Interactions between rumen bacterial strains during the degradation and utilization of the monosaccharides of barley straw cell-walls

Pure cultures and pair-combinations of strains representative of the rumen cellulolytic species Ruminococcus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisovens were grown on cell-wall materials from barley straw. Of the pure cultures, R. flavefaciens solubilized straw most rapidly. The presence of B. fibrisolvens , which was unable to degrade straw extensively in pure culture, increased the solubilization of dry matter by R. flavefaciens and the solubilization of cell-wall carbohydrates by both R. flavefaciens and F. succinogenes. During fermentation, both R. flavefaciens and F. succinogenes released bound glucose and free and bound arabinose and xylose into solution. The accumulation of these sugars, especially arabinose and xylose, was greatly reduced in co-cultures containing B. fibrisolvens , suggesting that significant interspecies cross feeding of the products of hemicellulose hydrolysis (particularly soluble bound xylose released by F. succinogenes ) occurs during straw degradation by mixed cultures containing this species.     

Microcalorimetric evaluation of the efficacy of the alkaline treatment on the fermentation of straw

Summary A microcalorimetric study is proposed to follow the degradation of straw by a mixed bacterial culture. The effect of an alkali pretreatment of straw is described.     

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.     

植物生物质生物降解组合菌株的筛选研究

本研究用平板混合培养和实际降解相结合的方法筛选了较优的降解植物生物质的菌株组合。首先将6株纤维素降解菌和7株木质素降解菌两两平行划线接种到基础平板上,通过观察菌落形态,挑选出了9对能较好共存的菌株组合。随后对这9个组合进行实际降解稻草的研究,培养结束后体系中半纤维素、纤维素和木质素的含量分析结果表明AF93252 M5和XP M5这两个菌株组合较理想,它们混合培养时对稻草的降解效果均优于其单独降解稻草的效果。     

Development of a method for the application of solid-phase microextraction to monitor biodegradation of volatile hydrocarbons during bacterial growth on crude oil

A quantitative solid-phase microextraction, gas chromatography, flame ionization detector (SPME-GC-FID) method for low-molecular-weight hydrocarbons from crude oil was developed and applied to live biodegradation samples. Repeated sampling was achieved through headspace extractions at 30°C for 45 min from flasks sealed with Teflon Mininert. Quantification without detailed knowledge of oil–water–air partition coefficients required the preparation of standard curves. An inverse relationship between retention time and mass accumulated on the SPME fibre was noted. Hydrocarbons from C₅ to C₁₆ were dated and those up to C₁₁ were quantified. Total volatiles were quantified using six calibration curves. Biodegradation of volatile hydrocarbons during growth on crude oil was faster and more complete with a mixed culture than pure isolates derived therefrom. The mixed culture degraded 55% of the compounds by weight in 4 days versus 30–35% by pure cultures of Pseudomonas aeruginosa, Rhodococcus globerulus or a co-culture of the two. The initial degradation rate was threefold higher for the mixed culture, reaching 45% degradation after 48 h. For the mixed culture, the degradation rate of individual alkanes was proportional to the initial concentration, decreasing from hexane to undecane. P. fluorescens was unable to degrade any of the low-molecular-weight hydrocarbons and methylcyclohexane was recalcitrant in all cases. Overall, the method was found to be reliable and cost-effective. Journal of Industrial Microbiology & Biotechnology (2000) 25, 155–162. Received 04 March 2000/ Accepted in revised form 25 June 2000     

木质素降解菌的筛选及混合菌发酵降解秸秆的研究

农作物秸秆是农业生产的副产品,也是一项重要的生物资源。由于其成分结构的特殊性所导致的难降解问题,一直成为了转化利用秸秆的难题。目前,利用混合菌将秸秆纤维素转化为蛋白质、乙醇、乙酸、乳酸等研究已逐渐为人们所重视。本文通过马铃薯琼脂平板培养、马铃薯液体摇瓶培养和稻草秸秆固态发酵,从6株常见的食用白腐菌中筛选出了生长优势较强、产漆酶酶活高的平菇HF。为了让秸秆得到更好的降解和利用,采用平菇和康氏木霉二步混合发酵法;通过不同的组合方式,发现H6-T10组合得出的降解效果最好,其木质素降解率达到44.77%,纤维素降解率达到41.48%。     

