Extensive biodegradation of polychlorinated biphenyls in Aroclor 1242 and electrical transformer fluid (Askarel) by natural strains of microorganisms indigenous to contaminated African systems

Evidence for substantial aerobic degradation of Aroclor 1242 and Askarel fluid by newly characterized bacterial strains belonging to the Enterobacter, Ralstonia and Pseudomonas genera is presented. The organisms exhibited degradative activity in terms of total PCB/Askarel degradation, degradation of individual congeners and diversity of congeners attacked. Maximal degradation by the various isolates of Askarel ranged from 69% to 86% whereas, Aroclor 1242, with the exception of Ralstonia sp. SA-4 (9.7%), was degraded by 37% to 91%. PCB analysis showed that at least 45 of the representative congeners in Aroclor 1242 were extensively transformed by benzoate-grown cells without the need for biphenyl as an inducer of the upper degradation pathway. In incubations with Aroclor 1242, no clear correlation was observed between percentage of congener transformed and the degree of chlorination, regardless of the presence or absence of biphenyl. Recovery of significant but nonstoichiometric amounts of chloride from the culture media showed partial dechlorination of congeners and suggested production of partial degradation products. Addition of biphenyl evidently enhanced dechlorination of the mixture by some isolates. With the exception of Ralstonia sp. SA-5, chloride released ranged from 24% to 60% in the presence of biphenyl versus 0.35% to 15% without biphenyl.     

Aerobic degradation of di- and trichlorobenzenes by two bacteria isolated from polluted tropical soils

Two polychlorinated biphenyl (PCBs)-degrading bacteria were isolated by traditional enrichment technique from electrical transformer fluid (Askarel)-contaminated soils in Lagos, Nigeria. They were classified and identified as Enterobacter sp. SA-2 and Pseudomonas sp. SA-6 on the basis of 16S rRNA gene analysis, in addition to standard cultural and biochemical techniques. The strains were able to grow extensively on dichloro- and trichlorobenzenes. Although they failed to grow on tetrachlorobenzenes, monochloro- and dichlorobenzoic acids, they were able to utilize all monochlorobiphenyls, and some dichlorobiphenyls as sole sources of carbon and energy. The effect of incubation with axenic cultures on the degradation of 0.9 mM 1,4-dichlorobenzene, 0.44 mM 1,2,3- and 0.43 mM 1,3,5-trichlorobenzene in mineral salts medium was studied. Approximately, 80-90% of these xenobiotics were degraded in 200 h, concomitant with cell increase of up to three orders of magnitude, while generation times ranged significantly (P<0.05) from 17-32 h. Catechol 1,2-dioxygenase and catechol 2,3-dioxygenase activities were detected in crude cell-free extracts of cultures pre-grown with benzoate, with the latter enzyme exhibiting a slightly higher activity (0.15-0.17 micromolmin(-1) mg of protein(-1)) with catechol, suggesting that the meta-cleavage pathway is the most readily available catabolic route in the SA strains. The wider substrate specificity of these tropical isolates may help in assessing natural detoxification processes and in designing bioremediation and bioaugmentation methods.     

An anaerobic continuous-flow fixed-bed reactor sustaining a 3-chlorobenzoate-degrading denitrifying population utilizing versatile electron donors and acceptors

An anaerobic continuous-flow fixed-bed column reactor capable of degrading 3-chlorobenzoate (3-CBA) under denitrifying conditions was established, and its rate reached 2.26 mM d(-1). The denitrifying population completely degraded 3-CBA when supplied at 0.1-0.54 mM, but its activity was partly suppressed when 3-CBA was supplied at 0.89 mM. Nitrate was concomitantly consumed throughout the operation of the reactor, the amount of which was similar to or up to 35% higher than the theoretical stoichiometric value that was calculated by assuming that 3-CBA degradation is coupled with denitrification. Batch incubation experiments proved that nitrate is strictly required for 3-CBA degradation in the absence of molecular oxygen. The population also degraded 3-CBA aerobically. Benzoate and 4-CBA were degraded under denitrifying conditions as well as 3-CBA, but 2-CBA was not. Considering that the previously reported denitrifying 3-CBA-degrading cultures do not exhibit 4-CBA degradation under denitrifying conditions, nor aerobic 3-CBA degradation [FEMS Microbiol. Lett. 144 (1996) 213, Appl. Environ. Microbiol. 66 (2000) 3446], the microbial population developed in this experiment was physiologically versatile with respect to the utilization of both electron donors and electron acceptors.     

