Using population dynamics analysis by DGGE to design the bacterial consortium isolated from mangrove sediments for biodegradation of PAHs

There are many PAH-degrading bacteria in mangrove sediments and in order to explore their degradation potential, surface sediment samples were collected from a mangrove area in Fugong, Longhai, Fujian Province of China. A total of 53 strains of PAH-degrading bacteria were isolated from the mangrove sediments, consisting of 14 strains of phenanthrene (Phe), 13 strains of pyrene (Pyr), 13 strains of benzo[a]pyrene (Bap) and 13 strains of mixed PAH (Phe + Pyr + Bap)-degrading bacteria. All of the individual colonies were identified by 16S rDNA sequencing. Based on the information of bacterial PCR-DGGE profiles obtained during enrichment batch culture, Phe, Pyr, Bap and mixed PAH-degrading consortia consisted of F1, F2, F3, F4 and F15 strains, B1, B3, B6, B7 and B13 strains, P1, P2, P3, P5 and P7 strains, M1, M2, M4, M12 and M13 strains, respectively. In addition, the degradation ability of these consortia was also determined. The results showed that both Phe and mixed PAH-degrading consortia had the highest ability to degrade the Phe in a liquid medium, with more than 91% being degraded in 3 days. But the biodegradation percentages of Pyr by Pyr-degrading consortium and Bap by Bap-degrading consortium were relatively lower than that of the Phe-degrading consortium. These results suggested that a higher degradation of PAHs depended on both the bacterial consortium present and the type of PAH compound. Moreover, using the bacterial community structure analysis method, where the consortia consist of different PAH-degrading bacteria, the information from the PCR-DGGE profiles could be used in the bioremediation of PAHs in the future.     

Design of bacterial defined mixed cultures for biodegradation of specific crude oil fractions, using population dynamics analysis by DGGE

Hydrocarbon-degrading bacteria isolated from oil-polluted soils, were used to design three defined mixed cultures (DMC) for biodegradation of Maya crude oil fractions. The first degrading culture, DMC A was made up with 10 strains. Design of DMC B (six strains) and DMC C (three strains) was based on DGGE profiles obtained throughout biodegradation assays of different petroleum fractions. Biodegradation of the aliphatic fraction (10 000 mg l⁻¹) and an aromatic–polar mixture (5000 mg l⁻¹) was evaluated for the DMC B. Biodegradation of total hydrocarbons (10 000 mg l⁻¹) and its fractions was evaluated for DMC B and DMC C. During biodegradation assays, O₂ consumption and CO₂ production were assessed by respirometry, while population dynamics of predominant strains was based on PCR-DGGE profiles of partial 16S rDNA. Aliphatic fraction was completely biodegraded by DMC B, while degradation of the aromatic–polar mixture was 12.5% and for total hydrocarbons 40.5%. DMC B was able to degrade the aromatic fraction (31%) and even the polar fraction (19.6%) present in total hydrocarbons. DMC C degraded the aromatic and polar fractions (5.6% and 2%, respectively) present in total hydrocarbons. DGGE profiles of the DMCs indicated that Pseudomonas sp., Gordonia rubripertincta and a non-identified strain were predominant and probably responsible of the hydrocarbons biodegradation. The use of DGGE-fingerprinting to track microbial populations, allowed selecting strains to design efficient oil-degrading defined mixed cultures.     

Effect of surfactants on PAH biodegradation by a bacterial consortium and on the dynamics of the bacterial community during the process

The aim of this work was to evaluate the effect of a non-biodegradable (Tergitol NP-10) and a biodegradable (Tween-80) surfactant on growth, degradation rate and microbial dynamics of a polycyclic aromatic hydrocarbon (PAHs) degrading consortium (C2PL05) from a petroleum polluted soil, applying cultivable and non cultivable techniques. Growth and degradation rate were significantly lower with Tergitol NP-10 than that with Tween-80. Toxicity did not show any significant reduction with Tergitol NP-10 whereas with Tween-80 toxicity was almost depleted (30%) after 40 days. Regarding to the cultured bacteria, Pseudomonas and Stenotrophomonas groups were dominant during PAH degradation with Tergitol NP-10, whereas Enterobacter and Stenotrophomonas were dominant with Tween-80. DGGE analyses (PRIMER and MDS) showed that bacteria composition was more similar between treatments when PAHs were consumed than when PAHs concentration was still high. Community changes between treatments were a consequence of Pseudomonas sp., Sphingomonas sp., Sphingobium sp. and Agromonas sp.     

