Advance on the production of polyhydroxyalkanoates by mixed cultures

Polyhydroxyalkanoates (PHAs)are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms.PHAs have attracted considerable attention as biodegradable substitutes for conventional polymers.Until now,however,industrial production of PHAs has encountered only limited success.The main barrier to the replacement of synthetic plastics by PHAs has been the higher cost.The use of mixed cultures and renewable sources obtained from waste organic carbon can substantially decrease the cost of PHA and increase their market potential.This work reviews two main methods of PHA production by mixed cultures,anaerobicaerobic processing and aerobic transient feeding processing,and analyzed the metabolic and effective factors.     

Advance on the production of polyhydroxyalkanoates by mixed cultures

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms. PHAs have attracted considerable attention as biodegradable substitutes for conventional polymers. Until now, however, industrial production of PHAs has encountered only limited success. The main barrier to the replacement of synthetic plastics by PHAs has been the higher cost. The use of mixed cultures and renewable sources obtained from waste organic carbon can substantially decrease the cost of PHA and increase their market potential. This work reviews two main methods of PHA production by mixed cultures, anaerobic-aerobic processing and aerobic transient feeding processing, and analyzed the metabolic and effective factors.     

Production of medium-chain-length polyhydroxyalkanoates by activated sludge enriched under periodic feeding with nonanoic acid

The potential use of activated sludge for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was investigated. The enrichment of bacterial populations capable of producing MCL-PHAs was achieved by periodic feeding with nonanoic acid in a sequencing batch reactor (SBR). Denaturing gradient gel electrophoresis analysis revealed Pseudomonas aeruginosa strains to be predominant in the bacterial community during the SBR process. The composition of PHA synthesized by the enriched biomass from nonanoic acid consisted of a large concentration (>89 mol%) of MCL monomer units and a small amount of short-chain-length monomer units. Under fed-batch fermentation with continuous feeding of nonanoic acid at a flow rate of 0.225 g/L/h and a C/N ratio of 40, a maximum PHA content of 48.6% dry cell weight and a conversion yield (Y_p/s) of 0.94 g/g were achieved. These results indicate that MCL-PHA production by activated sludge is a promising alternative to typical pure culture approaches.     

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.     

Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding

The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well adapted to each of these substrates was evaluated. Sludge fed with acetate (A), produced a homopolymer of hydroxybutyrate (HB), whereas sludge fed with propionate (P) produced a copolymer of HB and HV (hydoxyvalerate). Switching the substrate feeds, propionate to sludge A and acetate to culture P, a terpolymer of HB, HV and hydroxymethylvalerate (HMV) was obtained with culture A and a copolymer of P(HB/HV) by sludge P. Regardless of the population used, the polymer yield and productivity were much higher for acetate than for propionate. Feeding a mixture of acetate and propionate, in equal parts, to both cultures resulted in an increase of HV units produced per C mol of propionate consumed, relative to the situation where only propionate was used. The individual use of butyrate and valerate by culture A was also studied. Butyrate produced a homopolymer whereas valerate was stored as a terpolymer of P(HB/HV/HMV). The polymer yields on acetate and butyrate were higher than those on propionate and valerate. The polymer productivity was higher for acetate and propionate than for butyrate and valerate. Results showed that the polymer composition, and consequently the polymer properties, could be manipulated by varying the volatile fatty acid feed composition and/or the population.     

