Mixed cultures of Serratia marcescens and Kluyveromyces fragilis for simultaneous protease production and COD removal of whey

AIMS: The aim of this study was to investigate the behaviour of a Serratia marcescens-Kluyveromyces fragilis mixed culture in whey, with the objective of proteases production and organic waste reduction. METHODS AND RESULTS: Discontinuous aerobic fermentations in whey were carried out using individual pure cultures and mixed cultures of S. marcescens and K. fragilis. Cell growth, protease production, lactose and proteins consumption and COD/TOC reduction were monitored. Lactose and protein content of the fermenting medium was almost depleted in the mixed cultures, achieving a reduction in the organic content much higher than in both pure cultures. Interestingly, proteolytic activity in the mixed cultures was similar to that obtained for S. marcescens in pure culture. In addition, protease stability was increased in the mixed cultures. Kinetic models were developed fitting well with the experimental results. CONCLUSIONS: Mixed cultures were found to maintain the achievements of each individual fermentation, yielding a high and stable production of proteases and a significant reduction of COD/TOC. SIGNIFICANCE AND IMPACT OF THE STUDY: Mixed cultures tested in this work have shown a synergistic effect with possible industrial applications. These results lead to a gain in the chain value for enzyme production with an environmentally friendly operation.     

A method for high throughput bioelectrochemical research based on small scale microbial electrolysis cells

There is great interest in studying exoelectrogenic microorganisms, but existing methods can require expensive electrochemical equipment and specialized reactors. We developed a simple system for conducting high throughput bioelectrochemical research using multiple inexpensive microbial electrolysis cells (MECs) built with commercially available materials and operated using a single power source. MECs were small crimp top serum bottles (5 mL) with a graphite plate anode (92 m2/m(3)) and a cathode of stainless steel (SS) mesh (86 m2/m3), graphite plate, SS wire, or platinum wire. The highest volumetric current density (240 A/m3, applied potential of 0.7 V) was obtained using a SS mesh cathode and a wastewater inoculum (acetate electron donor). Parallel operated MECs (single power source) did not lead to differences in performance compared to non-parallel operated MECs, which can allow for high throughput reactor operation (>1000 reactors) using a single power supply. The utility of this method for cultivating exoelectrogenic microorganisms was demonstrated through comparison of buffer effects on pure (Geobacter sulfurreducens and Geobacter metallireducens) and mixed cultures. Mixed cultures produced current densities equal to or higher than pure cultures in the different media, and current densities for all cultures were higher using a 50 mM phosphate buffer than a 30 mM bicarbonate buffer. Only the mixed culture was capable of sustained current generation with a 200 mM phosphate buffer. These results demonstrate the usefulness of this inexpensive method for conducting in-depth examinations of pure and mixed exoelectrogenic cultures.     

Interspecies interaction based on transfer of a thioredoxin-like compound in anaerobic chitin-degrading mixed cultures

Abstract Fermentation of chitin by mixed cultures of the chitinolytic Clostridium sp. strain 9.1 and various non-chitinolytic bacteria proceeded up to eight times faster than in pure cultures. The addition of spent media of such mixed cultures also resulted in a marked stimulation of chitinolysis in pure cultures of strain 9.1. Pure cultures fermented chitin much faster if supplemented with either spent media or cell-free extracts of the non-chitinolytic bacteria. The compound responsible for this stimulation was thermostable (10 min at 85° C) and could not be removed by passage over Sephadex G-25, indicating a molecular weight of more than 1500. The heat stable enzyme thioredoxin (from Saccharomyces cerevisiae ) was shown to stimulate the chitin fermentation in a similar manner. Alkylation of this enzyme reduced its stimulatory action significantly indicating its (di)thiol: disulfide interchanging activity.
It is hypothesized that essential sulfhydryl groups in the chitinolytic system of strain 9.1 are reduced by thioredoxin and/or similar thiol: disulfide transhydrogenases present in the cell-free extracts and spent media, resulting in an acceleration of chitin hydrolysis and fermentation. This stimulation may thus be the result of a new type of interspecies interaction in anaerobic mixed cultures.     

Production of polyhydroxyalkanoates by mixed microbial cultures

Polyhydroxyalkanoates (PHAs) are biodegradable bioplastics formed from renewable resources, like sugars, with similar characteristics of polypropylene. These bioplastics are industrially produced by pure cultures using expensive pure substrates. These factors lead to a much higher selling price of PHAs compared to petroleum-based plastics, like polypropylene. The use of mixed cultures and cheap substrates (waste materials) can reduce costs of PHA production by more than 50%. Storage of PHAs by mixed populations occurs under transient conditions mainly caused by discontinuous feeding and variation in the electron donor/acceptor presence. In the last years the mechanisms of storage, metabolism and kinetics of mixed cultures have been studied. The maximum capacity of PHA storage and production rate is dependent on the substrate and on the operating conditions used. In this paper an overview and discussion of various mechanisms and processes for PHA production by mixed cultures is presented.     