Change in bacterial community during biodegradation of aniline

The response of river water microbial communities to chemical compounds was monitored under laboratory conditions using aniline as a model. Bacteria were collected from unpolluted and polluted sites. Bacterial abundance (plate and total direct counting) and its relation to aniline biodegradation was examined. Colony hybridization with 16S rRNA oligonucleotide probes was used to study the changes in microbial community structure during biodegradation of aniline. The changes in bacterial abundance and community structure were related to biodegradation of aniline. Burkholderia–Pseudomonas (rRNA group III), an authentic Alcaligenes group became dominant despite the initial differences in the microbial communities, suggesting that these genera are the main aniline degraders in the aquatic environment.     

Selective enrichment and characterization of a phosphorus-removing bacterial consortium from activated sludge

Under alternating aerobic/anaerobic conditions and without additional carbon sources, a bacterial consortium consisting initially of 18 bacterial strains was obtained in a sequence batch reactor. The phosphorus removal capability could only be maintained using sterile filtrate of activated sludge as medium. The addition of calcium and magnesium salts, as well as vitamins and trace elements, to autoclaved sterile filtrate of activated sludge was not sufficient to achieve stable phosphorus removal. A further enrichment by subcultivation on solid, agar, freezing, and shortening of the aerobic and anaerobic phases led to a defined bacterial consortium consisting of four strains. On the basis of physiological and chemotaxonomic characterization, and partial 16S rRNA sequencing, one of the organisms was identified as Delftia acidovorans. A further isolate belonged to the Bacillus cereus group, and the third isolate was identified as Microbacterium sp.. The remaining strain seems to represent a new genus within the Flavobacteriaceae. Under continuous chemostat conditions, this consortium was able to remove up to 9.6 mg P/l phosphate in the aerobic phase and released up to 8.5 mg/l in the anaerobic phase. Up to 25 mg P-polyphosphate/g dry mass was stored under aerobic conditions.     

Decolorization and biodegradation of reactive dyes and dye wastewater by a developed bacterial consortium

A bacterial consortium (consortium GR) consisting of Proteus vulgaris NCIM-2027 and Micrococcus glutamicus NCIM-2168 could rapidly decolorize and degrade commonly-used sulfonated reactive dye Green HE4BD and many other reactive dyes. Consortium GR shows markedly higher decolorization activity than that of the individual strains. The preferable physicochemical parameters were identified to achieve higher dye degradation and decolorization efficiency. The supplementation of cheap co-substrates (e.g., extracts of agricultural wastes) could enhance the decolorization performance of consortium GR. Extent of mineralization was determined with TOC and COD measurements, showing nearly complete mineralization of Green HE4BD by consortium GR (up to 90% TOC and COD reduction) within 24 h. Oxidoreductive enzymes seemed to be involved in fast decolorization/degradation process with the evidence of enzymes induction in the bacterial consortium. Phytotoxicity and microbial toxicity studies confirm that the biodegraded products of Green HE4BD by consortium GR are non-toxic. Consortium GR also shows significant biodegradation and decolorization activities for mixture of reactive dyes as well as the effluent from actual dye manufacturing industry. This confers the possibility of applying consortium GR for the treatment of industrial wastewaters containing dye pollutants.     

