Mixed Culture

Hydrogen gas can be recovered from the microbial fermentation of organic substrates at high concentrations when interspecies hydrogen transfer to methanogens is prevented. Two techniques that have been used to limit methanogenesis in mixed cultures are heat treatment, to remove nonsporeforming methanogens from an inoculum, and low pH during culture growth. We found that high hydrogen gas concentrations (57-72%) were produced in all tests and that heat treatment (HT) of the inoculum (pH 6.2 or 7.5) produced greater hydrogen yields than low pH (6.2) conditions with a nonheat-treated inoculum (NHT). Conversion efficiencies of glucose to hydrogen (based on a theoretical yield of 4 mol-H 2 /mol-glucose) were as follows: 24.2% (HT, pH ) 6.2), 18.5% (HT, pH ) 7.5), 14.9% (NHT, pH ) 6.2), and 12.1% (NHT, pH ) 7.5). The main products of glucose (3 g-COD/L) utilization (g99%) in batch tests were acetate (3.4-24.1%), butyrate (6.4-29.4%), propionate (0.3-12.8%), ethanol (15.4-28.8%), and hydrogen (4.0-8.1%), with lesser amounts of acetone, propanol, and butanol (COD basis). Hydrogen gas phase concentrations in all batch cultures reached a maximum of 57-72% after 30 h but thereafter rapidly declined to nondetectable levels within 80 h. Separate experiments showed substantial hydrogen losses could occur via acetogenesis and that heat treatment did not prevent acetogenesis. Heat treatment consistently eliminated the production of measurable concentrations of methane. The disappearance of ethanol produced during hydrogen production was likely due to acetic acid production as thermodynamic calculations show that this reaction is spontaneous once hydrogen is depleted. Overall, these results show that low pH was, without heat treatment, sufficient to control hydrogen losses to methanogens in mixed batch cultures and suggest that methods will need to be found to limit acetogenesis in order to increase hydrogen gas yields by batch cultures.

Thiobacillus denitrijicans is a strictly autotrophic and facultative anaerobe first described in detail by Baalsrud and Baalsrud. ' Under anaerobic conditions nitrate may be used as a terminal electron acceptor with reduction to elemental nitrogen. Thiosulfate, elemental sulfur, and sulfide may be used as energy sources with oxidation to sulfate; however, sulfide is an inhibitory substrate. Sublette and S y l v e~t e r~.~ have demonstrated that T. denitrijicans may be readily cultured anaerobically in batch and continuous reactors on H2S(g) under sulfide limiting conditions. Under these conditions sulfide concentrations in the culture medium were less than IpM resulting in very low concentrations in the reactor outlet gas (less than 0.05pM). The stoichiometry of the anaerobic oxidation of H2S(g) in batch and continuous cultures was reported and stable reactor operation demonstrated at reactor loadings as high as 4-5 mmol H2S oxidized/h/g biomass. Maximum H2S loading of the biomass was estimated at 5.4-7.7 mmol H2S/h/g biomass under sulfide limiting conditions. Sublette and S y l v e~t e r~,~ have proposed a process for the microbial desulfurization of natural gas based on the oxidation of H2S(g) by Thiobacillus denitrijicans. The proposed process could be greatly simplified if aseptic operation of the reactor was not required. Accordingly, we report here a study of the effects of heterotrophic contaminants on H2S(g) oxidation by T. denitrijicans.

To explain patterns and variations in concentration of defence-related carbon-based secondary compounds in plant tissues, different complementary and in some parts contradictory plant-defence hypotheses were developed since several years. These conceptual hypotheses cannot be fully evaluated without a plant growth simulation model and consequently, the discussion about adequacy of plantdefence hypotheses neglects to a large extent the dynamics of plant growth. To get a more realistic view of the volatile dynamics of plant internal source and sink strengths of carbon and nitrogen during different phenological growth stages, a modelling approach for allocation of carbohydrates to a pool of carbon-based secondary compounds was integrated into the functional generic plant growth model PLATHO. In this paper, we explain the fundamental assumptions of our modelling approach and present the results of a simulation study for juvenile beech and spruce trees growing in mono-and mixed cultures under ambient and elevated atmospheric CO 2 .

Inulin was used as a prebiotic to improve the quality and consistency of skim milk fermented by Lactobacillus acidophilus (La), Lactobacillus rhamnosus (Lr), Lactobacillus bulgaricus (Lb) and Bifidobacterium lactis (Bl) with Streptococcus thermophilus (St), either in binary co-cultures or in cocktail containing all microorganisms. We compared, either in the presence of 40 mg inulin g À1 or not, the results of the maximum acidification rate (V max ) and the times to reach it (t max ), to reach pH 5.0 (t pH5.0 ) and to complete the fermentation (t f ). Post-acidification, lactic acid formation and cell counts were also compared after either 1 day (D1) or 7 days of storage at 4°C (D7). In co-culture, inulin addition to the milk increased V max , decreased t max and t f , favored post-acidification and exerted a bifidogenic effect. S. thermophilus proved to stimulate the metabolism of the other lactic bacteria and enhanced the product features. After D7, a significant prebiotic effect of inulin was observed in all co-cultures. Either after D1 or D7, the enumerations of Lr and Bl in mixed culture markedly decreased compared to their respective co-cultures because of greater competition for the same substrates.

Catabolic products from anaerobic fermentation processes are potentially of industrial interest. The volatile fatty acids and alcohols produced can be used as building blocks in chemical processes or applied directly as substrates in a mixed culture process to produce bioplastics. Development of such applications requires a predictable and controllable product spectrum of the fermentation process. The aim of the research described in this paper was (i) to investigate the product spectrum of an open mixed culture fermentation (MCF) process as a function of the pH, using glucose as substrate, and (ii) to relate the product spectrum obtained to generalized biochemical and thermodynamic considerations. A chemostat was operated under carbon and energy limitation in order to investigate the pH effect on the product spectrum in a MCF process. A transition from CO2/H2 production at lower pH values to formate production at higher pH values was observed. The ratio of CO2/H2 versus formate production was found to be related to the thermodynamics of formate dehydrogenation to CO2/H2. This transition was associated with a shift in the catabolic products, from butyrate and acetate to ethanol and acetate, likely due to a decrease in the oxidation state of the electron carriers in the cell. The product spectrum of the MCF process as a function of the pH could largely be explained using general biochemical considerations. Biotechnol. Bioeng. 2007; 98: 69–79. © 2007 Wiley Periodicals, Inc.

