Investigation of the biotransformation of pentachlorophenol and pulp paper mill effluent decolorisation by the bacterial strains in a mixed culture

Mixed culture of two bacterial strains Bacillus sp. and Serratia marcescens showed potential pentachlorophenol (PCP) degradation and decolorisation of pulp paper mill effluent. The physico-chemical quality of pulp paper mill effluent has been analyzed after 168 h incubation period degraded by mixed culture. The study revealed that it has decreased high load of BOD, COD, TS, TDS, TSS, sulphate, phosphate, total nitrogen, total phenols, metals and different salts (i.e. chloride, sodium, nitrate, potassium) at 168 h incubation period. PCP degradation in pulp paper mill effluent was confirmed by HPLC analysis. Mixed culture was found to degrade PCP up to (94%) present in pulp paper mill effluent with 1% glucose and 0.5% peptone (w/v) at 30 ± 1 °C, pH 8.0 ± 0.2 at 120 rpm in 168 h incubation period. The simultaneous release of chloride ion up to 1200 mg/l at 168 h emphasized the bacterial dechlorination in the medium. The pulp paper mill effluent degradation was also supported by decline in pH, AOX (absorbable organic halides), color, D.O., BOD, COD and PCP. The analysis of pulp paper mill effluent degradation products by GC–MS analysis revealed the formation of low molecular weight compound like 2-chlorophenol (RT = 3.8 min) and tetrachlorohydroquinone (RT = 11.86 min) from PCP extracted degraded sample. Further, mixed culture may be used for bioremediation of PCP containing pulp paper mill waste in the environment.     

Primary biodegradation of a series of alkyl sulfosuccinates by mixed bacterial culture

A mixed bacterial culture capable of primary biodegradation of sodium alkyl sulfosuccinates R¹-OOC−CH(SO₃Na)−CH₂−COO−R² was obtained from soil microorganisms by enrichment cultivation and adaptation in the presence of mono-n-dodecyl sulfosuccinate. Gram-negative psychrophilic bacteria with proteolytic, lipolytic and ammonifying activities were prevalent in the culture. The process of primary biodegradation of alkyl sulfosuccinates can be described by firstorder reaction kinetics. The rate constants for linear esters were ascending in the order C₄<C₅<C₆ (45 μmol/min per g cell protein) and further descending with increasing length of the carbon chain C₆>C₈>=C₁₃. Substitution of cyclohexyl for n-hexyl group resulted in fourfold decrease in biodegradation rate. Terminal branching of alkyl chain does not affect the rate of primary biodegradation.     

Simultaneous biodegradation of pyridine and quinoline by two mixed bacterial strains

Experiments were conducted to provide data on the effectiveness of bioaugmentation in the removal of pyridine and quinoline from different wastewaters. A pyridine-degrading bacterial strain (Paracoccus sp. BW001) and a quinoline-degrading strain (Pseudomonas sp. BW003) were isolated from the activated sludge of a coking wastewater treatment plant. In this study, a consortium of these two bacterial strains was used as inoculum to simultaneously degrade pyridine and quinoline in three types of wastewaters: sterile synthetic, domestic, and industrial. In addition, variation of the bacterial community structures during degradation was monitored by denaturing gradient gel electrophoresis and amplicon length heterogeneity polymerase chain reaction techniques. The results of our experiments indicate that pyridine and quinoline can be removed efficiently using this inoculum but that the degradation process results in the production of ammonium as a by-product. Also, in the two actual wastewaters investigated, we observed that several autochthonous strains of bacteria in both the domestic and industrial wastewater were tolerant of pyridine and quinoline and grew rapidly.     

Influence of chemical surfactants on the biodegradation of crude oil by a mixed bacterial culture.

The effects of surfactant physicochemical properties, such as the hydrophile-lipophile balance (HLB) and molecular structure, on the biodegradation of 2% w/v Bow River crude oil by a mixed-bacterial culture were examined. Viable counts increased 4.6-fold and total petroleum hydrocarbon (TPH) biodegradation increased 57% in the presence of Igepal CO-630, a nonylphenol ethoxylate (HLB 13, 0.625 g/L). Only the nonylphenol ethoxylate with an HLB value of 13 substantially enhanced biodegradation. The surfactants from other chemical classes with HLB values of 13 (0.625 g/L) had no effect or were inhibitory. TPH biodegradation enhancement by Igepal CO-630 occurred at concentrations above the critical micelle concentration. When the effect of surfactant on individual oil fractions was examined, the biodegradation enhancement for the saturate and aromatic fractions was the same. In all cases, biodegradation resulted in increased resin and asphaltene concentrations. Optimal surfactant concentrations for TPH biodegradation reduced resin and asphaltene formation. Chemical surfactants have the potential to improve crude oil biodegradation in complex microbial systems, and surfactant selection should consider factors such as molecular structure, HLB, and surfactant concentration.     