Microcalorimetric study on the growth and metabolism of microencapsulated microbial cell culture

The rate of heat output is one of the suitable measurements of metabolic activity of the organism or its parts, down to the cellular or even the sub-cellular levels. In this paper, microcalorimetry was first applied to study the metabolic activity of microbial in both alginate-polylysine-alginate and alginate-chitosan-alginate microencapsulated cultures as well as in free non-encapsulated culture. The organisms used for the measurements were Escherichia coli and Saccharomyces cerevisiae. As a result of this work, it was found that, despite E. coli cell in free non-encapsulated culture has the highest metabolic rate due to the highest value of heat output, the proliferation of the cells terminates quickly with a lowest biomass formed. And we found also an obviously longer stationary phase in microencapsulated culture. As far as S. cerevisiae was concerned, it was found that there was also the highest value of heat output in free non-encapsulated culture, but the cell density was lower than that in microencapsulated culture. On account of the microcalorimetric and metabolic measurements, it can be concluded that more substrate can be used to convert to biomass in microencapsulated culture which means a higher biomass yield existed.     

Flow microcalorimetry investigation of the influence of surfactants on a heterogeneous aerobic culture

The influence of various surfactants on the biological activity of a mixed aerobic culture has been investigated by using flow microcalorimetry. The response of the culture to the addition of homologous n-alkylcarboxylates (C2 to C16) and n-alkylpyridinium bromides (C11 to C14) has been examined under endogenous and substrate saturation conditions, and inhibitory concentrations (MIC or the concentration which decreased the initial activity (heat flux) of the culture by 50%) were determined for each state. Under both conditions, the n-alkylpyridinium bromides were found to be more toxic than the n-alkylcarboxylates of identical chain length, thus confirming that the head group of the amphiphiles plays an important role in the microbial toxicity of surfactants. The relationship observed between the concentration at which 50% of the activity is lost and the chain length of the surfactant further confirms that cellular toxicity is also dependent on surfactant hydrophobicity. In relation to the biodegradability of surfactants in mixed aerobic cultures, the low concentration effects of n-alkylcarboxylates on endogenous culture were investigated in some detail. There appear to be compounded indications that these surfactants are rapidly metabolized by the microorganisms of the mixed culture, at least for homologs lower than C10.     

Flow microcalorimetry investigation of the influence of surfactants on a heterogeneous aerobic culture.

The influence of various surfactants on the biological activity of a mixed aerobic culture has been investigated by using flow microcalorimetry. The response of the culture to the addition of homologous n-alkylcarboxylates (C2 to C16) and n-alkylpyridinium bromides (C11 to C14) has been examined under endogenous and substrate saturation conditions, and inhibitory concentrations (MIC or the concentration which decreased the initial activity (heat flux) of the culture by 50%) were determined for each state. Under both conditions, the n-alkylpyridinium bromides were found to be more toxic than the n-alkylcarboxylates of identical chain length, thus confirming that the head group of the amphiphiles plays an important role in the microbial toxicity of surfactants. The relationship observed between the concentration at which 50% of the activity is lost and the chain length of the surfactant further confirms that cellular toxicity is also dependent on surfactant hydrophobicity. In relation to the biodegradability of surfactants in mixed aerobic cultures, the low concentration effects of n-alkylcarboxylates on endogenous culture were investigated in some detail. There appear to be compounded indications that these surfactants are rapidly metabolized by the microorganisms of the mixed culture, at least for homologs lower than C10.     