This study developed a procedure for the production of sour bread from 100% maize meal. The modified method combined sponge and dough methods in two mixing stages to form a batter rather than stiff dough. The baking quality of the maize meal was improved by using starter cultures of lactic acid bacteria selected from indigenous micro-flora of the maize meal. Lactobacillus plantarum lowered the pH of fermenting maize meal (3.1) more than other starters; Lactobacillus brevis produced the highest amount of diacetyl (180.8 mg) while Leuconostoc mesenteroides recorded the highest final viscosity value of 144.0 RVU. The sour maize bread fermented with a mixed culture of the three selected cultures had the best physical (weight 122 g, height 3.7 cm, crumb dry matter 69%) and sensory properties (taste and overall acceptability 4.8 on a five-point hedonic scale), which were significantly different (P ≤ 0.05) from bread samples produced with other test starters). The mixed cultures are recommended for sour maize bread production for good rheological properties, proper acidification, and acceptable flavor development.

AbstractÐA mixed culture bio®lm was developed with a sulfur oxidising, heterotrophic bacterium Thiosphaera pantotropha, autotrophic nitri®ers and other heterotrophs in a three stage rotating biological contactor (RBC). Speci®c bene®ts due to peculiar properties of T. pantotropha of simultaneous heterotrophic nitri®cation and aerobic denitri®cation were derived in the reactor investigated for a combined carbon and nitrogen removal from a synthetic domestic sewage. The ®rst stage bio®lm which contained T. pantotropha showed high COD and NH 4 + -N removal rates of 5.8±14.1 g COD/m 3 Ád and 0.47±1.1 g N/m 2 Ád for the corresponding loading rates of 6.9±20.7 g COD/m 3 Ád and 0.69±2.09 g N/m 2 Ád, respectively. Contrary to the conventional units designed for a concurrent carbon removal and nitri®cation, the nitri®cation rates increased linearly with an increase in organic loading rate before stabilising at about 1 g N/m 2 Ád corresponding to a COD loading rate of about 15 g/m 2 Ád and a nitrogen loading rate of 1.5 g N/m 2 Ád showing a change in the order of reaction from ®rst to zero. A simultaneous nitrogen removal of 20±68% was also obtained. The system's performance indicated that a single stage aerobic bio®lm can be developed to meet the increasingly stringent regulations on euent nitrogen discharges aording several advantages over the conventional systems, e.g. low buer requirements, no need for external carbon source for denitri®cation, etc., which may result in substantial reduction in the treatment cost. #

The freshwater microalga Chlorella vulgaris was grown heterotrophically in fed-batch 50-600-L fermenters at 36°C, on aerated and mixed nutrient solution with urea as a nitrogen and glucose as a carbon and energy source. Cell density increased from the initial value 6.25 to 117.18 g DW L −1 in 32 h in the fermenter 50 L at a mean growth rate 3.52 g DW L −1 h −1 . The DW increase in the fermenter 200 L was from 7.25 to 94.82 g DW L −1 in 26.5 h at a mean growth rate 3.37 g DW L −1 h −1 . Mean specific growth rate μ was about 0.1 h −1 in the both fermenters, if nutrients and oxygen were adequately supplied. The DW increase in the fermenter 600 L was from 0.8 to 81.6 g DW L −1 in 66.5 h at a mean growth rate 1.22 g DW L −1 h −1 and μ=0.07 h −1 . A limitation of the cell growth rate in 600 L fermenter caused by a low dissolved oxygen concentration above cell densities higher than 10 g DW L −1 ) occurred. Specific growth rate decreased approximately linearly with increasing glucose concentration (25-80 g glucose L −1 ) at the beginning of cultivation and decreased with the time of cultivation. The cell yield was 0.55-0.69 g DW (g glucose) −1 . The content of proteins, β-carotene, and chlorophylls in the cells steadily increased and starch content decreased, by keeping aerated and mixed culture another 12 h in fermenter after the cell growth was stopped due to glucose deficiency.

Growth and sugars utilization by mixed cultures of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp, thermophilus isolated from Argentina yogurt M.J. Amoroso and M.C. Manca de Nadra* Four different strains of Lactobacillus delbrueckii subsp, bulgaricus (Ss 1 and Yop12) and Streptococcus salivarius subsp, thermophilus (Ss z and YopO were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yoplz + Ss~) shows stimulation of S. thermophilus and inhibition of L. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated.

Comparative'phagocytic activity ofbuffalo blood neutrophils incubated simultaneodv with ~u r e and mixed cultures of Escherichia cdi. Staphylococcus aureus, &d strepcrgtdudue was studied. Flmgo&iC activity for pure bacterial cultures of Staph. aureua, Strep. agahctiae and E. d i were 90.8%, 86.4% and 76.246, respectively. The percent phagocytosis of neutrophils incubated with mixed bacterial cultures was similar to that with pure bacterial cuhres. However. with mixed bacterial cdtures. a maioritv of heutrophils showed preferential ihagocytosia of one species of ba-&ria: l%urc, neutrophds incubated with a mixed bacterial culture ofE. mli and Staph. a m showed a significantly higher phagocytosis of Staph rrureus, with a per cent phagocytosia of approximately 40%. Neutrophils incubated with a mixed bacterial culture of E. coli and Strep. walactiue showed a significantly higher --phagocyt~sia of Strep. ag&tiae, G t h apercent phagoqtnsi~of appmximat+ly 33%. These observations indicate that when buffalo neutrophils are exposed to a mixed bacterial culture the &ity for phagocytizingE. &li was much lees in the presence of Staph. aurells than in the presence of Strep. agalactiae. Key w o r k Buffalo, mixed bacterial cultures, neutmphil, pha@xybsis.