Total biodegradation of the oestrogenic mycotoxin zearalenone by a bacterial culture

A mixed culture of bacteria, enriched from soil collected at a coal gasification site, proved capable of removing the potent oestrogenic mycotoxin zearalenone from culture media. The bacteria grew rapidly when zearalenone was provided as the sole source of carbon and energy. HPLC and ELISA analysis of culture extracts revealed no zearalenone or zearalenone-like products. Fourteen bacterial isolates from the mixed culture were identified and purified. The ability to degrade zearalenone was lost upon purification and recombination of the bacterial members of the mixed culture. A strain of Pseudomonas fluorescens capable of degrading polychlorinated biphenyls was unable to degrade zearalenone. This is the first report of the complete degradation of zearalenone by bacteria. The present study suggests the potential of mixed cultures in the biodegradation of zearalenone.     

Cellulose degradation by a mixed bacterial culture

Summary An integrated mixed bacterial culture consisting of four strains has been isolated by a batch enrichment technique. The cellulolytic member (strain D) is aCellulomonas sp. and the others are non-cellulolytic. The interaction between strains D and C is pronounced and appears to involve an exchange of reducing sugars and growth factors. The symbiotic relationship of this naturally occurring mixed culture is therefore one of mutualism. The filter paper cellulase and carboxymethyl cellulase activities in extracellular fluid are high, while -glucosidase activity is low. The mixed culture digests a variety of lignocellulosics efficiently and is of fundamental interest in the study of microbial interrelationships.     

Anaerobic biodegradation of pentachlorophenol by a methanogenic consortium

An anaerobic consortium degrading pentachlorophenol (PCP) by methanogenic fermentation was enriched from PCP-contaminated soils. In a semi-continuous reactor, PCP biodegradation was unstable and necessitated periodic additions of unacclimated anaerobic sludge waste to restore the activity. In continuous-flow reactors, PCP degradation activity was more stable when a mixture of glucose and sodium formate was used as secondary carbon source instead of glucose. The analysis of the chlorophenol intermediates suggested that the main pathway of PCP dechlorination was PCP 2,3,5,6-tetrachlorophenol 2,3,5-trichlorophenol 3,5-dichlorophenol 3-chlorophenol phenol. In a laboratory-scale continuous-upflow fixed-film column reactor, a PCP removal of more than 99% was achieved at a PCP loading rate of 60 mol (1 reactor volume)^–1 day^–1 for a hydraulic retention time of 0.7 day. Analysis of culture samples taken at different levels in the reactor have shown that, at this PCP loading rate, only the lower part of the reactor was active. 3-chlorophenol and 3,5- and 3,4-dichlorophenol were detected at the different levels of the reactor. A study of the microorganisms in the biofilm was carried out by scanning electron microscopy and suggested that the microorganisms involved in the consortium were present as a well-structured arrangement. Methanosaeta-like microorganisms were observed mainly at the base of the biofilm whereas, at the surface, a larger diversity of morphotypes was observed in which coccoid or small rod organisms were dominant. This work shows the importance of the design and the control of the operation parameters on the efficiency of the fixed-film reactor.     