Monitoring impact of in situ biostimulation treatment on groundwater bacterial community by DGGE

Changes in bacterial diversity during the field experiment on biostimulation were monitored by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments. The results revealed that the bacterial community was disturbed after the start of treatment, continued to change for 45 days or 60 days and then formed a relatively stable community different from the original community structure. DGGE analysis of soluble methane monooxygenase (sMMO) hydroxylase gene fragments, mmoX, was performed to monitor the shifts in the numerically dominant sMMO-containing methanotrophs during the field experiment. Sequence analysis on the mmoX gene fragments from the DGGE bands implied that the biostimulation treatment caused a shift of potential dominant sMMO-containing methanotrophs from type I methanotrophs to type II methanotrophs.     

Microbial community and function of enrichment cultures with methane and toluene

The interaction effect of co-existence of toluene and CH₄ on community and activity of methanotrophs and toluene-degrading bacteria was characterized in three consortia enriched with CH₄ and toluene (MT), toluene (T), and CH₄ (M), respectively, in this study. The CH₄ oxidation activity in the enrichment culture of MT was significantly lower than that of M at the end of the experiment (P?=?0.001). The toluene degradation rate could be enhanced by continuous addition of CH₄ and toluene in the initial days, but it was inhibited in the later days. Phylogenetic analysis of 16S rRNA genes showed that Proteobacteria and Bacteroidetes were dominant in the three enriched consortia, but the community of methanotrophs and toluene-degrading bacteria was significantly affected by the co-existence of CH₄ and toluene. Both Methylosinus (91.8 %) and Methylocystis (8.2 %) were detected in the enrichment culture of MT, while only Methylocystis species were detected in M. The toluene-degrading bacteria including Burkholderia, Flavobacteria, Microbacterium, and Azoarcus were all detected in the enrichment culture of T. However, only Azoarcus was found in the enrichment culture of MT. Significantly higher contents of extracellular polymeric substances polysaccharose and protein in the enrichment culture of MT than that of T and M suggested that a higher environmental stress occurred in the enrichment culture of MT.     

Anaerobic degradation of p-cresol in batch and continuous cultures by a denitrifying bacterial consortium

Summary The anaerobic degradation of p-cresol under denitrifying conditions by a bacterial consortium was studied in batch and continuous cultures. Concentrations up to 3 mm were degraded within 5–6 days with 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde and 4-hydroxybenzoate as intermediates. Steady states could be maintained at only one dilution rate, D=0.04 h^–1. A further increase in the dilution rate to 0.0 8 h^–1 resulted in culture wash-out. An estimation of the Saturation constant was made (<1 mg/l), taking the maximum specific growth rate as 0.045 h^–1, thus yielding a value of 0.125 mg p-cresol/l. Correspondence to: N. Khoury     

Effect of immobilization of a bacterial consortium on diuron dissipation and community dynamics

This work intended to study the relationship between diuron herbicide dissipation and the population dynamics of co-cultivated Delftia acidovorans WDL34 (WDL34) and Arthrobacter sp. N4 (N4) for different cell formulations: free cells or immobilization in Ca-alginate beads of one or both strains. GFP-tagged WDL34 and N4 Gram staining allowed analyzing the cell growth and distribution of each strain in both beads and culture medium in the course of the time. Compared to the free cell co-culture of WDL34 and N4, immobilization of WDL34 in Ca-alginate beads co-cultivated with free N4 increased the dissipation rate of diuron by 53% (0.141 mg ml⁻¹ h⁻¹). In that case, immobilization strongly modified the final equilibrium among both strains (highest total N4 to WDL34 ratio). Our results demonstrated that the inoculant formulation played a major role in the cell growth of each cultivated strain possibly increasing diuron dissipation. This optimized cell formulation may allow improving water and soil treatment.     

A proteomics strategy for the analysis of bacterial biodegradation pathways

Bacterial biodegradation (bioremediation) is the use of microorganisms to break down organic materials into simpler compounds; it plays a pivotal role in the clean-up of hazardous wastes in the environment. Following the completion of genome sequencing in bacteria capable of biodegradation, functional genomic studies have played a major role in obtaining information on bacterial biodegradation pathways. Novel proteomics technologies have recently been developed to make it possible to analyze global protein expression. Proteomics can also provide important information on the life cycle, regulation, and post-translational modification of proteins induced under specific conditions. Proteomics technologies have been applied to the comprehensive study of bacterial biodegradation. In this paper, we introduce the proteomics technologies applicable to bacterial biodegradation studies, review the results of the proteomics analysis of representative biodegrading bacteria, and discuss the potential use of proteomics technologies in future biodegradation studies.     