子午岭林区白桦-辽东栎混交林光合生理生态特征研究

对黄土高原子午岭次生林区白桦林、辽东栎林和白桦-辽东栎混交林3种林分的土壤物理特性和叶片光合特性进行了研究。结果表明:(1)白桦-辽东栎混交林地的土壤水分明显改善,其土壤容重最小、土壤孔隙度最大,且均优于纯林,即混交林地有深层次的土壤水分可供利用,并改善了土壤的物理结构;(2)辽东栎林的光合速率和气孔导度最大,其次为白桦-辽东栎混交林,水分利用率(WUE)为混交林白桦>混交林辽东栎>辽东栎林>白桦林;(3)混交林中白桦、辽东栎的Fv/Fm和Fv/Fo值均较大,与纯林差异不显著;白桦林和辽东栎林的qP和NPQ值均大于混交林。     

Effect of ellagitannin acyl hydrolase,xylanase and cellulase on ellagic acid production from cups extract of valonia acorns

Ellagic acid production from cups extract of valonia acorns by pure and mixed cultures of Aspergillus oryzae and Trichoderma reesei was investigated. Ellagitannin acyl hydrolase and xylanase as well as cellulase during the pure and mixed cultures were also determined. The results revealed that mixed culture could produce higher ellagic acid yield (23%) than either of pure culture. And it was found that the three enzymes from mixed culture appeared synergistic effect on ellagic acid production. Statistical analysis showed that ellagic acid yield was correlated very well with the three enzymes activities, resulting in the model for ellagic acid production with high R² value of 0.998 and significant level p < 0.004.     

Possible use of Biolog methodology for monitoring yeast presence in alcoholic fermentation for wine‐making

Aims: A research was undertaken to explore the possibility to use Biolog system of microbial metabolic characterization for the monitoring of yeast population evolution during alcoholic fermentation for wine production. Methods and Results: An application of Biolog system was employed for the characterization of yeasts of oenological interest, in pure cultures and mixed consortia, in various cell concentrations. The system’s capacity to discriminate among different cell concentrations of the same yeast strain was ascertained, along with the capacity to discriminate between mixed and pure populations. Conclusions: The tested application of Biolog system resulted suitable for a quick recognition (24 h) of the presence of starter cultures within mixed populations of autochthonous yeasts. Such discrimination was confirmed with the one resulting from molecular techniques. Significance and Impact of the Study: The study suggests the possibility to employ Biolog system for an early monitoring of yeast evolution in modern wine‐making fermentations, where specialized yeasts are more and more frequently used as starters and their ability to overcome autochthonous yeast populations is crucial.     

Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons

Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch-induced injury alters mitochondrial membrane potential (delta(psi)m) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, delta(psi)m was measured using rhodamine-123, and ATP was quantified with a luciferin-luciferase assay. In astrocytes, delta(psi)m dropped significantly, and ATP content declined 43-52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, delta(psi)m declined at 15 min only in the severely stretched group. At 48 h postinjury, delta(psi)m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, delta(psi)m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24-48 h. ATP content in mixed cultures declined 22-28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.     

Analytical differentiation of wine fermentations using pure and mixed yeast cultures

Summary Thirty-three fermentations of Pedro Ximénez grapes, collected in three degrees of ripeness, were carried out by inoculation with three types of inoculum: pure cultures ofSaccharomyces cerevisiae races and ofTorulaspora delbrueckii, indigenous yeasts, and mixed cultures of indigenous yeasts enriched with the pure cultures. By means of variance analysis 21 compounds were determined whose final concentrations in the wines significantly depended on the musts, the inocula or both. Eleven products that depended significantly on the inocula were subjected to a discriminant analysis in which most of the pure cultures gathered in a discriminant space area different from that occupied by the indigenous yeasts. The centroids corresponding to most of the mixed cultures were shifted to the central area of the discriminant space, moved away from their corresponding pure cultures and approached the indigenous yeasts. The results show a high similarity between the fermentations carried out with mixed cultures with the addedS. cerevisiae races and those fermentations carried out with the indigenous yeasts, with regard to those compounds which were significantly dependent on the inocula.     