Isolation of novel bacteria able to degrade alpha-halocarboxylic acids by enrichment from environmental samples

In order to isolate novel bacteria able to degrade alpha-halocarboxylic acids a variety of culturing strategies were implemented. Eight pure cultures were obtained and were found to be associated with the Gram negative Proteobacteria and the Gram positive Bacillus and Enterococcus genera. Furthermore, several strains were obtained which were able to degrade the DL-halocarboxylic acids anaerobically. Molecular analysis of the pure cultures led us to conclude that they may possess novel enzymes involved in the biodegradation of the alpha-halocarboxylic acids. These results are the first for nearly 40 years to describe the isolation of Gram positive isolates on an alpha-halocarboxylic acid as the sole source of carbon and energy, which also show the ability to de-toxify the test substrate by releasing chloride.     

Biodegradation of aromatic compounds by white rot and ectomycorrhizal fungal species and the accumulation of chlorinated benzoic acid in ectomycorrhizal pine seedlings

The capability of different white rot (WR, Heterobasidion annosum, Phanerochaete chrysosporium, Trametes versicolor) and ectomycorrhizal (ECM, Paxillus involutus, Suillus bovinus) fungal species to degrade different aromatic compounds and the absorption of 3-chlorobenzoic acid (3-CBA) by ECM pine seedlings was examined. The effect of aromatic compounds on the fungal biomass development varied considerably and depended on (a) the compound, (b) the external concentration, and (c) the fungal species. The highest effect on the fungal biomass development was observed for 3-CBA. Generally the tolerance of WR fungi against aromatic compounds was higher than that of the biotrophic fungal species. The capability of different fungi to degrade aromatic substances varied between the species but not generally between biotrophic and saprotrophic fungi. The highest degradation capability for aromatic compounds was detected for T. versicolor and H. annosum, whereas for Phanerochaete chrysosporium and the ECM fungi lower degradation rates were found. However, Paxillus involutus and S. bovinus showed comparable degradation rates at low concentrations of benzoic acid and 4-hydroxybenzoic acid. In contrast to liquid cultures, where no biodegradation of 3-CBA by S. bovinus was observed, mycorrhizal pines inoculated with S. bovinus showed a low capability to remove 3-CBA from soil substrates. Additional X-ray microanalytical investigations showed, that 3-CBA supplied to mycorrhizal plants was accumulated in the root cell cytoplasm and is translocated across the endodermis to the shoot of mycorrhizal pine seedlings.     

初级基质对降解菌Pseudomonasputida共代谢苯扎贝特的影响

通过对3,5-二硝基水杨酸（DNS）法测定还原糖含量各影响因素的筛选,优化确定了显色时间8min,稳定时间10min,最佳波长490nm下进行吸光度测定的检测方法;分析分别以葡萄糖和麦芽糖作为外加碳源时,降解菌Pseudo—monasputidaB-31的生长情况和共代谢降解典型药物苯扎贝特（BZF）的过程。结果表明,降解菌只有在外加碳源的条件下才可正常生长,而且其在葡萄糖环境中生长得更好;拟合得到的葡萄糖、麦芽糖和BZF代谢动力学结果显示,葡萄糖对BZF去除的促进作用更为明显,同时从葡萄糖培养基中降解菌所提取的酶比活力要高于麦芽糖培养基,分析原因可能是葡萄糖所诱导的降解菌关键酶活力更强,而且还可能会产生不同的蛋白质点位。     

华北某油田石油降解菌的筛选及降解特性

以石油为唯一碳源,从华北某油田污染土壤中筛选出石油降解菌12株。其中5株菌有较强降解能力,分别编号为z-3、z-6、z-7、z-8和z-b,在30℃,160 r/min摇床培养10 d后,菌株对石油降解率分别为63.8%、34.2%、44.8%、50.5%和42.3%。通过生理生化和分子生物学鉴定,确定这5株菌分别属于假单胞菌属（Pseudomonas）、根瘤菌属（Rhizobium）、假黄单胞菌属（Pseudoxanthomonas）、假单胞菌属（Pseudomonas）、鞘氨醇单胞菌属（Sphingomonas）。通过对这5株菌生物量、脱氢酶及邻苯二酚 2,3-双加氧酶活性的监测,发现生物量及酶活性与石油降解能力具有直接关系。最后,对5株菌的生长条件进行优化,其中z-3、z-6、z-7和z-b菌株对石油降解率较优化前提高10%左右。     