Postharvest diseases cause considerable losses to harvested fruits and vegetables during transportation and storage. Synthetic fungicides are primarily used to control postharvest decay loss. However, the recent trend is shifting toward safer and more eco-friendly alternatives for the control of postharvest decays. Of various biological approaches, the use of antagonistic microorganisms is becoming popular throughout the world. Several postharvest diseases can now be controlled by microbial antagonists. Although the mechanism(s) by which microbial antagonists suppress the postharvest diseases is still unknown, competition for nutrients and space is most widely accepted mechanism of their action. In addition, production of antibiotics, direct parasitism, and possibly induced resistance in the harvested commodity are other modes of their actions by which they suppress the activity of postharvest pathogens in fruits and vegetables. Microbial antagonists are applied either before or after harvest, but postharvest applications are more effective than preharvest applications. Mixed cultures of the microbial antagonists appear to provide better control of postharvest diseases over individual cultures or strains. Similarly, the efficacy of the microbial antagonist(s) can be enhanced if they are used with low doses of fungicides, salt additives, and physical treatments like hot water dips, irradiation with ultraviolet light etc. At the international level, different microbial antagonists like Debaryomyces hansenii Lodder & Krejer-van Rij, Cryptococcus laurentii Kufferath & Skinner, Bacillus subtilis (Ehrenberg) Cohn, and Trichoderma harzianum Rifai, are being used. Biocontrol products like Aspire, BioSave, and Shemer etc., have also been developed and registered. Although the results of this technology are encouraging, we need to continue to explore potential uses on the commercial scale in different corners of the world.

One of the most important causes of decay of calcareous stones is due to the conversion of calcium carbonate into calcium sulphate (gypsum). In order to optimise a strategy for the removal of the sulphates from artistic stoneworks, a procedure based on the use of sulphate-reducing bacteria, has been established. Different strains of Desulfovibrio in pure and mixed cultures were tested in batch to verify their sulphate-reducing potentiality. The biomasses of the selected strains, D. desulfuricans 1 and D. vulgaris, were applied under anaerobic conditions to the sample surfaces directly and after adhesion to sepiolite used as a substratum. Stone samples artificially enriched with sulphates and real fragments of a marble column and a marble statue were treated. The results obtained show that sulphate removal was more effective on real samples than on artificially enriched samples and in the both cases when the treatment was performed using sepiolite as substratum. The best result was obtained on the statue fragment with 8 I % sulphate removal after 36 h (against 20% for control).

The biodegradability of water soluble poly(vinyl alcohol)(PVA) blown ®lms was investigated under dierent environmental and test conditions. In particular respirometric determinations were utilized in order to evaluate the biodegradability of polymer ®lms in composting, soil burial and aquatic degradation tests. Several microbial inocula derived from mature compost, forest and loamy soils as well as sewage sludges from municipal and paper mill waste-water treatments plants were matched with the PVA-based blown ®lms. A fairly active PVA-degrading bacterial mixed culture was obtained by starting from sewage sludge of a paper mill as inoculum. Limited rates and extents of mineralization were recorded in solid cultures in the presence of either soil or compost samples, whereas signi®cant biodegradation level within fairly short incubation time were obtained in liquid cultures in the presence of acclimated microbial populations. Experimental diculties encountered in trials aimed at isolating single degrading microbial species de®nitely indicated the existence of strong symbiotic or commensal interactions between the single components of the PVAdegrading mixed culture. A PVA degradation mechanism dierent from random scission or unzipping of carbon backbone has been suggested on the basis of viscometric behavior of PVA solution in the presence of mixed culture and its ®ltrate. #

The breakthroughs in microbiology have allowed us to come to terms with the microbial resources present in culture collections and in natural environments. The challenge at present is to manage these microbial resources, particularly when one deals with open systems where the dynamics of microbial ecology are predominant. Hence, to properly address the aspects of Microbial Resource Management (MRM), one needs to handle the questions of who is there, who is doing what with whom and how can one adjust, control and/or steer these mixed cultures and communities. It is argued that microbial ecologists and environmental microbiologists need to address a new mind-set. The Beijerinck axioma that all microorganisms are everywhere should not be presumed to be generally valid. The Darwin based niche assembly concept needs to be supplemented with the neutral theory of Hubbell. The Pareto 80/20 principle is also applicable to the macro-economies of microbial communities. Finally, the concept of a “stable” microbial community should be replaced by that of a cooperative community continuum. Overall, MRM is at the basis of a number of new developments in domains such as environmental safety and health, renewable energy production, closing environmental cycles and providing new materials. Specific examples such as, for example, pro-active immuno-stimulation by means of drinking water, electro-microbiology, decreasing global warming by implementation of methanotrophs and generation of nano-biocatalysts are discussed.

Purpose -The Food and Agriculture Organization (FAO) of the United Nations has established that vinegar is a liquid only obtained by fermentation. Although in Pakistan mostly synthetic vinegar is marketed, production of vinegar through fermentation at industrial level is also carried out. However, vinegar produced by this method tends to be of inferior quality due to uncontrolled conditions. In industry a mixed culture of Acetobacter is used for the production of acetic acid but no attention is given towards its proper maintenance and culture is contaminated with other kinds of microorganisms and still no work has been done along these lines in Pakistan. There is a need to develop pure vinegar cultures for vinegar production so that the use of synthetic vinegar may be avoided as it is prohibited in most countries. Keeping in view all of these points, the present work was conducted to isolate a pure culture of Acetobacter aceti, the maintenance of this culture and finally its utilization in fermentation for vinegar production. Design/methodology/approach -In this study efforts were made to isolate vinegar culture (Acetobacter aceti ) from sugar cane juice, rotten apples, flowers, wine, canal water and vinegar as a primary source for Acetobacter by continuous sub-culturing on standard medium glucose, yeast extract and calcium carbonate (GYC). The culture was identified on the basis of colony characteristics and morphology. It was finally confirmed by different biochemical/enzymatic tests and further specified by nutritional and temperature requirements for the growth. The isolated strain was later used for the production of vinegar through fermentation. Findings -Among canal water, crushed apples, sugar cane juice, alcohol, vinegar and flowers, the alcohol and vinegar were found to be the most suitable sources for isolation of Acetobacter spp. The colonies of purified culture were found to be pale to off-white, circular, raised, convex, smooth and not .3 mm in diameter with morphology of Gram-ve, ellipsoidal, rods, squat bacilli, roundish, single, in pairs and in chains. The isolated and identified spp. gave excellent results for the production of vinegar and this vinegar was more acceptable rather than commercially available fermented vinegar. Practical implication -At industrial level good quality vinegar can be produced by using pure culture of Acetobacter aceti for acetic acid fermentation. Originality/value -The research carried out is one of an original type as no work has been done in Pakistan previously. Further, for the accuracy of the results, all the practices were carried out in triplicates.