Processing and fermentation of Jerusalem artichoke for ethanol production

Summary Processing and fermentation trials on Jerusalem artichoke (Helianthus tuberosus L.) tubers, and on pure inulin media were carried out. Acid and thermal treatments, pure and mixed cultures of yeast, and enzyme preparations were investigated. Best ethanol yields on either substrate were obtained with pH 2 thermal treatments, resulting in 131.6 liters ethanol per metric ton fresh tuber.     

In vitro studies on bacterial colonization. The effects of beta-lactam antibiotics on the growth of Escherichia coli and Pseudomonas aeruginosa in mixed culture.

Many cases of Pseudomonas aeruginosa infection are considered to be secondary superinfections, resulting from bacterial colonization. Such cases of superinfection with P. aeruginosa developing after administration of cephalosporin or penicillin are offering serious clinical problems. To make a fundamental analysis of the development of such superinfections, attempts were made to compare the growth patterns of Escherichia coli and P. aeruginosa in pure and mixed cultures and to determine the effects of cephalothin, cefazolin, cephalexin, and ampicillin on the growth patterns. In mixed cultures, the growth of P. aeruginosa was markedly inhibited by E. coli. The higher the concentration of each of the cephalosporins and ampicillin added to the mixed culture, the smaller the population of E. coli sensitive to these agents. When the population of E. coli became smaller than that of P. aeruginosa, which is resistant to these agents, the latter was restored to the same population level as that in pure cultures. Experimental bacterial colonization, by which the predominant population of E. coli was replaced by that of P. aeruginosa in mixed culture, was brought about more efficiently with the cephalosporins than with ampicillin. This might be accounted for by the difference in minimal inhibitory concentration for P. aeruginosa between ampicillin and the other three agents.     

Biooxidation of pyrite by defined mixed cultures of moderately thermophilic acidophiles in pH-controlled bioreactors: significance of microbial interactions

The oxidative dissolution of pyrite (FeS2) by pure and mixed cultures of moderately thermophilic acidophiles was studied in shake flask cultures and in pH-controlled bioreactors, incubated at 45 degrees C. Various combinations of seven eubacteria (a Leptospirillum sp. (MT6), Acidimicrobium ferrooxidans, Acidithiobacillus caldus, an Alicyclobacillus sp. (Y004), and three Sulfobacillus spp.) and one archaeon (Ferroplasma sp. MT17) were examined. Pyrite dissolution was determined by measuring changes in soluble iron and generation of acidity, and microbial populations were monitored using a combined culture-dependent (plate counts) and culture-independent (fluorescent in situ hybridization) approach. In pure cultures, the most efficient pyrite-oxidizing acidophile was Leptospirillum MT6, which was unique among the prokaryotes used in being obligately autotrophic. Mixed cultures of Leptospirillum MT6 and the sulfur-oxidizer At. caldus generated more acidity than pure cultures of the iron-oxidizer, though this did not necessarily enhance pyrite dissolution. In contrast, a mixed culture of Leptospirillum MT6 and the obligate heterotroph Alicyclobacillus Y004 oxidized pyrite more rapidly and more completely than a pure culture of Leptospirillum MT6, in synchronized bioreactors. Although the autotroph, At. caldus, and the "heterotrophically inclined" iron-oxidizer, Am. ferrooxidans, were both ineffective at leaching pyrite in pure culture, a mixed culture of the two bacteria was able to accelerate dissolution of the mineral. Concentrations of dissolved organic carbon accumulated to >100 mg/L in some mixed cultures, and the most effective bioleaching systems were found to be consortia containing both autotrophic and heterotrophic moderate thermophiles. A mixed culture comprising the autotrophs Leptospirillum MT6 and At. caldus, and the heterotroph Ferroplasma MT17, was the most efficient of all of those examined. Mutualistic interactions between physiologically distinct moderately thermophilic acidophiles, involving transfer of organic and inorganic carbon and transformations of iron and sulfur, were considered to have critical roles in optimizing pyrite dissolution.     

Kinetics of pure cultures of hydrogen-oxidizing denitrifying bacteria and modeling of the interactions among them in mixed cultures

In this study we report the isolation of four denitrifying bacteria from a batch reactor, where the progress of hydrogenotrophic denitrification was examined. Only three of the strains had the ability to use hydrogen as electron donor. In the present work, kinetic batch experiments were carried out in order to study the dynamic characteristics of pure and defined mixed cultures of hydrogen-oxidizing denitrifying bacteria, under anoxic conditions, in a defined synthetic medium, in the presence of nitrates. Kinetic models were developed and the kinetic parameters were determined from the batch experiments for each bacterium separately. The behavior of mixed cultures and the interactions between the bacteria were described using kinetic models based on the kinetic models developed for each bacterium separately and their predictions were compared with the results from mixed culture experiments. The mathematical models that were developed and validated in the present work are capable of describing the behavior of the bacteria in pure and mixed cultures, and in particular, the kinetics of nitrate and nitrite reduction and cell growth.     