Isolation and characterization of mesotrione-degrading Bacillus sp. from soil

Dissipation kinetics of mesotrione, a new triketone herbicide, sprayed on soil from Limagne (Puy-de-Dôme, France) showed that the soil microflora were able to biotransform it.Bacteria from this soil were cultured in mineral salt solution supplemented with mesotrione as sole source of carbon for the isolation of mesotrione-degrading bacteria. The bacterial community structure of the enrichment cultures was analyzed by temporal temperature gradient gel electrophoresis (TTGE). The TTGE fingerprints revealed that mesotrione had an impact on bacterial community structure only at its highest concentrations and showed mesotrione-sensitive and mesotrione-adapted strains. Two adapted strains, identified as Bacillus sp. and Arthrobacter sp., were isolated by colony hybridization methods.Biodegradation assays showed that only the Bacillus sp. strain was able to completely and rapidly biotransform mesotrione. Among several metabolites formed, 2-amino-4-methylsulfonylbenzoic acid (AMBA) accumulated in the medium. Although sulcotrione has a chemical structure closely resembling that of mesotrione, the isolates were unable to degrade it.     

Biodegradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) by using Serratia marcescens NCIM 2919

A solvent tolerant bacterium Serratia marcescens NCIM 2919 has been evaluated for degradation of DDT (1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane). The bacterium was able to degrade up to 42% of initial 50 mg L^?1 of DDT within 10 days of incubation. The highlight of the work was the elucidation of DDT degradation pathway in S. marcescens. A total of four intermediates metabolites viz. 2,2-bis (chlorophenyl)-1,1-dichloroethane (DDD), 2,2-bis (chlorophenyl)-1,1-dichloroethylene (DDE), 2,2-bis (chlorophenyl)-1-chloroethylene (DDMU), and 4-chlorobenzoic acid (4-CBA) were identified by GC-Mass and FTIR. 4-CBA was found to be the stable product of DDT degradation. Metabolites preceding 4-CBA were not toxic to strain as reveled through luxuriant growth in presence of varying concentrations of exogenous DDD and DDE. However, 4-CBA was observed to inhibit the growth of bacterium. The DDT degrading efficiency of S. marcescens NCIM 2919 hence could be used in combination with 4-CBA utilizing strains either as binary culture or consortia for mineralization of DDT. Application of S. marcescens NCIM 2919 to DDT contaminated soil, showed 74.7% reduction of initial 12.0 mg kg^?1 of DDT after 18-days of treatment.     

Microbial and kinetic characterization of pure and mixed cultures aerobically degrading 4-nitrophenol

The molecular and kinetic characterization of a microorganism able to aerobically degrade 4-nitrophenol (4NP) is presented. The microorganism was isolated from a mixed culture operating in a laboratory-scale sequencing batch reactor with an aerobic anoxic cycle. It was identified as a member of Ralstonia genus within Betaproteobacteria. It is a gram negative coccobacillum (cell length of 2-3 microm) able to aerobically store lipid inclusions when grown aerobically on nitrophenol as the sole carbon source in the range of tested concentrations (80-320 mg l(-1)). Batch kinetic tests were performed with the pure culture, while the kinetics of the mixed biomass was directly investigated in the reactor. For pure cultures exponential growth was observed, with growth rate values in the range of 2-6 d(-1); in experiments with the mixed cultures 4NP concentrations were correlated with growth using the Haldane equation (k(max) = 0.30 mg 4NP mg(-1) VSSh(-1); K(s) = 55 mg 4NPl(-1) and K(I) = 15 mg 4NPl(-1)). Observed pure culture growth rates were higher than those of mixed cultures. This result can be explained by considering that in mixed culture the biomass is evaluated as volatile suspended solids, including both specialized biomass for 4NP removal and denitrifying bacteria.     