The current study aimed to compare lindane degradation by pure and mixed cultures of Streptomyces sp. Cell-free extracts were assayed for potentiating dechlorinase activity and, based on these results, consortia of two to six microorganisms were assayed for their growth on and degradation of lindane. Furthermore, the role of bacterial consortia of lindane-degrading strains was examined in lindane decontamination soil assays. Four actinobacteria, previously isolated from a pesticide-contaminated area, were selected because of their tolerance to lindane and their ability to use the pesticide as sole carbon source. These strains as well as Streptomyces sp. M7 and Streptomyces coelicolor A3 were used to study specific dechlorinase activity (SDA) and lindane removal in mixed cultures. Pure cultures presented SDA in the presence of 1.66 mg L -1 lindane as carbon source. SDA was improved by certain mixed cultures until 12 times compared with pure cultures. Mixed cultures with two, three, and four strains showed maximum lindane removal of 46% to 68%, whereas combinations of five and six strains did not efficiently remove the pesticide from the culture medium. The Streptomyces sp. A2, A5, M7, and A11 consortium presented the lowest ratio between residual lindane concentration and SDA and could be a promising tool for lindane biodegradation.

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10 6 viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45°C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO 2 atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37°C for 72 h.

A typical munkoyo beverage was made by fermenting Rhynchosia heterophylla root extract-cooked maize meal mixture with Lactobacillus confusus LZ1 and Saecharomyces cerevisiae YZ20. The fermented munkoyo beverage had a pH of 3.3, lactic acid content of 60 mmol/l, ethanol 320-410 mmol/1 and a characteristic 'munkoyo' aroma. L. confusus, used alone, produced a beverage with a faint munkoyo flavour note whilst beverage produced with S. cerevisiae alone seemed not to have a typical munkoyo note. R. heterophylla root extract converted 75% of the starch in sterile cooked maize meal to maltose (80% of total sugars), maltotriose (17%) and glucose (3%) in 1 h at 45°C. During fermentation by the mixed culture or the yeast alone most of the maltose was utilised but little or none of the maltotriose. The ratio of yeast to lactic acid bacteria in the starter culture affected the rate of production of ethanol but had no effect on the growth or acid production by the bacterium. To obtain a munkoyo beverage with the desired low alcohol concentration ( < 100 mmol/1), the ratio of yeast concentration (cfu/ml) to Lactobacillus concentration in the starter culture should be 1:1000 or less and the beverage should be fermented for 24 h only.

Lactobacillus delbrueckii subsp. lactis R0187, Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011 and Bifidobacterium longum R0175 were examined for their ability to grow in combination with Streptococcus thermophilus cultures in milk and a laboratory soy beverage (LSB; both standardized to 4.5% protein and 2.3% fat). Strains R0011 and R0187 did not rapidly acidify the soy beverage despite good growth rates on soy carbohydrates. The S. thermophilus populations in the LSB were similar to that of milk even though milk had 30% more buffering capacity. In milk but not in soy, symbiosis with respect to acidification rate was observed between S. thermophilus and L. helveticus or B. longum. The populations of L. helveticus in the fermented products were similar in pure cultures or in the presence of the streptococci. However B. longum did not compete well in the mixed culture. Fermentation conditions varied as a function of the ability of S. thermophilus strains to acidify media to a pH of 4.65 (between 8 and 24 h). The probiotic populations in the mixed culture were influenced by the S. thermophilus strain and by the time of fermentation. Variations in growth rates of the bacteria did not appear to be linked to differences in initial redox or a-amino nitrogen levels. Strain selection enabled the preparation of a mixed starter, probiotic-fermented soy beverage containing 1.1 Â 10 8 CFU/mL of L. helveticus R0052, which represented approximately 13% of the total final population.

Postharvest diseases cause considerable losses to harvested fruits and vegetables during transportation and storage. Synthetic fungicides are primarily used to control postharvest decay loss. However, the recent trend is shifting toward safer and more eco-friendly alternatives for the control of postharvest decays. Of various biological approaches, the use of antagonistic microorganisms is becoming popular throughout the world. Several postharvest diseases can now be controlled by microbial antagonists. Although the mechanism(s) by which microbial antagonists suppress the postharvest diseases is still unknown, competition for nutrients and space is most widely accepted mechanism of their action. In addition, production of antibiotics, direct parasitism, and possibly induced resistance in the harvested commodity are other modes of their actions by which they suppress the activity of postharvest pathogens in fruits and vegetables. Microbial antagonists are applied either before or after harvest, but postharvest applications are more effective than preharvest applications. Mixed cultures of the microbial antagonists appear to provide better control of postharvest diseases over individual cultures or strains. Similarly, the efficacy of the microbial antagonist(s) can be enhanced if they are used with low doses of fungicides, salt additives, and physical treatments like hot water dips, irradiation with ultraviolet light etc. At the international level, different microbial antagonists like Debaryomyces hansenii Lodder & Krejer-van Rij, Cryptococcus laurentii Kufferath & Skinner, Bacillus subtilis (Ehrenberg) Cohn, and Trichoderma harzianum Rifai, are being used. Biocontrol products like Aspire, BioSave, and Shemer etc., have also been developed and registered. Although the results of this technology are encouraging, we need to continue to explore potential uses on the commercial scale in different corners of the world.