Aerobic biodegradation of vinyl chloride by a highly enriched mixed culture

Lower chlorinated compounds such as cis-dichloroethene (cis-DCE) and vinyl chloride (VC) often accumulate in chloroethene-contaminated aquifers due to incomplete reductive dechlorination of higher chlorinated compounds. A highly enriched aerobic culture that degrades VC as a growth substrate was obtained from a chloroethene-contaminated aquifer material. The culture rapidly degraded 50-250 microM aqueous VC to below GC detection limit with a first-order rate constant of 0.2 day(-1). Besides VC, the culture also degraded ethene as the sole carbon source. In addition, the culture degraded cis-DCE, but only in the presence of VC. However, no degradation of trans-DCE or TCE occurred either in the presence or absence of VC. The ability of the TRW culture to degrade cis-DCE is significant for natural attenuation since both VC and cis-DCE are often found in chloroethene-contaminated groundwater. Experiments examining the effect of oxygen threshold on VC degradation showed that the culture was able to metabolize VC efficiently at extremely low concentrations of dissolved oxygen (DO). Complete removal of 150 micromoles of VC occurred in the presence of only 0.2 mmol of oxygen (1.8 mg/L DO). This is important since most groundwater environments contain low DO (1-2 mg/L). Studies showed that the culture was able to withstand long periods of VC starvation. For example, the culture was able to assimilate VC with minimal lag time even after 5 months of starvation. This is impressive from the point of its sustenance under field conditions. Overall the culture is robust and degrades VC to below the detection limit rendering this culture suitable for field application.     

Kinetic analysis of high-concentration isopropanol biodegradation by a solvent-tolerant mixed microbial culture

The ability of a previously enriched microbial population to utilize isopropanol (IPA) as the sole carbon source within a minimal salts medium is studied. The advantage of prior enrichment procedures for the improvement of IPA biodegradation performance is demonstrated for an IPA concentration of up to 24 g L(-1). Results showing the interrelationship between temperature and substrate utilization and inhibition levels at temperatures of between 2 degrees C and 45 degrees C are examined. Models of inhibition based on enzyme kinetics are assessed via nonlinear analysis, in order to accurately represent the growth kinetics of this solvent-tolerant mixed culture. The model that best describes the data is the Levenspiel substrate inhibition model, which can predict the maximum substrate level above which growth is completely limited. This is the first report of IPA treatment of up to 24 g L(-1) by an aerobic solvent-tolerant population.     

Rapid biodegradation of calcium lignosulfonate by means of a mixed culture of microorganisms

In this paper we discuss the aerobic biodegradation of calcium libnosulfonate (CLS) in a beechwood sulfite waste liquor by means of a mixed culture of microorganisms consisting of two Trichosporn Years and bacteria in the Arthrobacter (two species), Psedomonas, and Chromobacterium genera. Under the established parameters 50% CLS was biodegraded in 24 hr accompanied by the demethylation of methoxyl groups, the splitting of sulpher–carbon bonds, and the appearance of carbonyl and carboxyl groups. The achieved results by determination of phenolic OH groups, as well as established changes of the absorption bands of IR spectra of the CLS molecule and the results of the shortening of the analyses of the C, H, O, and S, show that the degradation of the aromatic nuclei-culture biodegradation, which confirms the increase in the concentration of conjugated carbonyl groups and carboxyl groups.     

共基质对10株细菌降解芘的作用

从石油污染的污泥中分离出10株细菌(SB01—SB10),研究了有(或无)共基质(葡萄糖Glu,或菲PHE)对细菌降解芘(PYR)的影响.结果表明：当以PYR为唯一碳源和能源时(MS₁),SB01的PYR降解率最高,5 d可降解30.4%;以Glu为共代谢基质时(MS₂),SB09的PYR降解率最高,可达37.7%;以PHE为共代谢基质时(MS₃),SB10的PYR降解率为50.2%.Glu抑制SB01、SB03对PYR的降解,对SB01抑制作用最明显,使SB01的PYR降解率降低7.9%;Glu对SB02、SB07、SB08、SB10降解率无明显促进或抑制作用.PHE对细菌降解PYR均有促进作用,对SB10的促进作用最明显,使其降解率提高298%.Glu与PHE对SB04和SB09降解PYR的促进作用无显著差异,而对其它各菌株而言,PHE对PYR降解的促进作用大于Glu.     

Aerobic biodegradation of N-nitrosodimethylamine (NDMA) by axenic bacterial strains