Degradation of 2,4-dichlorophenoxyacetic acid by mixed cultures of bacteria

Summary We explored the feasibility of using mixed cultures for herbicide degradation, with the ultimate aim of application for effluent treatment. The present study reports on mixed cultures which were developed to grow aerobically with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon substrate. Degradation of 2,4-D was verified by HPLC and UV-spectroscopic analysis of the residual 2,4-D concentration in the test cultures. Cultures that were initially developed with 2,4-D also grew readily with glucose, but the degradation of 2,4-D was effectively prevented under mixed substrate conditions. Mamor intermediates or metabolites resulting from 2,4-D degradation were not detected with the HPLC methodology except 2,4-dichlorophenol which appeared to accumulate transiently in the growth medium.     

Development of a miniaturized selective counting strategy of lactic acid bacteria for evaluation of mixed starter in a model cassava fermentation

A miniaturized most probable number (MPN) method for the selective enumeration of three bacteria species ( Lactobacillus plantarum A6, Leuconostoc mesenteroides and Lactococcus lactis ) is described. This selective count method, based on specific consumption of carbon substrate and resistance to antibiotics, was used for the quantitative assessment of the three bacteria during mixed cultures in a model cassava fermentation. A typical microbial succession pattern was observed: (i) Lactococcus lactis and Leuc. mesenteroides dominated during the first hours of fermentation as their growth was very rapid ; (ii) from hour 12, Lactobacillus plantarum replaced the two latter strains and Lactococcus lactis disappeared gradually, followed by Leuc. mesenteroides . The growth rates of each strain appeared to be independent of the others, while acidification rates increased strongly in mixed cultures compared with pure cultures. No positive interactions resulting from the amylolytic character of Lactobacillus plantarum A6, and no negative interactions resulting from the Nis⁺ property of Lactococcus lactis , were revealed between the three strains under the model conditions used.     

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.     

Comparison of growth,acidification and productivity of pure and mixed cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398

Pure and mixed controlled-pH batch cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398 have been conducted. The characteristics of growth and acidification and the productivity of the cultures were compared. During the mixed cultures, the growth characteristics revealed a pronounced stimulation of S. thermophilus whereas L. bulgaricus metabolism was not significantly improved. The final total population was 1.4 to 4.9 higher than in pure cultures. The acidification characteristics were not enhanced by the mixed culture conditions. The productivity of mixed cultures was 1.7 to 2.4 times higher as compared to an equivalent mixing of pure cultures.Correspondence to: C. Béal     

A method for the selective enumeration and isolation of ruminal Lactobacillus and Streptococcus

Ruminal lactic acid-producing bacteria were selectively isolated and enumerated using a one hour aerobic exposure prior to incubation on a semi-selective Lactobacillus medium, MRS, under anaerobic conditions. The technique allowed growth of pure cultures of ruminal Lactobacillus spp. and Streptococcus bovis without supporting the growth of pure cultures of any of the prominent ruminal bacterial species. In mixed cultures, the one hour aerobic pre-incubation inhibited the growth of the obligate anaerobic ruminal bacteria which can otherwise grow on the MRS medium, and the subsequent anaerobic incubation permitted maximal recovery of the weakly aerotolerant ruminal lactic acid-producing Lactobacillus spp. and Streptococcus spp. The efficacy of this technique in selecting exclusively for the lactic acid-producing bacteria was also demonstrated from populations of rumen bacteria from mixed culture end-point in vitro fermentation, continuous in vitro culture and isolations from fresh ruminal samples.     

Interspecific interactions in a methane-utilizing mixed culture

A two-member methane-utilizing mixed culture of bacteria, formed by combining two pure cultures isolated from a naturally occurring methane-utilizing mixed culture, was studied in continuous culture. From the nutritional requirements and substrate ranges of the pure cultures, a mechanism for the interspecific interactions occurring in the mixed culture was proposed. Product formation kinetics were determined in continuous culture for each product involved in the proposed mechanism. From this proposed mechanism a mathematical model was derived based on simple material balance equations around a single-stage chemostat. The steady-state predictions of this model were compared to experimental results obtained from continuous-culture experiments with the two-member methane-utilizing mixed culture. Interspecific interactions occurring in two-member methanol-utilizing and three-member methane-utilizing mixed cultures have also been discussed.