硝化菌的分离及特性分析

从联合脱硫脱氮填料塔中的陶粒上分离出两株硝化菌,命名为NS2和NS5,这2种菌均为革兰氏阴性菌。硝化能力测试结果表明:NS2和NS5能利用亚硝酸盐作为氮源,将亚硝酸盐转化成硝酸盐,硝化速率都达到60%以上。部分长度的16S rDNA序列同源性分析表明,NS2和NS5与Delftia sp.的同源性高达99%,并用MEGA程序对该菌株进行了系统发育进化分析。结合形态观察和生理生化鉴定,初步鉴定NS2,NS5为戴尔福特菌(Delftia sp.)。     

Biodegradation of s-triazine herbicide atrazine by Enterobacter cloacae and Burkholderia cepacia sp. from long-term treated sugarcane-cultivated soils in Kenya

In this study soils from sugarcane-cultivated fields were screened for bacterial species capable of atrazine (6-chloro-N²-ethyl-N⁴-isopropyl-1,3,5-triazine-2,4-diamine) degradation due to long exposure of the soils to this herbicide. To enrich for atrazine degraders, Minimal Salt Medium containing atrazine as the sole N source and glucose as the C source was inoculated with soils impacted with this herbicide and incubated. Bacterial growth was monitored by measuring optical density. The degradation of atrazine was followed by measuring residual atrazine in liquid cultures over a given time period by high performance liquid chromatography. Bacterial strains isolated from the enrichment cultures were characterized by biochemical tests and identified by 16S rRNA gene sequencing. Two bacterial strains coded ISL 8 and ISL 15 isolated from two different fields were shown to have 94 and 96% 16S rRNA gene sequence similarity to Burkholderia cepacia respectively. Another bacterial sp., ISL 14 was closely related to Enterobacter cloacae with a 96% 16S rRNA gene sequence similarity. There was not much difference between the extents of atrazine degradation by the enrichment cultures with communities (79–82% applied amount) from which pure strains were isolated and the pure strains themselves in liquid cultures that showed a degradation of 53–83% of applied amount. The study showed existence of bacterial strains in different sugarcane-cultivated fields which can use atrazine as a nitrogen source. The bacterial strains isolated can be used to enhance the degradation of atrazine in contaminated soils where atrazine is still considered to be recalcitrant.     

Biodegradation of s-triazine herbicide atrazine by Enterobacter cloacae and Burkholderia cepacia sp. from long-term treated sugarcane-cultivated soils in Kenya

In this study soils from sugarcane-cultivated fields were screened for bacterial species capable of atrazine (6-chloro-N2-ethyl-N?-isopropyl-1,3,5-triazine-2,4-diamine) degradation due to long exposure of the soils to this herbicide. To enrich for atrazine degraders, Minimal Salt Medium containing atrazine as the sole N source and glucose as the C source was inoculated with soils impacted with this herbicide and incubated. Bacterial growth was monitored by measuring optical density. The degradation of atrazine was followed by measuring residual atrazine in liquid cultures over a given time period by high performance liquid chromatography. Bacterial strains isolated from the enrichment cultures were characterized by biochemical tests and identified by 16S rRNA gene sequencing. Two bacterial strains coded ISL 8 and ISL 15 isolated from two different fields were shown to have 94 and 96% 16S rRNA gene sequence similarity to Burkholderia cepacia respectively. Another bacterial sp., ISL 14 was closely related to Enterobacter cloacae with a 96% 16S rRNA gene sequence similarity. There was not much difference between the extents of atrazine degradation by the enrichment cultures with communities (79-82% applied amount) from which pure strains were isolated and the pure strains themselves in liquid cultures that showed a degradation of 53-83% of applied amount. The study showed existence of bacterial strains in different sugarcane-cultivated fields which can use atrazine as a nitrogen source. The bacterial strains isolated can be used to enhance the degradation of atrazine in contaminated soils where atrazine is still considered to be recalcitrant.     