Biodegradability of bacterial surfactants

This work aimed at evaluating the biodegradability of different bacterial surfactants in liquid medium and in soil microcosms. The biodegradability of biosurfactants by pure and mixed bacterial cultures was evaluated through CO₂ evolution. Three bacterial strains, Acinetobacter baumanni LBBMA ES11, Acinetobacter haemolyticus LBBMA 53 and Pseudomonas sp. LBBMA 101B, used the biosurfactants produced by Bacillus sp. LBBMA 111A (mixed lipopeptide), Bacillus subtilis LBBMA 155 (lipopeptide), Flavobacterium sp. LBBMA 168 (mixture of flavolipids), Dietzia Maris LBBMA 191(glycolipid) and Arthrobacter oxydans LBBMA 201(lipopeptide) as carbon sources in minimal medium. The synthetic surfactant sodium dodecyl sulfate (SDS) was also mineralized by these microorganisms, but at a lower rate. CO₂ emitted by a mixed bacterial culture in soil microcosms with biosurfactants was higher than in the microcosm containing SDS. Biosurfactant mineralization in soil was confirmed by the increase in surface tension of the soil aqueous extracts after incubation with the mixed bacterial culture. It can be concluded that, in terms of biodegradability and environmental security, these compounds are more suitable for applications in remediation technologies in comparison to synthetic surfactants. However, more information is needed on structure of biosurfactants, their interaction with soil and contaminants and scale up and cost for biosurfactant production.     

Degradation of the herbicide bentazon as related to enzyme production by Phanerochaete chrysosporium in two solid substrate fermentation systems

Enzyme production and degradation of the herbicide bentazon by Phanerochaete chrysosporium growing on straw (solid substrate fermentation, SSF) and the effect of nitrogen and the hydraulic retention time (HRT) were studied using a small bioreactor and batch cultures. The best degradation of bentazon was obtained in the low nitrogen treatments, indicating participation of the ligninolytic system of the fungus. The treatments that degraded bentazon also had manganese peroxidase (MnP) activity, which seemed to be necessary for degradation. Pure MnP (with Mn(II) and H₂O₂) did not oxidize bentazon. However, in the presence of MnP, Mn(II) and Tween 80, bentazon was slowly oxidized in a H₂O₂-independent reaction. Bentazon was a substrate of pure lignin peroxidase (LiP) and was oxidized significantly faster (22,000–29,000 times) as compared to the MnP-Tween 80 system. Although LiP was a better enzyme for bentazon oxidation in vitro, its role in the SSF systems remains unclear since it was detected only in treatments with high nitrogen and high HRT where no degradation of bentazon occurred. Inhibition of LiP activity may be due to phenols and extractives present in the straw.     

Cometabolic degradation of chloroallyl alcohols in batch and continuous cultures

The biodegradation of chloroallyl alcohols by pure and mixed bacterial cultures was investigated. Only 2-chloroallyl alcohol and cis- and trans-3-chloroallyl alcohol served as growth substrate for pure cultures. The other chloroallyl alcohols could be cometabolically degraded during growth on 2-chloroallyl alcohol. Cometabolic degradation of trichloroallyl alcohol, which was the most recalcitrant congener, by a Pseudomonas strain isolated on 2-chloroallyl alcohol resulted in 60% dechlorination. Efficient degradation of a mixture of chloroallyl alcohols in continuous culture could only be achieved in the presence of a satellite population. The mixed culture degraded 99% of the total chloroallyl alcohols added with 71% chloride release. The culture contained strains with a new catabolic potential. The results indicate the importance of mixed cultures and genetic adaptation for efficient chloroallyl alcohol removal.     

Influence of wine-related physicochemical factors on the growth and metabolism of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts in mixed culture

The influence of two physicochemical factors involved in winemaking, temperature and SO₂, on the kinetics and metabolic behavior of Kloeckera apiculata and Saccharomyces cerevisiae was examined. Highest biomass was reached at 15 and 25°C for K. apiculata and S. cerevisiae, respectively. Pure cultures of K. apiculata died off early with increasing temperature, but in co-culture with S. cerevisiae it showed higher viability and a change in the death curve from exponential to linear. Statistical analysis revealed that metabolite production was significantly different for the three cultures and also at the different fermentation temperatures. Besides, the interaction between culture type and temperature was significant. At temperatures from 15 to 30°C the mixed culture showed similar ethanol and lower acetic acid production compared with a pure culture of K. apiculata. SO₂ addition slightly increased survival of the non-Saccharomyces species in pure and mixed cultures. Statistical evaluation indicated that culture type and SO₂ addition significantly affected metabolite production, but the interaction between culture and SO₂ was not significant. These results contribute to current knowledge of enological factors and their effect on prevalence and fermentative activities of the composite yeast flora and the statistical significance emphasizes the importance of the combined influence of the culture type and physicochemical factors on the production of fermentation metabolites.     