Simultaneous degradation of the pesticides methyl parathion and chlorpyrifos by an isolated bacterial consortium from a contaminated site

The simultaneous degradation of the pesticide methyl parathion and chlorpyrifos was tested using a bacterial consortium obtained by selective enrichment from highly contaminated soils in Moravia (Medellin, Colombia). Microorganisms identified in the consortium were Acinetobacter sp, Pseudomonas putida, Bacillus sp, Pseudomonas aeruginosa, Citrobacter freundii, Stenotrophomonas sp, Flavobacterium sp, Proteus vulgaris, Pseudomonas sp, Acinetobacter sp, Klebsiella sp and Proteus sp. In culture medium enriched with each of the pesticides, the consortium was able to degrade 150 mg l⁻¹ of methyl parathion and chlorpyrifos in 120 h. When a mixture of 150 mg l⁻¹ of both pesticides was used the percentage decreased to 72% for methyl parathion and 39% for chlorpyrifos. With the addition of glucose to the culture medium, the consortium simultaneously degraded 150 mg l⁻¹ of the pesticides in the mixture. 4 treatments were carried out in soil that included the addition of glucose with microorganisms, the addition of sugar cane with microorganisms, microorganisms without nutrient addition and without the addition of any item. In the treatment in which glucose was used, degradation percentages of methyl parathion and chlorpyrifos of 98% and 97% respectively were obtained in 120 h. This treatment also achieved the highest percentage of reduction in toxicity, monitored with Vibrio fischeri.     

Anaerobic biodegradation of pentachlorophenol in mixtures containing cadmium by two physiologically distinct microbial enrichment cultures

Anaerobic biodegradation of pentachlorophenol (PCP), in mixtures containing cadmium (Cd), by sulfidogenic (SRB) and methanogenic (MET) enrichment cultures, was studied. Removal of 91–93% of PCP occurred in both SRB- and MET-enriched cultures, in the absence of Cd, within 82 days. The presence of soluble Cd initially decreased the rate of PCP removal by the enrichment cultures, but PCP removal rates improved as the Cd precipitated. GC-MS, ¹⁴C-PCP, and ¹³C-PCP studies confirmed mineralization of PCP by both enrichment cultures, as well as the incorporation of PCP carbon into specific phospholipid fatty acids (PLFAs) of the cell membranes of PCP-degrading anaerobes. This is the first report on anaerobic biodegradation of PCP by SRB- and MET-enriched cultures in the presence, with simultaneous precipitation, of the toxic heavy metal Cd, and of the incorporation of PCP carbons into specific PLFAs of the anaerobic bacterial cells. Journal of Industrial Microbiology & Biotechnology (2001) 27, 11–17. Received 22 May 2000/ Accepted in revised form 17 March 2001     

应用DGGE法对青海相邻两盐湖中细菌多样性的快速检测

分别对青海相邻两盐湖柯柯盐湖、茶卡盐湖土样、泥样进行富集培养后,从中提取的DNA用两套不同的细菌通用引物进行扩增,分别得到包含V8和V9高变区的16SrDNA片断。经变性梯度凝胶电泳(DGGE)分析,结果显示这两个盐湖富集样品中细菌多样性具有较大的差异,而且同一盐湖不同性质样品中的细菌多样性差异也较大,两湖的泥样富集样品中均表现出了稍丰富的多样性。     

Bacterial community diversity assessment in municipal solid waste compost amended soil using DGGE and ARISA fingerprinting methods