In this study the degree of bacterial attachment of the main microorganisms involved in the bioleaching of metal sulfides and their influence on the dissolution rate and final metal extractions were determined. Three different mineral sulfides were bioleached: chalcopyrite (CuFeS 2 ), sphalerite (ZnS) and pyrite (FeS 2 ). A mixed culture of mesophilic bacteria (Acidithiobacillus and Leptospirillum spp.) was tested at 35 • C and thermophilic bacteria (Sulfolobus spp.) were tested at 68 • C. The results confirmed that a relationship exists between attachment and mineral dissolution rates. The bioleaching process can be divided into three stages. An initial stage with extensive bacterial attachment to the pyritic phase, which is of major importance in order to obtain high dissolution rates (since attached cells liberate Fe 2+ by contact bioleaching and oxidize Fe 2+ to Fe 3+ avoiding iron accumulation at the surface). In a second stage the bacterial attachment diminishes due to the saturation of the surface by the attached cells. This limitation, together with the presence of Fe 2+ in solution, produces an increase in the concentration of free cells, which use Fe 2+ as an energy source. Finally, in a third stage, a balance between free and attached cells is reached, giving rise to a cooperative mechanism. In this case, the attached cells attack the pyrite phase of the mineral generating Fe 2+ . This in turn is oxidized by the free cells in solution, regenerating the oxidizing agent (Fe 3+ ) for the indirect bioleaching of the mineral.

The population dynamics of mixed-culture and sequential-cultures in fruit-must fermentation is a very interesting problem from both the theoretical and technological stand point. By mixed-culture we refer to those fermentations in which more that one strain/species are present from the beginning of the process; and by sequential to those in which different microorganisms are added along the process. These kinds of fermentations play a key role in industry of fermented beverages. The mathematical models of such processes should represent the dynamics of multiple species growing in a mixture of substrates as the fruit must composition includes an important proportion of hexoses and pentoses. Since the flavor is a basic aspect of beverages, and in general of all fermented foods, it is fundamental to study the population dynamics and its impact in the organoleptic properties, through the influence in the volatile compound profile. The goal of this paper is to present a panorama of the investigations of these fermentative ecosystems from a biological, mathematical, and technological stand point.

This study compares the capacity of pure and mixed cultures of mesophilic bacteria for bioleaching of a low grade, pyritic chalcopyrite concentrate. In pure culture form, Acidithiobacillus ferrooxidans was found to have a higher bioleaching capacity than Leptospirillum ferrooxidans and Acidithiobacillus thiooxidans with the capability of the latter to bioleach copper being very limited. Mixed cultures, MixA (At. ferrooxidans, L. ferrooxidans and At. thiooxidans) and MixB (L. ferrooxidans and At. thiooxidans) were shown to perform better than the pure cultures with the highest extraction of copper (62.1% Cu) being achieved by MixA. Copper bioleaching performances of the cultures were observed to agree with their respective growth pattern. The results also indicated that the increase in the pulp density (1-5% wt/vol) adversely affected bioleaching process regardless of the pure and mixed cultures used having led to the decrease in the extent of final copper extraction i.e. 50.3% Cu recovery at 1% wt/vol for At. ferrooxidans compared with 38.6% Cu at 5% wt/vol. This study underlines the importance of mixed cultures and, iron and sulphur-oxidising activity of a bacterial culture to efficiently oxidise chalcopyrite.

Postharvest diseases cause considerable losses to harvested fruits and vegetables during transportation and storage. Synthetic fungicides are primarily used to control postharvest decay loss. However, the recent trend is shifting toward safer and more eco-friendly alternatives for the control of postharvest decays. Of various biological approaches, the use of antagonistic microorganisms is becoming popular throughout the world. Several postharvest diseases can now be controlled by microbial antagonists. Although the mechanism(s) by which microbial antagonists suppress the postharvest diseases is still unknown, competition for nutrients and space is most widely accepted mechanism of their action. In addition, production of antibiotics, direct parasitism, and possibly induced resistance in the harvested commodity are other modes of their actions by which they suppress the activity of postharvest pathogens in fruits and vegetables. Microbial antagonists are applied either before or after harvest, but postharvest applications are more effective than preharvest applications. Mixed cultures of the microbial antagonists appear to provide better control of postharvest diseases over individual cultures or strains. Similarly, the efficacy of the microbial antagonist(s) can be enhanced if they are used with low doses of fungicides, salt additives, and physical treatments like hot water dips, irradiation with ultraviolet light etc. At the international level, different microbial antagonists like Debaryomyces hansenii Lodder & Krejer-van Rij, Cryptococcus laurentii Kufferath & Skinner, Bacillus subtilis (Ehrenberg) Cohn, and Trichoderma harzianum Rifai, are being used. Biocontrol products like Aspire, BioSave, and Shemer etc., have also been developed and registered. Although the results of this technology are encouraging, we need to continue to explore potential uses on the commercial scale in different corners of the world.

Production of polyhydroxyalkanoates (PHA) by mixed cultures has been widely studied in the last decade. Storage of PHA by mixed microbial cultures occurs under transient conditions of carbon or oxygen availability, known respectively as aerobic dynamic feeding and anaerobic/aerobic process. In these processes, PHA-accumulating organisms, which are quite diverse in terms of phenotype, are selected by the dynamic operating conditions imposed to the reactor. The stability of these processes during long-time operation and the similarity of the polymer physical/chemical properties to the one produced by pure cultures were demonstrated. This process could be implemented at industrial scale, providing that some technological aspects are solved. This review summarizes the relevant research carried out with mixed cultures for PHA production, with main focus on the use of wastes or industrial surplus as feedstocks. Basic concepts, regarding the metabolism and microbiology, and technological approaches, with emphasis on the kind of feedstock and reactor operating conditions for culture selection and PHA accumulation, are described. Challenges for the process optimization are also discussed.

