-containing medium for biohydrogen production. The strain can effectively degrade cellobiose free of severe inhibitory effects at up to 200 mg l⁻¹ sulfide or to 5 mg l⁻¹ nitrite, yielding hydrogen at >2.0 mol H₂ mol⁻¹ cellobiose. Principal metabolites of cellobiose fermentation are acetate and butyrate, with the concentration of the former increases with increasing sulfide and nitrite concentrations. The isolated strain can yield hydrogen from cellobiose in sulfide-laden wastewaters. However, the present of nitrite significantly limit the efficiency of the biohydrogen harvesting process.     

分别利用产甘油假丝酵母抗逆转录因子CgSTB5和CgSEF1对酿酒酵母菌株糠醛耐受改造

【背景】纤维素是生物转化解决能源问题的主要原料之一,其水解物中存在严重影响抑制菌株生长的糠醛,需脱毒才可应用于发酵,提高菌株耐受性是解决纤维素水解液实际生产应用的关键。【目的】酿酒酵母(Saccharomyces cerevisiae)是主要的纤维素水解液发酵工业菌株,但糠醛耐受性较低,通过分子改造获得具有高糠醛耐受性的菌株。【方法】利用新获得的产甘油假丝酵母(Candidaglycerinogenes)的相关抗逆转录因子CgSTB5、CgSEF1和CgCAS5,通过分子技术进行S.cerevisiae改造,考察其对酿酒酵母糠醛耐受性的影响,并尝试应用于未脱毒纤维素乙醇发酵。【结果】单个表达CgSTB5和CgSEF1的酿酒酵母,通过菌株点板实验表明菌株的糠醛耐受性提高25%以上,并且摇瓶发酵结果显示糠醛降解性能明显提高,生长延滞期明显缩短,S.cerevisiae W303/p414-CgSTB5的未脱毒纤维素乙醇发酵生产效率提高12.5%左右。【结论】转录因子CgSTB5和CgSEF1均能对提高酿酒酵母糠醛耐受性起到重要作用,并且有助于提高酿酒酵母菌株未脱毒纤维素乙醇发酵性能。     

Genetic engineering of <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> strain JDR-1 for efficient <Emphasis Type="SmallCaps">d</Emphasis>(−) lactic acid production from hemicellulose hydrolysate

In the dilute acid pretreatment of lignocellulose, xylose substituted with α-1,2-methylglucuronate is released as methylglucuronoxylose (MeGAX), which cannot be fermented by biocatalysts currently used to produce biofuels and chemicals. Enterobacter asburiae JDR-1, isolated from colonized wood, efficiently fermented both MeGAX and xylose in acid hydrolysates of sweetgum xylan. Deletion of pflB and als genes in this bacterium modified the native mixed acid fermentation pathways to one for homolactate production. The resulting strain, Enterobacter asburiae L1, completely utilized both xylose and MeGAX in a dilute acid hydrolysate of sweetgum xylan and produced lactate approximating 100% of the theoretical maximum yield. Enterobacter asburiae JDR-1 offers a platform to develop efficient biocatalysts for production of fuels and chemicals from hemicellulose hydrolysates of hardwood and agricultural residues.     

Biohydrogen production from pretreated lignocellulose by <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

In consolidated bioprocessing (CBP), the difference in optimum temperature between saccharification and fermentation poses a significant technical challenge to producing bioenergy efficiently with lignocellulose. The thermophilic anaerobic strain of Clostridium thermocellum has the potential to overcome this challenge if hydrolysis and fermentation is performed at an elevated temperature. However, this strain is sensitive to structure and components of lignocellulosic materials. To understand biohydrogen production from lignocellulosic materials, C. thermocellum was examined for biohydrogen production as well as bioconversion from different cellulosic materials (Avicel, filter paper and sugarcane bagasse (SCB)). We investigated hydrolysis-inhibitory effects of the cellulosic material types on the substrate degradation and biohydrogen production of C. thermocellum 27405. Within 168 h, the substrate degradation ratios of Avicel, filter paper, and SCB were 83.01, 51.78, and 42.19%, respectively. The substrate utilization and biohydrogen production of SCB reached 81 and 89.77% those of filter paper, respectively, indicating that SCB is a feasible substrate for biohydrogen production. Additionally, optimizing fermentation conditions can improve biohydrogen production, with the optimal conditions being an inoculum size of 7%, substrate concentration of 2%, particle size of 0.074 mm, and yeast extract concentration of 1%. This research provides important clues in relation to the low-cost conversion of renewable biomass to biohydrogen.     