Comparative bioelectrochemical analysis of Pseudomonas aeruginosa and Escherichia coli with anaerobic consortia as anodic biocatalyst for biofuel cell application

Aims: To study the bioelectrochemical behaviour of Pseudomonas aeruginosa (MTCC 17702) and Escherichia coli (MTCC 10436) and to assess their potential to act as anodic biocatalyst with the function of anaerobic consortia for microbial (bio) fuel cell (BFC) application. Methods and Results: Three BFCs (single chamber; open‐air cathode; noncatalysed electrodes) were operated simultaneously in acidophilic microenvironments. Pseudomonas aeruginosa (BFC_P) showed higher current density (264 mA m^?2) followed by mixed culture (BFC_M; 166 mA m^?2) and E. coli (BFC_E; 147 mA m^?2). However, total operating period and substrate degradation were relatively found to be effective with mixed culture (58%; 72 h) followed by BFC_P (39%; 60 h) and BFC_E (31%; 48 h). Higher electron discharge (ED) was observed with Ps. aeruginosa while mixed culture showed the involvement of redox mediators in the ED process. Conclusions: Mixed culture showed to sustain biopotential for longer periods along with a stable ED. The presence of redox signals and high substrate degradation was also evidencing its performance compared to the pure strains studied. This supports the practical utility of mixed culture over the pure cultures for real‐field BFC applications especially while operating with wastewater. Significance and Impact of the Study: This study revealed the efficiency and viability of mixed consortia in comparison with pure strains for microbial (bio) fuel cell applications.     

Effect of inoculation on the biodegradation of wathered Prudhoe Bay crude oil

Summary Enrichment cultures from oil-contaminated beach material from Prince William Sound, Alaska, generated both a mixed bacterial community of indigenous, oil-degrading marine microorganisms and a pure culture oil-degrader, strain EI2V. The mixed and axenic cultures were used in comparative shake flask studies of inoculation on biodegradation of Prudhoe Bay crude oil. Within 12 h following inoculation of homogenized, oiled beach material with the mixed culture, total CO₂ production was increased 2-fold relative to a noninoculated control. Moreover, measurements of phenanthrene degradation (as determined by the release of¹⁴CO₂ from [9-¹⁴C]phenanthrene) showed a 2-or 3-fold greater degradation when inoculated with either strain EI2V or with the mixed culture, respectively. However, as medium was replaced by a simulated tidal cycle, the observed stimulation of CO₂ production decreased, and the addition of strain EI2V had no greater effect on total CO₂ production than the addition of inorganic nutrients alone. Chemical analysis of oil recovered after 7 days incubation also suggested that, while these cultures are capable of efficient biodegradation of Prudhoe Bay crude in liquid culture, inoculation of beach material with high numbers of these microorganisms had little effect on the rate and extent of biodegradation of weathered crude oil. Overall, the sustained stimulatory effect was no greater than that observed with the addition of inorganic nutrients alone.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

Enhanced hydrogen and 1,3‐propanediol production from glycerol by fermentation using mixed cultures

The conversion of glycerol into high value products, such as hydrogen gas and 1,3‐propanediol (PD), was examined using anaerobic fermentation with heat‐treated mixed cultures. Glycerol fermentation produced 0.28 mol‐H₂/mol‐glycerol (72 mL‐H₂/g‐COD) and 0.69 mol‐PD/mol‐glycerol. Glucose fermentation using the same mixed cultures produced more hydrogen gas (1.06 mol‐H₂/mol‐glucose) but no PD. Changing the source of inoculum affected gas production likely due to prior acclimation of bacteria to this type of substrate. Fermentation of the glycerol produced from biodiesel fuel production (70% glycerol content) produced 0.31 mol‐H₂/mol‐glycerol (43 mL H₂/g‐COD) and 0.59 mol‐PD/mol‐glycerol. These are the highest yields yet reported for both hydrogen and 1,3‐propanediol production from pure glycerol and the glycerol byproduct from biodiesel fuel production by fermentation using mixed cultures. These results demonstrate that production of biodiesel can be combined with production of hydrogen and 1,3‐propanediol for maximum utilization of resources and minimization of waste. Biotechnol. Bioeng. 2009; 104: 1098–1106. © 2009 Wiley Periodicals, Inc.     