Microbial fuel cells (MFCs) can be used to directly generate electricity from the oxidation of dissolved organic matter, but optimization of MFCs will require that we know more about the factors that can increase power output such as the type of proton exchange system which can affect the system internal resistance. Power output in a MFC containing a proton exchange membrane was compared using a pure culture (Geobacter metallireducens) or a mixed culture (wastewater inoculum). Power output with either inoculum was essentially the same, with 4071 mW/m 2 for G. metallireducens and 3871 mW/m 2 for the wastewater inoculum. We also examined power output in a MFC with a salt bridge instead of a membrane system. Power output by the salt bridge MFC (inoculated with G. metallireducens) was 2.2 mW/m 2 . The low power output was directly attributed to the higher internal resistance of the salt bridge system ð19920 AE 50 OÞ compared to that of the membrane system ð1286 AE 1 OÞ based on measurements using impedance spectroscopy. In both systems, it was observed that oxygen diffusion from the cathode chamber into the anode chamber was a factor in power generation. Nitrogen gas sparging, L-cysteine (a chemical oxygen scavenger), or suspended cells (biological oxygen scavenger) were used to limit the effects of gas diffusion into the anode chamber. Nitrogen gas sparging, for example, increased overall Coulombic efficiency (47% or 55%) compared to that obtained without gas sparging (19%). These results show that increasing power densities in MFCs will require reducing the internal resistance of the system, and that methods are needed to control the dissolved oxygen flux into the anode chamber in order to increase overall Coulombic efficiency. r

Several studies have reported the beneficial influence of non-Saccharomyces yeasts and their potential applications in the wine industry, mainly in mixed-culture fermentation with S. cerevisiae. The potential impact of 15 non-Saccharomyces strains from 7 species on 4-methyl-4-sulfanylpentan-2-one (4MSP) and 3-sulfanylhexan-1-ol (3SH) release in model medium and Sauvignon Blanc must was evaluated after partial fermentation. Whereas the impact of non-Saccharomyces on 4MSP release in both media was low, some M. pulcherrima, T. delbrueckii and K. thermotolerans strains had a high capacity to release 3SH, despite their minimal fermentation activity. As previously demonstrated for Saccharomyces yeast, this contribution is strain dependant. Taking into account their dynamic and quantitative presence during the whole process, the real impact of non-Saccharomyces yeast on 4MSP and 3SH release was evaluated using a recreated community simulating the yeast ecosystem. Our results revealed a positive impact on 3SH release in Sauvignon Blanc wines by promoting non-Saccharomyces yeast activity and delaying the growth of S. cerevisiae. Some non-Saccharomyces yeast strains are capable of making a positive contribution to volatile thiol release in wines, essentially during the prefermentation stage in winemaking, when this microbiological sub-population is dominant.

In search of the main properties of phthalocyanines participating in toxicity against cyanobacteria. Chemosphere 77(11): 1520-1525 VII Jančula, D., Bláhová, L., Karásková, M., Maršálek, B. Degradation of natural toxins by phthalocyanines -example of cyanobacterial toxin, microcystin. Accepted, Water Science and Technology VIII Jančula, D., Maršálková, E., Maršálek, B. Organic flocculants for the removal of phytoplankton biomass. Submitted, Aquaculture International IX Mikula, P., Zezulka, Š., Jančula, D., Maršálek B. Photodynamic impact of hydrogen peroxide -complex assessment of algicidal agent on Microcystis aeruginosa physiology. Submitted, Chemosphere X Jančula, D., Mikula P., Maršálek B. Effects of water treatment by polyaluminium chloride on freshwater invertebrate (Daphnia magna) -laboratory study.

A hydrogen utilizing exoelectrogenic bacterium (Geobacter sulfurreducens) was compared to both a nonhydrogen oxidizer (Geobacter metallireducens) and a mixed consortium in order to compare the hydrogen production rates and hydrogen recoveries of pure and mixed cultures in microbial electrolysis cells (MECs). At an applied voltage of 0.7 V, both G. sulfurreducens and the mixed culture generated similar current densities (ca. 160 A/m 3 ), resulting in hydrogen production rates of ca. 1.9 m 3 H 2 /m 3 /day, whereas G. metallireducens exhibited lower current densities and production rates of 110 ؎ 7 A/m 3 and 1.3 ؎ 0.1 m 3 H 2 /m 3 /day, respectively. Before methane was detected in the mixed-culture MEC, the mixed consortium achieved the highest overall energy recovery (relative to both electricity and substrate energy inputs) of 82% ؎ 8% compared to G. sulfurreducens (77% ؎ 2%) and G. metallireducens (78% ؎ 5%), due to the higher coulombic efficiency of the mixed consortium. At an applied voltage of 0.4 V, methane production increased in the mixed-culture MEC and, as a result, the hydrogen recovery decreased and the overall energy recovery dropped to 38% ؎ 16% compared to 80% ؎ 5% for G. sulfurreducens and 76% ؎ 0% for G. metallireducens. Internal hydrogen recycling was confirmed since the mixed culture generated a stable current density of 31 ؎ 0 A/m 3 when fed hydrogen gas, whereas G. sulfurreducens exhibited a steady decrease in current production. Community analysis suggested that G. sulfurreducens was predominant in the mixed-culture MEC (72% of clones) despite its relative absence in the mixed-culture inoculum obtained from a microbial fuel cell reactor (2% of clones). These results demonstrate that Geobacter species are capable of obtaining similar hydrogen production rates and energy recoveries as mixed cultures in an MEC and that high coulombic efficiencies in mixed culture MECs can be attributed in part to the recycling of hydrogen into current.

Syntrophobacter Methanogens a b s t r a c t Inocula play an important role in anaerobic reactor startup by balancing the populations of Syntrophobacter and methanogens. Such balances make syntrophic metabolism thermodynamically feasible in anaerobic digestion. In this study, the effect of inocula on the performance of dairy manure digestion was investigated by analyzing the change in volatile fatty acids (VFA), total solids (TS), volatile solids (VS), specific biogas production (SGPR), and specific methane production (SPMP) as well as scanning and transmission electron micrographs. The study was performed at four treatments. Treatment one was granular sludge (GM); treatment two was non-granular sludge (SM); treatment three was mixed culture from an anaerobic lagoon (LM); while the fourth treatment (the control denoted MM) did not receive any exogenous inocula. In addition, stirred and unstirred conditions were maintained in the reactors to determine their effect on reactor startup. Performance ranking based on the SGPR and SPMP of treatments (in descending order) was: GM, SM, LM and MM under stirred conditions. Under unstirred conditions, performance ranking (also in descending order) was: SM, GM, LM, and MM. Results of the examination of microcolonies in the granular, non-granular sludge, and dairy manure suggest that syntrophic juxtaposition of methanogens and Syntrophobacter in granular inoculum was common while it was less visible in non-granular sludge, and completely absent in dairy manure. (P.K. Pandey).