Haloalkane and haloacid dehalogenases from aerobic bacterial isolates indigenous to contaminated sites in Africa demonstrate diverse substrate specificities

Five bacteria were isolated from contaminated sites in Nigeria and South Africa using the culture enrichment technique. They were subjected to standard cultural, biochemical and microbiological techniques and identified to be species of Bacillus, Burkholderia, Corynebacterium, Micrococcus and Pseudomonas. Axenic cultures of the bacterial isolates utilized 1,2-dichloroethane (1,2-DCE) as the sole carbon source up to a final substrate concentration of 10 mM. Their mean generation time in 1,2-DCE ranged significantly (P<0.05) from 9.77 to 15.72 h with the maximum chloride release ranging between 59% and 86%. All the bacterial isolates produced two different dehalogenases, viz. one which is heat labile and specific for halogenated alkanes with optimum activity at a pH of 7.5 and the other which is more heat stable with a higher pH optimum of 9.0 and specific for halogenated alkanoic acids. However, the two enzyme types when tested demonstrated wide substrate specificities. It is therefore adjudged that these organisms may play a vital role in the bioremediation of sites polluted with chlorinated hydrocarbons.     

Characterisation and optimisation of three potential aerobic bacterial strains for kraft lignin degradation from pulp paper waste

Eight aerobic bacterial strains were isolated from pulp paper mill effluent sludge. Out of eight through nutrient enrichment technique three potential aerobic bacterial strains ITRC S(6), ITRC S(7) and ITRC S(8) were found capable to effectively degrade the kraft lignin (KL), a major byproduct of the chemical pulping process and main contributor to the colour and toxicity of effluent. Further, these potential strains (ITRC S(6), ITRC S(7) and ITRC S(8)) were biochemically characterised as Gram variable small rod, Gram negative rod and Gram positive rod respectively. Subsequently, 16S rRNA sequencing showed 95% base sequence homology and it was identified as Paenibacillus sp. (AY952466), Aneurinibacillus aneurinilyticus (AY856831), Bacillus sp. (AY952465) for ITRC S(6), IITRC S(7) and ITRC S(8), respectively. In batch decolourization experiments Bacillus sp. ITRC S(8) reduced the colour of lignin amended mineral salt medium, pH 7.6 by 65% after 6th d, at 30 degrees C, A. aneurinilyticus ITRC S(7) by 56% and Paenibacillus ITRC S(6) 43%. Under these conditions the three strains degraded the KL by 37%, 33% and 30%, respectively while the mixed culture of these three bacteria reduced colour by 69%, lignin by 40% and total substrate by 50% under same conditions. Biodegradation of the KL was not affected by low (<0.2 mg l(-1)) dissolved oxygen content; thus oxygen inhibition is more likely to be a metabolism-dependent event. Initially with 48 h incubation the decolourization was slow with decreased pH. Further incubation there was rapid decolourization with slight increase in pH at 6d compared with initial pH by increasing culture optical density. The lignin analysis from medium with HPLC indicated complete degradation rather than biotransformation with complete loss of absorbance peak at 280 nm.     

Chlorometabolite production by the ecologically important white rot fungus Bjerkandera adusta.

Two strains of the basidiomycete, Bjerkandera adusta (DAOM 215869 and BOS55) produce in static liquid culture, phenyl, veratryl, anisyl and chloroanisyl metabolites (CAM's) (alcohols, acids and aldehydes) as well as a series of compounds not previously known to be produced by Bjerkandera species: 1-phenyl, 1-anisyl, 1-(3-chloro-4-methoxy) and 1-(3,5-dichloro-4-methoxy) propan-1,2-diols, predominantly as erythro diastereomers with IR, 2S absolute configurations. 1-Anisyl-propan-1,2-diol and 1-(3,5-dichloro-4-methoxy)-propan-1,2-diol are new metabolites for which the names Bjerkanderol A and B, respectively, are proposed. Experiments with static liquid cultures supplied with 13C6- and 13C9-L-phenylalanine showed that all identified aromatic compounds (with the exception of phenol) can be derived from L-phenylalanine. For the aryl propane diols, the 13C label appeared only in the phenyl ring and the benzylic carbon, suggesting a stereoselective re-synthesis from a C7 and a C2-unit, likely aromatic aldehyde and decarboxylated pyruvate, respectively. Other compounds newly discovered to be derived from phenylalanine by this white rot fungus include phenylacetaldehyde and phenylpyruvic, phenylacetic, phenyllactic, mandelic and phenyl glyoxylic (benzoyl formic) acids. For both strains, cultures supplied with Na37Cl showed incorporation of 37Cl in all identified chlorometabolites. Veratryl alcohol and the CAM alcohols, which occur in both strains and can be derived from L-phenylalanine (all 13C-labelled), have reported important physiological functions in this white rot fungus. Possible mechanisms for their formation through the newly discovered compounds are discussed.     