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen whose appearance in the environment is related to the release of rocket fuel and to chlorine-based disinfection of water and wastewater. Although this compound has been shown to be biodegradable, there is minimal information about the organisms capable of this degradation, and little is understood of the mechanisms or biochemistry involved. This study shows that bacteria expressing monooxygenase enzymes functionally similar to those demonstrated to degrade NDMA in eukaryotes have the capability to degrade NDMA. Specifically, induction of the soluble methane monooxygenase (sMMO) expressed by Methylosinus trichosporium OB3b, the propane monooxygenase (PMO) enzyme of Mycobacterium vaccae JOB-5, and the toluene 4-monooxygenases found in Ralstonia pickettii PKO1 and Pseudomonas mendocina KR1 resulted in NDMA degradation by these strains. In each of these cases, brief exposure to acetylene gas, a suicide substrate for certain monooxygenases, inhibited the degradation of NDMA. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene monooxygenases found in strains PKO1 and KR1 mimicked the behavior of the parent strains. In contrast, M. trichosporium OB3b expressing the particulate form of MMO, Burkholderia cepacia G4 expressing the toluene 2-monooxygenase, and Pseudomonas putida mt-2 expressing the toluene sidechain monooxygenase were not capable of NDMA degradation. In addition, bacteria expressing aromatic dioxygenases were not capable of NDMA degradation. Finally, Rhodococcus sp. RR1 exhibited the ability to degrade NDMA by an unidentified, constitutively expressed enzyme that, unlike the confirmed monooxygenases, was not inhibited by acetylene exposure.     

Thermophilic methanogenesis from gelatin by a mixed defined bacterial culture

Summary The fermentation of gelatin by different associations of bacteria, including Thermobacteroides proteolyticus, Methanobacterium sp. and Methanosarcina MP was studied. Experimental vessels were incubated at 55°C. T. proteolyticus growing axenically produced acetate, isovalerate, H₂ and CO₂. Traces of propionate and isobutyrate were detected. Cocultures of T. proteolyticus and Methanobacterium sp. showed an increase in propionate and isobutyrate production. The Thermobacteroides-Methanosarcina association had no effect on metabolism of T. proteolyticus, and acetate was not used.In triculture, growth of Methanosarcina MP occurred on acetate in coculture with T. proteolyticus and Methanobacterium sp. Utilization of H₂ by Methanobacterium sp. in the triculture lowered the H₂ concentration sufficiently to permit acetate utilization by Methanosarcina. Maximum methane production was obtained with the triculture system.     

Thermophilic methanogenesis from pectin by a mixed defined bacterial culture

Thermophilic degradation of pectin was studied in batch cultures at 55°C by different associations of anaerobic bacteria, includingClostridium thermocellum, Methanobacterium sp., andMethanosarcina sp.Clostridium thermocellum alone produced large amounts of methanol along with some isopropanol and H₂. The inoculation ofMethanobacterium sp. in the culture did not affect the metabolism ofC. thermocellum; this demonstrates the absence of interspecies hydrogen transfer. In the presence of the methylotrophicMethanosarcina sp., methanol was reduced to methane without effect on pectin hydrolysis; a small amount of the H₂ produced was also used to reduce methanol.     

Aerobic biodegradation of trichloroethylene using a consortium of five bacterial strains

Degradation with an aerobic consortium was used to evaluate the bioremediation trichloroethylene (TCE) as a model substrate. After one week, 228-1186 mg TCE l(-1) was degraded at rates of 20-50 microg TCE l(-1) h(-1). The introduction of 10 mg toluene l(-1) enhanced the degradation rates for TCE when greater than 600 mg l(-1). Using isolated enzymes, a TCE degradation intermediate(s) appears inhibitory to the oxygenase enzymes thereby diminishing the overall degradation.     

Total biodegradation of 4-chlorobiphenyl (4 CB) by a two-membered bacterial culture

Summary Several bacterial strains that can oxidize mono- and dichlorinated biphenyls with one unsubstituted ring have already been described. The major route for this biodegradation leads ultimately to the corresponding chlorobenzoic acid, but several other minor chlorinated metabolites that might possibly be of concern for the environment have also been described previously. Since none of the bacterial strains that are able to oxidize these chlorinated biphenyls in pure culture are known to degrade chlorobenzoic acid, the oxidation of these substrates by axenic cultures always generates chlorobenzoates plus several other metabolites. In the present study, we have estimated the biodegradation of 4-chlorobiphenyl (4CB) by a two-membered bacterial culture containing one strain able to grow on 4CB and to transform it into 4-chlorobenzoate (4CBA) and one strain able to degrade 4CBA. The results were encouraging, since it was shown that the degradation of 4CB was more rapid and complete with the double bacterial culture.     