Fermentation of xylose to succinate by enhancement of ATP supply in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

In Escherichia coli K12, succinate was not the dominant fermentation product from xylose. To reduce by-product formation and increase succinate accumulation, pyruvate formate lyase and lactate dehydrogenase, encoded by pflB and ldhA genes, were inactivated. However, these mutations eliminated cell growth and xylose utilization. During anaerobic growth of bacteria, organic intermediates, such as pyruvate, serve as electron acceptors to maintain the overall redox balance. Under these conditions, the ATP needed for cell growth is derived from substrate level phosphorylation. In E. coli K12, conversion of xylose to pyruvate only yielded 0.67 net ATP per xylose during anaerobic fermentation. However, E. coli produces equimolar amounts of acetate and ethanol from two pyruvates, and these reactions generate one additional ATP. Conversion of xylose to acetate and ethanol increases the net ATP yield from 0.67 to 1.5 per xylose, which could meet the ATP needed for xylose metabolism. A pflB deletion strain cannot convert pyruvate to acetyl coenzyme A, the precursor for acetate and ethanol production, and could not produce the additional ATP. Thus, the double mutations eliminated cell growth and xylose utilization. To supply the sufficient ATPs, overexpression of ATP-forming phosphoenolpyruvate-carboxykinase from Bacillus subtilis 168 in an ldhA, pflB, and ppc deletion strain resulted in a significant increase in cell mass and succinate production. In addition, fermentation of corn stalk hydrolysate containing a high percentage of xylose and glucose produced a final succinate concentration of 11.13 g l⁻¹ with a yield of 1.02 g g⁻¹ total sugars during anaerobic fermentation.     

Calcium gluconate production by a nonconventional fermentation method

Summary A strain of Aspergillus niger was grown in still (liquid), shake and semi-solid fermentation for calcium gluconate production from glucose, starch, or molasses. The yield from glucose or starch hydrolysate was acceptably high in both shake and semi-solid fermentation indicating that the semi-solid fermentation process offers a promising practical alternative.     

Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor

Fermentation of wood hydrolysates to desirable products, such as fuel ethanol, is made difficult by the presence of inhibitory compounds in the hydrolysates. Here we present a novel method to increase the fermentability of lignocellulosic hydrolysates: enzymatic detoxification. Besides the detoxification effect, treatment with purified enzymes provides a new way to identify inhibitors by assaying the effect of enzymatic attack on specific compounds in the hydrolysate. Laccase, a phenol oxidase, and lignin peroxidase purified from the ligninolytic basidiomycete fungus Trametes versicolor were studied using a lignocellulosic hydrolysate from willow pretreated with steam and SO₂. Saccharomyces cerevisiae was employed for ethanolic fermentation of the hydrolysates. The results show more rapid consumption of glucose and increased ethanol productivity for samples treated with laccase. Treatment of the hydrolysate with lignin peroxidase also resulted in improved fermentability. Analyses by GC-MS indicated that the mechanism of laccase detoxification involves removal of monoaromatic phenolic compounds present in the hydrolysate. The results support the suggestion that phenolic compounds are important inhibitors of the fermentation process. Received: 3 November 1997 / Received revision: 4 February 1998 / Accepted: 6 February 1998     

Isolation and characterisation of lactic acid bacterium for effective fermentation of cellobiose into optically pure homo <Emphasis Type="SmallCaps">l</Emphasis>-(+)-lactic acid

Effective utilisation of cellulosic biomasses for economical lactic acid production requires a microorganism with potential ability to utilise efficiently its major components, glucose and cellobiose. Amongst 631 strains isolated from different environmental samples, strain QU 25 produced high yields of l-(+)-lactic acid of high optical purity from cellobiose. The QU 25 strain was identified as Enterococcus mundtii based on its sugar fermentation pattern and 16S rDNA sequence. The production of lactate by fermentation was optimised for the E. mundtii QU25 strain. The optimal pH and temperature for batch culturing were found to be 7.0°C and 43°C, respectively. E. mundtii QU 25 was able to metabolise a mixture of glucose and cellobiose simultaneously without apparent carbon catabolite repression. Moreover, under the optimised culture conditions, production of optically pure l-lactic acid (99.9%) increased with increasing cellobiose concentrations. This indicates that E. mundtii QU 25 is a potential candidate for effective lactic acid production from cellulosic hydrolysate materials.     

Extractive fermentation of lactic acid by immobilizedLactobacillus casei using ion—exchange resin

Summary A novel method of lactic acid fermentation byLactobacillus casei immobilized in Ca—alginate gels is described, in which an ion—exchange resin packed column is attached to a fermentor for separation of lactic acid from fermentative broth. The technique successfully alleviated the restriction imposed by lactic acid on bacterial growth and product formation. As compared to the conventional batch fermentation, the new fermentation technique enhanced the lactic acid productivity and sugar conversion rate from 0.328g/L·h and 88. 2% to 0.482g/L·h and 98.6%, respectively.     

Enzymatic saccharification of cornstalk by onsite cellulases produced by Trichoderma viride for enhanced biohydrogen production

Lignocellulosic biomass, if properly saccharified, could be an ideal feedstock for biohydrogen production. However, the high cellulases cost is the key obstacle to its development. In this work, cost‐effective enzyme produced by Trichoderma viride was used to saccharify cornstalk. To obtain high sugar yield, a central composite design of response surface method was used to optimize enzymatic saccharification process. Experimental results showed that the enzymatic saccharification rate reached the highest of 81.2% when pH, temperature, cellulases and substrate concentration were 5, 49.7 °C, 35.7 IU g^?1, and 38.5 g L^?1, respectively. The cornstalk hydrolysate was subsequently introduced to fermentation by Thermoanaerobacterium thermosaccharolyticum W16, the yield of hydrogen reached the highest level of 90.6 ml H₂ g^?1 pretreated cornstalk. The present results indicate the potential of using T. thermosaccharolyticum W16 for high yield conversion of cornstalk hydrolysate, which was saccharified by onsite enzyme produced by T. viride.