The effects of different stocking densities of freshwater prawn (Macrobrachium rosenbergii) on its growth and production in relation to the presence of small self-recruiting species mola (Amblypharyngodon mola) were investigated in modified rice fields after rice harvest at Mymensingh, Bangladesh. Keeping the stocking density of mola fixed at 20 000 ha−1 in each treatment, four densities of freshwater prawn (treatments) were maintained: 10 000, 15 000, 20 000 and 25 000 ha−1 respectively. The rice plots were limed (CaCO3) and fertilized with urea, triple super phosphate and cowdung regularly. The prawns were fed daily with commercial pellets. Water quality parameters such as temperature, dissolved oxygen, transparency, pH, total alkalinity, inorganic nitrogen (nitrate, nitrite and total ammonia), chlorophyll a and orthophosphate were determined fortnightly. Numerical analysis of plankton communities was performed monthly. All water quality parameters were found to be within the suitable range for freshwater prawn culture, except high temperature, in the peak summer months. There was a homogenous abundance of plankton communities in all treatments throughout the experimental period. Mola started to breed in the second month of the original stocking with partial harvesting after the second month and continued until the final harvest. The total production of mola ranged between 124 and 152 kg ha−1 during the 4-month culture period. The average prawn survival ranged from 49% to 57% without any significant difference among treatments. Freshwater prawn production ranged from 294 to 596 kg ha−1 with significantly higher production in the treatment where 20 000 ha−1 freshwater prawn were stocked. This treatment also resulted in a higher net profit margin (74%), indicating that stocking at a combination of 20 000 ha−1 freshwater prawn and 20 000 ha−1 mola could be the optimum proposition for prawn–mola culture in modified rain-fed rice fields after rice cultivation.

The effects of selenium-containing green tea (SGT; 1.4 mg selenium/kg) and China green tea (CGT; 0.13 mg selenium/kg) on the in vitro growth of lactobacilli and bifidobacteria were investigated using pure and mixed cultures. SGT had significantly higher phenolic contents (TPC), higher reducing activity, higher DPPH free-radical scavenging activity, and higher ferrous-ion chelating activity (P < 0.05-0.0001) than CGT. The addition of aqueous extracts from CGT to Mann-Rogosa-Sharpe (MRS) broth at 10% and 25% (v/v) resulted in small but nonsignificant (P > 0.05) increases in the numbers of Lactobacillus rhamnosus and Bifidobacterium breve over the control incubations (without tea). Addition of 10% and 25% of SGT extract resulted in a significant increase (P < 0.05-0.0001) in the number of lactobacilli and bifidobacteria recovered from batch fermentation while CGT did not increase the number of bifidobacteria. The higher prebiotic activity of SGT over CGT may be related to the higher TPC or minerals, notably selenium or a combination of these factors. To test whether selenium itself has an effect on bacterial growth, Na-selenite and Na-selenate were added alone or in combination with CGT to the MRS broth containing pure culture of L. rhamnosus. Growth of this bacterium was enhanced relative to the control incubation of MRS only. When added in combination with CGT, Na-selenate was more effective at enhancing the growth of L. rhamnosus than Na-selenite. The prebiotic effect of SGT could be largely explained by its selenium content. . Effect of sodium (Na) selenite and Na-selenate on the growth and viability of Lactobacillus rhamnosus when added alone to MRS broth (Mean ± standard error). Statistical analysis based on ANOVA, * : P < 0.05 and ** : P < 0.01 compared with the control group.

The overall program objective is to develop and evaluate environmentally benign agents or products that are effective in the prevention, inhibition, and mitigation of microbially influenced corrosion (MIC) in the internal surfaces of metallic natural gas pipelines. The goal is to develop one or more environmentally benign (a.k.a. "green") products that can be applied to maintain the structure and dependability of the natural gas infrastructure. Approach: The technical approach for this quarter included the fractionation of extracts prepared from several varieties of pepper plants, and using several solvents, by high performance liquid chromatography (HPLC). A preliminary determination of antimicrobial activities of the new extracts and fractions using a growth inhibition assay, and evaluation of the extracts' ability to inhibit biofilm formation was also performed. Results: The analysis of multiple extracts of pepper plants and fractions of extracts of pepper plants obtained by HPLC illustrated that these extracts and fractions are extremely complex mixtures of chemicals. Gas chromatography-mass spectrometry was used to identify the chemical constituents of these extracts and fractions to the greatest degree possible. Conclusions: Analysis of the chemical composition of various extracts of pepper plants has illustrated the complexity of the chemical mixtures present, and while additional work will be performed to further characterize the extracts to identify bioactive compounds the focus of efforts should now shift to an evaluation of the ability of extracts to inhibit corrosion in mixed culture biofilms, and in pure cultures of bacterial types which are known or believed to be important in corrosion.

This study assessed the optimal conditions for PHA production using saponified fatty acid, derived from sunflower oil (SO). A fed-batch reactor was used to produce PHA. A mixture of sewage and semi-treated oil palm effluent from a facultative pond was cultured for six months to obtain a steady-state condition. The culture was then transferred to the fed-batch system. SO mainly contains long-chain-fatty-acid (LCFA) with unsaturated fatty acid fractions of C 14:1 -C 18:3 , therefore the PHA production was assessed under feast-famine condition. The main purpose of supplementing saponified SO was to improve the production of PHA constituents (copolymer of hydroxyl-unit) such as hydroxybutyrates (HBs), hydroxyvalerates (HVs) and hydroxyhexanoates (HHs) in mixed cultures. Fed-batch operation under aerobic-mixed cultures increased the PHA production up to 33% of the dried cell. Although sludge submitted to aerobic condition in mixed cultures could improve the PHA production, the production rates are still low. This study found that the HBs constituent in the sludge is always higher compared to HVs and HHs. Saponified SO has high specific PHA storage rates (q p feast = 0.5 C-mol/C-mol. h) which are comparable to other vegetable oils (e.g. corn oil, soy bean oil, etc.) Abstrak: Kajian telah dijalankan bagi menilai keadaan optima penghasilan PHA menggunakan asid lemak tersaponifikasi yang diubahsuai daripada minyak bunga matahari (SO). Sebuah reaktor suapan-kelompok telah digunakan untuk menghasilkan PHA. Campuran daripada airsisa dan efluen separa olahan daripada kolam pengoksidaan dikulturkan selama enam bulan untuk menghasilkan keadaan 'steady-state'. Kultur tersebut kemudiannya dipindahkan ke sistem suapan-kelompok. SO mengandungi asid lemak rantaian panjang dalam kumpulan asid lemak tak tepu (C 14:1 -C 18:3 ), oleh itu penghasilan PHA telah diteliti semasa keadaan 'feast-famine'. Tujuan utama membekalkan SO yang tersaponifikasi adalah untuk menambah kualiti kandungan PHA (kopolimer unit hidroksil) seperti hidroksibutirat (HB), hidroksivalerat (HV) dan hidroksiheksanoat (HH) di dalam kultur campuran. Operasi suapan berkelompok menggunakan kultur campuran secara aerobik dapat meningkatkan penghasilan PHA sehingga 33% daripada Malaysian Journal of Civil Engineering 18(2) : 109-128 (2006) 110 berat sel kering. Walaupun enapcemar yang dikulturkan secara campuran dapat meningkatkan kualiti penghasilan PHA, kadarnya masih rendah. Kajian ini mendapati kandungan HB dalam enapcemar sentiasa lebih tinggi berbanding HV dan HH. SO yang tersaponifikasi mempunyai kadar simpanan spesifik PHA yang tinggi (q p feast = 0.5 C-mol/C-mol. h) dan setanding dengan minyak sayuran lain (seperti minyak jagung, minyak soya, dan sebagainya) Kata Kunci: Kultur Campuran Aerobik; Kondungan PHA; Asid Lemak Tersaponifikasi; Minyak Bunga Matahari.