Bacterial community structure and diversity of Tunisian agricultural soil treated with different amounts of municipal solid waste compost (MSWC) and other fertilizers were studied using DGGE and ARISA fingerprinting methods. Sequence analysis of dominant DGGE bands revealed the presence of three major clusters, Cytophaga/Flexibacter/Bacteroides (CFB) group, Proteobacteria and Acidobacteria group. Using ARISA profiles, dominant populations were assigned to low and high GC Gram positive bacteria, Cyanobacteria, Spirochetes and Cytophagales. The two methods revealed the absence of significant bacterial community shifts related to the different MSWC applications. Moreover, indigenous bacterial population of the used loam-clayey soil was observed to limit proliferation and survival of Proteobacteria, initially dominant in MSWC and farmyard manure. Effectiveness of the two methods for soil bacterial community studying was shown. While DGGE was more accurate for bacterial identification, ARISA was more practical for handling and rapid estimation of dominant bacteria.     

The metabolic pathway of metamifop degradation by consortium ME-1 and its bacterial community structure

Metamifop is universally used in agriculture as a post-emergence aryloxyphenoxy propionate herbicide (AOPP), however its microbial degradation mechanism remains unclear. Consortium ME-1 isolated from AOPP-contaminated soil can degrade metamifop completely after 6 days and utilize it as the carbon source for bacterial growth. Meanwhile, consortium ME-1 possessed the ability to degrade metamifop stably under a wide range of pH (6.0–10.0) or temperature (20–42 °C). HPLC–MS analysis shows that N-(2-fluorophenyl)-2-(4-hydroxyphenoxy)-N-methyl propionamide, 2-(4-hydroxyphenoxy)-propionic acid, 6-chloro-2-benzoxazolinone and N-methyl-2-fluoroaniline, were detected and identified as four intermediate metabolites. Based on the metabolites identified, a putative metabolic pathway of metamifop was proposed for the first time. In addition, the consortium ME-1 was also able to transform or degrade other AOPP such as fenoxaprop-p-ethyl, clodinafop-propargyl, quizalofop-p-ethyl and cyhalofop-butyl. Moreover, the community structure of ME-1 with lower microbial diversity compared with the initial soil sample was investigated by high throughput sequencing. β-Proteobacteria and Sphingobacteria were the largest class with sequence percentages of 46.6% and 27.55% at the class level. In addition, 50 genera were classified in consortium ME-1, of which Methylobacillus, Sphingobacterium, Bordetella and Flavobacterium were the dominant genera with sequence percentages of 25.79, 25.61, 14.68 and 9.55%, respectively.     

Characterization of the biodegradation,bioremediation and detoxification capacity of a bacterial consortium able to degrade the fungicide thiabendazole

Thiabendazole (TBZ) is a persistent fungicide used in the post-harvest treatment of fruits. Its application results in the production of contaminated effluents which should be treated before their environmental discharge. In the absence of efficient treatment methods in place, biological systems based on microbial inocula with specialized degrading capacities against TBZ could be a feasible treatment approach. Only recently the first bacterial consortium able to rapidly transform TBZ was isolated. This study aimed to characterize its biodegradation, bioremediation and detoxification potential. The capacity of the consortium to mineralize ¹⁴C-benzyl-ring labelled TBZ was initially assessed. Subsequent tests evaluated its degradation capacity under various conditions (range of pH, temperatures and TBZ concentration levels) and relevant practical scenarios (simultaneous presence of other postharvest compounds) and its bioaugmentation potential in soils contaminated with increasing TBZ levels. Finally cytotoxicity assays explored its detoxification potential. The consortium effectively mineralized the benzoyl ring of the benzimidazole moiety of TBZ and degraded spillage level concentrations of the fungicide in aqueous cultures (750 mg L^?1) and in soil (500 mg kg^?1). It maintained its high degradation capacity in a wide range of pH (4.5–7.5) and temperatures (15–37 °C) and in the presence of other pesticides (ortho-phenylphenol and diphenylamine). Toxicity assays using the human liver cancer cell line HepG2 showed a progressive decrease in cytotoxicity, concomitantly with the biodegradation of TBZ, pointing to a detoxification process. Overall, the bacterial consortium showed high potential for future implementation in bioremediation and biodepuration applications.