2-Chlorobenzoate biodegradation by recombinant Burkholderia cepacia under hypoxic conditions in a membrane bioreactor.

The feasibility of applying bacterial hemoglobin technology to degrade 2-chlorobenzoate (2-CBA) through co-metabolism under hypoxic conditions in a membrane bioreactor (MBR) process has been studied in the laboratory. 2-chlorobenzoate removal and chloride release rates in the MBR system varied from 99 to 78% and 98 to 73%, respectively, depending on the operation conditions. Chemical oxygen demand (COD) removal efficiencies were more than 90% at food-to-microorganism ratios ranging from 0.32 to 0.62 g/g/d, and the observed yield was 0.13 to 0.20 g biomass/g COD. The bacterial cell-floc size-distribution analysis showed that there is a significant change in bacterial floc size due to high shear stress during operation of the MBR. To characterize growth kinetics of Burkholderia cepacia strain dinitrotoluene, a mathematical model that describes co-metabolic oxidation of 2-CBA in an MBR has been developed.     

巢湖低温高效聚磷菌的筛选及其特性

采用低温富集培养及混合平板分离技术,从巢湖底泥中分离筛选得到2株在低温下仍具有较高效能的菌株D3、D6,经过厌氧/好氧交替培养,2种菌株在低温下(8℃)的除磷率均达到80%以上。在低温下,研究了pH、微量元素对2株菌株生长及除磷率的影响,实验结果表明,2株菌都具有广泛的温度适应性,适宜其生长和除磷的pH为中性偏碱,微量元素的缺乏对2株菌株的生长和除磷效果有不同程度的不良影响。染色观察显示,厌氧培养时菌体内聚羟基丁酸(PHB)颗粒明显增多,转为好氧培养后异染颗粒增多,为典型的聚磷菌特征。经鉴定,2株菌均属假单胞菌属。     

Treatment of cork boiling wastewater using chemical oxidation and biodegradation

Three cultures were enriched from cork boiling wastewater using tannic acid as the selective carbon substrate, at 25 degrees C and pH 7.2, 25 degrees C and pH 4.7 and 50 degrees C and pH 4.7. The enrichment culture obtained at neutral pH was composed of five culturable isolates, whereas from each acidic enrichment two bacterial strains were isolated. Mesophilic isolates were Gram negative bacteria belonging to the genera Klebsiella, Pseudomonas, Stenotrophomonas and Burkholderia. Thermophilic isolates were members of the genus Bacillus. Despite the capability of the enrichment cultures to use tannic acid as single carbon and energy source, those cultures were unable to reduce the total polyphenols or the total organic carbon content of cork boiling wastewater. In order to increase the bioavailability of the organic carbon in cork boiling wastewater, biodegradation was preceded by Fenton oxidation. It was demonstrated that the combined process, using small amounts of Fenton reagents and biodegradative inoculum added almost simultaneously to cork boiling wastewater, leads to TOC reductions of more than 90%.     

Diversity of biphenyl degraders in a chlorobenzene polluted aquifer

Biphenyl degrading bacteria (40 strains) have been isolated along a gradient of chlorobenzene pollution from an aquifer which did not contain any PCB to answer the question of how metabolic/catabolic abilities exist in ecosystems that have not been stressed with the relevant substrates is important for intrinsic bioremediations. Only few of the isolates were characterized by 16S rRNA gene sequence analyses as Pseudomonas species while the majority were Gram-positive, belonging to the order Actinomycetales and representing the genera Rhodococcus and Arthrobacter. The strains could grow on a variety of chlorobenzoates but no pattern of substrate usage and phylogeny or pollution gradient could be found. Strains which were able to grow on 2,5-dichlorobenzoate were often also able to use 3,4- and 3,5-dichloro- and 2,3,5-trichlorobenzoate or those using 2-chlorobenzoate could usually use 2,6-dichlorobenzoate as well. From that results, it is concluded that a highly diverse, basic metabolic activity for PCB degradation existed at this site despite the absence of PCB.