The recovery on solid media of microorganisms able to utilize complex mixtures such as petroleum hydrocarbons (PHCs) as their sole source of carbon and energy can be difficult due to differing solubilities of the PHC components. The inability to get adequate amounts of these compounds to partition into the solid media during preparation, without the use of surfactants, is a problem. To deal with these problems, an easy, inexpensive, and rapid method was developed for recovery and cultivation of microorganisms that are capable of growing on PHCs and other types of volatile and relatively insoluble substrates as sole sources of carbon and energy. This method involves introduction of the substrate to the microorganisms in the vapor phase by placing the growth substrate on a filter pad in the lid of a petri dish containing a minimal agar medium and microorganisms. The plate is incubated inverted, so the substrate / filter pad is below the microorganisms; growth can occur only if the microbes can utilize the volatilized compounds. This method has been used with a variety of volatilized hydrocarbon compounds, such as benzene, naphthalene, and alpha-pinene, to cultivate and enumerate both pure and mixed cultures of microorganisms. The filter pad method may be of use in the evaluation of microorganisms for their application to biofiltration applications. © 1997 Elsevier Science B. V.

Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45°C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron-and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.

The aim of the study was to investigate petroleum waste remediation and toxicity reduction by five bacterial strains: Ralstonia picketti SRS (BP-20), Alcaligenes piechaudii SRS (CZOR L-1B), Bacillus subtilis (I 0 -1a), Bacillus sp. (T-1), and Bacillus sp. (T 0 -1), previously isolated from petroleum-contaminated soils. Petroleum hydrocarbons were significantly degraded (91%) by the mixed bacterial cultures in 30 days (reaching up to 29% in the first 72 h). Similarly, the toxicity of the biodegraded petroleum waste decreased 3-fold after 30 days. This work shows the influence of bacteria on hydrocarbon degradation and associated toxicity, and its dependence on the specific microorganisms present. The ability of these mixed cultures to degrade hydrocarbons and reduce toxicity makes them candidates for environmental restoration applications at other hydrocarbon-contaminated environments.

Purpose -The purpose of this paper is to examine pure culture isolates from traditionally fermented product of African oil bean seeds (ugba) with a view to identifying the appropriate starters. Design/methodology/approach -The isolates were purified and characterized, and used singly and in combination to ferment freshly prepared oil bean seed slices for 72 h at 37 C. The pH and microbial load of fermenting beans were monitored daily, and organoleptic tests using semi-trained panelists were employed to assess the fermented products based on quality attributes of flavour, appearance, texture and overall acceptability. Findings -The isolates included Bacillus subtilis, Bacillus licheniformis and Pseudomonas fluorescens. Total viable counts ranged from 1.3 Â 10 7 -9.3 Â 10 9 cfu/g, 7.0 Â 10 6 -4.0 Â 10 9 cfu/g, and 3.0 Â 10 6 -1.5 Â 10 9 cfu/g, respectively for the three isolates; while counts for the mixed culture fermentation ranged from 7.0 Â 10 6 -1.72 Â 10 10 cfu/g. The mixed culture fermentation gave the best rated product, indicating a synergy among the isolates. The pH values increased steadily in the samples fermented by Bacillus sp. and the mixed culture organisms, showing that the production of ugba follows alkaline fermentation pattern.

A continuously stirred tank bioreactor (CSTR) was used to optimize feasible and reliable bioprocess system in order to treat hydrocarbon-rich industrial wastewaters. A successful bioremediation was developed by an efficient acclimatized microbial consortium. After an experimental period of 225 days, the process was shown to be highly efficient in decontaminating the wastewater. The performance of the bioaugmented reactor was demonstrated by the reduction of COD rates up to 95%. The residual total petroleum hydrocarbon (TPH) decreased from 320 mg TPH l −1 to 8 mg TPH l −1. Analysis using gas chromatography-mass spectrometry (GC-MS) identified 26 hydrocarbons. The use of the mixed cultures demonstrated high degradation performance for hydrocarbons range n-alkanes (C10-C35). Six microbial isolates from the CSTR were characterized and species identification was confirmed by sequencing the 16S rRNA genes. The partial 16S rRNA gene sequences demonstrated that 5 strains were closely related to Aeromonas punctata (Aeromonas caviae), Bacillus cereus, Ochrobactrum intermedium, Stenotrophomonas maltophilia and Rhodococcus sp. The 6th isolate was affiliated to genera Achromobacter. Besides, the treated wastewater could be considered as non toxic according to the phytotoxicity test since the germination index of Lepidium sativum ranged between 57 and 95%. The treatment provided satisfactory results and presents a feasible technology for the treatment of hydrocarbon-rich wastewater from petrochemical industries and petroleum refineries.