Toxicity and kinetic parameters of the aerobic biodegradation of the phenol and alkylphenols by a mixed culture

A mixed culture aerobically metabolized phenol, cresol isomers (o-,m-,p-), 2-ethylphenol and xylenol isomers (2,5-DMP and 3,4-DMP) as the sole carbon and energy source. This culture had a high tolerance towards phenol with values of maximum degradation rate (V_\max) of 47 M phenol mg^–1 protein h^–1 and inhibition substrate constant (Ki) of 10 mM. These kinetic parameters were considerably diminished and the toxicity increased with the alkylphenols. For example with 2,5-xylenol, V_\max and Ki values of 0.8 M 2,5-xylenol mg^–1 protein h^–1 and 1.3 mM, respectively, were obtained. The cresols were 5-fold more toxic than phenol, whereas 2-ethylphenol and 3,4-xylenol were 11-fold more toxic, and 2,5-xylenol was 34-fold more toxic than phenol.     

Single cell protein from bagasse pith by a mixed bacterial culture

A spontaneous association of Cellulomomas sp. with another bacterial strain was studied for its capabilities for single cell protein (SCP) production from bagasse pith. The associated strain was identified as Pseudomonas sp. and further characterized for its physiological properties. The effect of the initial proportions of both strains, the way of propagation, and the effect of pH on the growth of the mixed culture on bagasse pith was studied. Separate propagation of both strains before the fermentation step (“controlled mixed culture”), a range of proportions Cellulomonas-Pseudomonas from 4:1 to 100: 1, and pH 7.0, were found to be the most appropriate conditions of growth. A mutualistic symbiotic relationship was demonstrated to take place between both strains during the mixed growth on bagasse pith, the Cellulomonas supplying the carbon source (glucose produced from bagasse degradation) to the Pseudomonas, and the latter producing the vitamin supplements necessary for the Cellulomonas growth, allowing the growth of the mixed culture in a minimal medium, without any growth factor supplement. Fed-batch cultivation of the mixed culture on this substrate was successful, giving rise to high biomass production (19.4 g/l), thus increasing the productivity of the system. Due to its improved productivity, high biomass production, inexpensiveness of the culture medium, (without any vitamin supplement), and good stability, this culture presents economical advantages and constitutes an attractive choice for lignocellulosic substrate utilization.     

Degradation and mineralization of 3-chlorobiphenyl by a mixed aerobic bacterial culture

Summary A mixed bacterial culture obtained from polychlorinated-biphenyl-contaminated river sediments proved capable of degrading 3-chlorobiphenyl (3-CB) under aerobic laboratory conditions. Almost total mineralization of 150 mg/l of 3-CB occurred when, after 3 days of incubation, the mineral medium was supplied with benzoic acid as a carbon source. Two strains of Pseudomonas capable of degrading the substrate to 3-chlorobenzoic acid and a strain of Pseudomonas fluorescens capable of co-metabolizing this metabolite were selected from the mixed culture. A nearly stoichiometric amount of chloride, which defines the percentage of total mineralization, was eliminated during mixed culture growth. Offprint requests to: F. Fava     

Aerobic mineralization of chlorobenzoates by a natural polychlorinated biphenyl-degrading mixed bacterial culture

A natural mixed aerobic bacterial culture, designated MIXE1, was found to be capable of degrading several low-chlorinated biphenyls when 4-chlorobiphenyl was used as a co-substrate. MIXE1 was capable of using all the three monochlorobenzoate (CBA) isomers tested as well as 2,5-, 3,4- and 3,5-dichlorobenzoate (dCBA) as the sole carbon and energy source. During MIXE1 growth on these substrates, a nearly stoichiometric amount of chloride was released: 0.5 g/l of each chlorobenzoate was completely mineralized by MIXE1 after 2 or 3 days of culture incubation. Two strains, namely CPE2 and CPE3, were selected from MIXE1: CPE2, referred to the Pseudomonas genus, was found to be capable of totally degrading both 2-CBA and 2,5-dCBA, whereas Alcaligenes strain CPE3 was capable of mineralizing 3-, 4-CBA and 3,4-dCBA. Substrate uptake studies carried out with whole cells of strain CPE2 suggested that 2-CBA was metabolized through catechol, while 2,5-dCBA was degraded via 4-chlorocatechol. 3-CBA, 4-CBA, and 3,4-dCBA appeared to be degraded through 3,4-dihydroxybenzoate by the CPE3 strain. MIXE1, which is capable of degrading several chlorobenzoates, should therefore be able to mineralize a number of low-chlorinated congeners of simple and complex polychlorinated biphenyl mixtures. Correspondence to: F. Fava