Effect of pH on bile salt degradation by mixed fecal cultures

Stool specimens from 3 healthy volunteers were cultured under anaerobic conditions in brain heart infusion broth with and without the addition of cholate, deoxycholate or chenodeoxycholate. The initial pH of the medium was adjusted to 5.5, 6.3, 7.3 (unadjusted), 8.0, and 9.0. Cell-free extracts prepared from the resulting bacterial growth contained increased levels of NAD- and NADP-dependent 3α-, 7α-, and 12α-hydroxysteroid oxidoreductases when the initial pH was 8.0 or 9.0 and depressed levels of these activities when the initial pH was 5.5 or 6.3 (as compared to control values obtained at 7.3). At pH 5.5 all activities except NAD-dependent 7α-hydroxysteroid oxidoreductase were absent. A powerful selective effect was imposed on NAD-dependent 7α-hydroxysteroid oxidoreductase when deoxycholate or chenodeoxycholate were incorporated into the medium. Thin-layer chromatography of ether extracts of cholate-containing, acidified spent bacterial medium showed measurable amounts of deoxycholate only when the initial culture pH was alkaline or neutral (optimal at pH 8). The percent hydroxyl group estimations at the 3α-, 7α-, and 12α-positions revealed an increase in disappearance of OH groups at all three positions with increasing initial pH value. The order of extent of bioconversion was 7α-OH > 12α-OH > 3α-OH; at pH 8 and 9, approximately 90% 7α-OH bioconversion was observed. Spent bacterial media and a number of commercial secondary bile salts were all negative in the Ames' assay for mutagenicity.     

Effect of ammonia concentration on fermentative hydrogen production by mixed cultures

The effect of ammonia concentration ranging from 0 to 10 g N/L on fermentative hydrogen production by mixed cultures was investigated in batch tests using glucose as substrate at 35 degrees C and initial pH 7.0. The experimental results showed that during the fermentative hydrogen production, the substrate degradation efficiency increased with increasing ammonia concentration from 0 to 0.01 g N/L. The hydrogen production potential, hydrogen yield and average hydrogen production rate increased with increasing ammonia concentration from 0 to 0.1g N/L. The maximum hydrogen production potential of 291.4 mL, maximum hydrogen yield of 298.8 mL/g glucose and maximum average hydrogen production rate of 8.5 mL/h were all obtained at the ammonia concentration of 0.1g N/L.     

Optimization of polyhydroxybutyrate production by mixed cultures submitted to aerobic dynamic feeding conditions

Activated sludge submitted to aerobic dynamic feeding conditions showed a good and stable capacity to store polyhydroxybutyrate (PHB). The system, working for 2 years, selected a microbial population with a high PHB storage capacity. The influence of carbon and nitrogen concentrations on the PHB accumulation yield was studied in a range of 15-180 Cmmol/l for acetate and 0-2.8 Nmmol/l for ammonia. Low ammonia concentrations favored PHB accumulation. The maximum PHB content, 67.5%, was obtained for 180 Cmmol/l of acetate supplied in one pulse. However, such high substrate concentration proved to be inhibitory for the storage mechanism, causing a slowdown of the specific PHB storage rate. In order to avoid substrate inhibition, 180 Cmmol/l of acetate was supplied in different ways: continuously fed and in three pulses of 60 Cmmol/l each. In both cases the specific PHB storage rate increased and the PHB content obtained were 56.2% and 78.5%, respectively. The latter value of PHB content is similar to that obtained by pure cultures and was never reported for mixed cultures. Addition of acetate by pulses controlled by the oxygen concentration was kept for 16 days, the PHB content being always above 70% of cell dry weight.     

Crystallisation and fractionation of selected polyhydroxyalkanoates produced from mixed cultures

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Effect of pH on growth of mixed cultures in batch reactor

This work has studied the effect of pH on specific growth rate mu, yield factor Y, and specific substrate consumption rate U for two mixed microbial populations (sludge A and B) in a batch reactor with a limiting substrate (phenol). The mathematical analysis of mu and U as a function of pH has been applied not only to the results of the present work but also to other published data. On the basis of the results obtained, the following remarks can be made: (a) The effect of pH on bacterial activity differed for the two sludges; (b) variations in pH of one unit more or less than optimum pH can give rise to appreciable variations in mu and U; and (c) with regard to the mixed populations used in this study, the mu or U and pH data for the range investigated can be suitably described by a parabolic relation.     