Fungal degradation of pine and straw alkali lignins

Summary Kraft pine and straw lignins were fractionated into aqueous soluble and organic soluble-ether insoluble parts. Chemical analysis, UV characteristics, and gel permeation chromatograms of crude and fractionated lignins were studied. Using pure and mixed, N-limited and non N-limited standing cultures of several fungal species, the biodegradability of curde and fractionated lignins was compared. Straw lignins, especially the aqueous fraction were degraded by most of the fungi studied. Except for Sporotrichum pulverulentum, nitrogen limitation did not seem to favour degradation. The best fungi for degradation under conditions of N-limitation were S. pulverulentum, Humicola fuscoatra, and Aspergillus wentii, under sufficient nitrogen: A. wentii, Chaetomium cellulolyticum and H. fuscoatra. The greatest percentage degradation, 55%, was obtained with S. pulverulentum under nitrogen limited conditions from 1 gl^–1 organic soluble-ether insoluble kraft lignin. Gel chromatography showed that the degradation was over the complete molecular size range.     

Alkali treatment and fermentation of straw for animal feed

The feed value of annual ryegrass straw was improved by treatment with various concentrations of NaOH or NH₃ followed by fermentation of the treated straw with a mixed culture of Cellulomonas sp. and Alcaligenes faecalis. Laboratory feeding trials with voles showed that NaOH or NH₃ treatment considerably increased the feed efficiency of straw, but apparently gave a poorly palatable product. Fermentation tended to decrease the in vitro rumen digestibility (IVRD) of alkali-treated straw. The fermentations were carried out aerobically on a semisolid straw matrix having 11–86% moisture. Treatment by both NaOH and NH₃ increased the IVRD of straw. NH₃ also increased the nitrogen content in straw. The optimum condition for alkaline treatment of the straw was 4–6% NaOH for 1 hr or with 3% NH₃ for four weeks at room temperature. A minimum of 63% moisture was needed for significant fermentation of the straw. The combined effects of NaOH treatment and fermentation more than doubled crude protein, doubled crude fat, and increased IVRD by 75%. The NH₃ plus fermentation treatment tripled crude protein, doubled crude fat, and increased IVRD by 60%. Acetic acid was the main volatile fatty acid in the fermented straw.     

Biodegradation of sulfamethoxazole by individual and mixed bacteria

Antibiotic compounds, like sulfamethoxazole (SMX), have become a concern in the aquatic environment due to the potential development of antibacterial resistances. Due to excretion and disposal, SMX has been frequently detected in wastewaters and surface waters. SMX removal in conventional wastewater treatment plants (WWTPs) ranges from 0% to 90%, and there are opposing results regarding its biodegradability at lab scale. The objective of this research was to determine the ability of pure cultures of individual and mixed consortia of bacteria (Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas putida, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus rhodocrous, and Rhodococcus zopfii) known to exist in WWTP activated sludge to remove SMX. Results showed that R. equi alone had the greatest ability to remove SMX leading to 29% removal (with glucose) and the formation of a metabolite. Degradation pathways and metabolite structures have been proposed based on the potential enzymes produced by R. equi. When R. equi was mixed with other microorganisms, a positive synergistic effect was not observed and the maximum SMX removal achieved was 5%. This indicates that pure culture results cannot be extrapolated to mixed culture conditions, and the methodology developed here to study the biodegradability of compounds under controlled mixed culture conditions offers an alternative to conventional studies using pure bacterial cultures or inocula from activated sludge sources consisting of unknown and variable microbial populations.