H2 production by mixed cultures

Production of H₂ from glucose by an anoxygenic phototrophic bacterium (Rhodobacter sphaeroides), a cyanobacterium (Synechococcus cedrorum) and a heterotrophic bacterium (Pseudomonas fluorescens) was tested individually and in mixed cultures of various combinations in light. H₂ production was maximal with a mixed culture of R. sphaeroides and P. fluorescens, which could be further enhanced by immobilization of the bacteria in alginate gel. Inhibition of H₂ photoproduction was observed in a mixture of S. cedrorum and P. fluorescens and a co-culture of all the three organisms.Ch. Sasikala and Ch. V. Ramana are and G. S. Prasad was with the Microbial Biotechnology Laboratory, Department of Botany, Osmania University, Hyderabad-500 007, India. G. S. Prasad is now with the Microbial Type Culture Collection Centre (MTCC), IMTECH, Chandigar, India.     

The effect of salinity on trichloroethylene co-metabolism by mixed cultures enriched on phenol

Summary This work examines the effects of salinity on the biodegradation of trichloroethylene (TCE) by four chemostat-cultivated cultures: LHPO-3, LHPO-6, HHPO-3 and HHPO-6, all of which had been enriched on phenol but grown under different conditions. Cultures LHPO-3 (with hydraulic retention time [HRT] of 3.1 days) and LHPO-6 (6.5-day HRT) were cultivated with fresh water, whereas cultures HHPO-3 (3.3-day HRT) and HHPO-6 (6.1-day HRT) were cultivated with seawater. Batch tests of TCE degradation by the four bacterial consortia in the absence of phenol were undertaken in solutions with salinities in the range 0–3.28% (w/v). Moreover, the effect of adding phenol on TCE degradation by LHPO-3 in 1.64% salinity solution was investigated. The results showed that the observed bacterial yields for the cultures LHPO-3, LHPO-6, HHPO-3 and HHPO-6 were 0.66, 0.47, 0.58 and 0.33 mg volatile suspended solids/mg phenol, respectively. In the absence of phenol, the extents of TCE degradation by cultures LHPO-3 and LHPO-6 increased with salinity stress, reaching 0.052 mg TCE/mg VSS for LHPO-3 and 0.033 mg TCE/mg VSS for LHPO-6, and then declined as salinity increased further. The tolerance of TCE degradation to salinity for culture LHPO-3 was around 3.28% and that for LHPO-6 was 1.64–2.33%. In the presence of phenol, the rate and extent of TCE degradation by LHPO-3 were enhanced when an optimal dosage of phenol of 10 mg phenol/mg TCE was applied. Degradation of TCE by cultures HHPO-3 and HHPO-6 was not observed.     

Effect of carbon source during enrichment on BTEX degradation by anaerobic mixed bacterial cultures

A comprehensive study on the effects of different carbon sources during the bacterial enrichment on the removal performances of benzene, toluene, ethylbenzene, and xylenes (BTEX) compounds when present as a mixture was conducted. Batch BTEX removal kinetic experiments were performed using cultures enriched with individual BTEX compounds or BTEX as a mixture or benzoate alone or benzoate–BTEX mixture. An integrated Monod-type non-linear model was developed and a ratio between maximum growth rate (μ _max) and half saturation constant (K_s) was used to fit the non-linear model. A higher μ _max/K_s indicates a higher affinity to degrade BTEX compounds. Complete removal of BTEX mixture was observed by all the enriched cultures; however, the removal rates for individual compounds varied. Degradation rate and the type of removal kinetics were found to be dependent on the type of carbon source during the enrichment. Cultures enriched on toluene and those enriched on BTEX mixture were found to have the greatest μ _max/K_s and cultures enriched on benzoate had the least μ _max/K_s. Removal performances of the cultures enriched on all different carbon sources, including the ones enriched on benzoate or benzoate–BTEX mixture were also improved during a second exposure to BTEX. A molecular analysis showed that after each exposure to the BTEX mixture, the cultures enriched on benzoate and those enriched on benzoate–BTEX mixture had increased similarities to the culture enriched on BTEX mixture.     

Recent developments in bioreactor scale production of bacterial polyhydroxyalkanoates

Bioprocess and Biosystems Engineering - Polyhydroxyalkanoates (PHAs) are biological plastics that are sustainable alternative to synthetic ones. Numerous microorganisms have been identified as PHAs...     

Response surface methodological analysis on biohydrogen production by enriched anaerobic cultures

The individual and interactive effects of pH, temperature and substrate concentration on the biohydrogen production from sucrose by mixed anaerobic cultures were investigated in this study. A central composite design and response surface methodology (RSM) were employed in planning the experiments, in order to determine the optimum conditions for biohydrogen production. Experimental results show that pH, temperature and substrate concentration all had a significant influence on specific hydrogen production potential (P_s) and the maximum hydrogen production rate (R_max) individually. Temperature and sucrose concentration, pH and temperature were interdependent or there was a significant interaction on P_s and R_max. Substrate concentration and pH were slightly interdependent, or their interactive effect on P_s and R_max was not significant. A maximum P_s of 252 mL H₂/g sucrose was estimated under the optimum conditions of pH 5.5, temperature 34.8 °C and sucrose concentration of 24.8 g/L, while a maximum R_max of 1511 mL H₂/h was calculated under the optimum conditions of pH 5.5, temperature 35.5 °C and sucrose concentration of 25.4 g/L. The experiment results show that the RSM with the central composite design was useful for optimizing the biohydrogen-producing process.     

Effect of culture medium pH on bacterial gellan production.

The effect of the initial pH of the culture medium used in the production of the exopolysaccharide gellan by the bacterium Pseudomonas species ATCC 31461, when glucose or corn syrup served as the carbon source, was investigated. With glucose as the carbon source, exopolysaccharide formation was highest after 72 h of growth when the initial pH of the culture medium was 6.8 to 7.4. Polysaccharide production by the bacterial cells grown on corn syrup for 72 h was maximal when the initial pH of the medium was 7.0 or 7.2. Cell weights of the strain after 72 h tended to be higher for the glucose-grown cells than for the corn syrup-grown cells.     

Biodegradable polymers from organic acids by using activated sludge enriched by aerobic periodic feeding

This article describes a new process for the production of biopolymers (polyhydroxyalkanoates, PHAs) based on the aerobic enrichment of activated sludge to obtain mixed cultures able to store PHAs at high rates and yields. Enrichment was obtained through the selective pressure established by feeding the carbon source in a periodic mode (feast and famine regime) in a sequencing batch reactor. A concentrated mixture of acetic, lactic, and propionic acids (overall concentration of 8.5 gCOD L(-1)) was fed every 2 h at 1 day(-1) overall dilution rate. Even at such high organic load (8.5 gCOD L(-1) day(-1)), the selective pressure due to periodic feeding was effective in obtaining a biomass with a storage ability much higher than activated sludges. The immediate biomass response to substrate excess (as determined thorough short-term batch tests) was characterized by a storage rate and yield of 649 mgPHA (as COD) g biomass (as COD)(-1) h(-1) and 0.45 mgPHA (as COD) mg removed substrates (as COD(-1)), respectively. When the substrate excess was present for more than 2 h (long-term batch tests), the storage rate and yield decreased, whereas growth rate and yield significantly increased due to biomass adaptation. A maximum polymer fraction in the biomass was therefore obtained at about 50% (on COD basis). As for the PHA composition, the copolymer poly(beta-hydroxybutyrate/beta-hydroxyvalerate) with 31% of hydroxyvalerate monomer was produced from the substrate mixture. Comparison of the tests with individual and mixed substrates seemed to indicate that, on removing the substrate mixture for copolymer production, propionic acid was fully utilized to produce propionylCoA, whereas the acetylCoA was fully provided by acetic and lactic acid.