Isolation and characterization of a diverse group of phenylacetic acid degrading microorganisms from pristine soil

A diverse range of microorganisms capable of growth on phenylacetic acid as the sole source of carbon and energy were isolated from soil. Sixty six different isolates were identified and grouped according to 16S rRNA gene RFLP analysis. Subsequent sequencing of 16S rDNA from selected strains allowed further characterization of the phenylacetic acid degrading population isolated from soil. Nearly half (30) of the isolates are Bacillus species while the rest of the isolates are strains from a variety of genera namely, Arthrobacter, Pseudomonas, Rhodococcus, Acinetobacter, Enterobacter, Flavobacterium, and Paenibacillus. Sixty-one of the sixty-six strains reproducibly grew in defined minimal liquid culture medium (E2). All strains isolated grew when at least one hydroxylated derivative of phenylacetic acid was supplied as the carbon source, while 59 out of the 61 strains tested, accumulated ortho-hydroxyphenylacetic acid in the assay buffer; when pulsed with phenylacetic acid. Oxygen consumption experiments failed to indicate a clear link between phenylacetic acid and hydroxy-substituted phenylacetic acid in isolates from a broad range of genera.     

Oxidation of polycyclic aromatic hydrocarbons by fungal isolates from an oil contaminated refinery soil

BACKGROUND AND OBJECTIVE: Indigenous soil microorganisms are used for the biodegradation of petroleum hydrocarbons in oily waste residues from the petroleum refining industry. The objective of this investigation was to determine the potential of indigenous strains of fungi in soil contaminated with petroleum hydrocarbons to biodegrade polycyclic aromatic hydrocarbons (PAH). MATERIALS AND METHODS: Twenty one fungal strains were isolated from a soil used for land-farming of oily waste residues from the petrochemical refining industry in Singapore and identified to genus level using laboratory culture and morphological techniques. Isolates were incubated in the presence of 30 mg/L of phenanthrene over a period of 28 days at 30 degrees C. The most effective strain was further evaluated to determine its ability to oxidise a wider range of PAH compounds of various molecular weight i.e acenaphthene, fluorene, fluoranthene, chrysene, benzo(a)pyrene and dibenz(ah)anthracene RESULTS AND DISCUSSION: After 28 days of incubation, 18 of the 21 fungal cultures were capable of oxidising over 50% of the phenanthrene present in culture medium, relative to abiotic controls. Fungal isolate, Penicillium sp. 06, was able to oxidise 89% of the phenanthrene present. This isolate could also oxidise more than 75% of the acenaphthene, fluorene and fluoranthene after 30 days of incubation. However, the oxidation of high molecular weight PAH i.e. chrysene, benzo(a)pyrene and dibenz(ah)anthracene by the Penicillium sp. 06 isolate was limited, where the extent of oxidation was inversely proportional to PAH molecular weight. CONCLUSIONS: Fungal isolate, Penicillium sp. 06, was effective at oxidising a range of PAH in petroleum contaminated soils, but higher molecular weight PAH were more recalcitrant. RECOMMENDATIONS AND OUTLOOK: There is potential for the re-application of this fungal strain to soil for bioremediation purposes.     

菌源对多环芳烃降解菌的筛选及降解性能的影响

高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。分别以石油污染土壤和焦化废水活性污泥为菌源,分离出芘降解菌和混合PAHs(菲、荧蒽和芘)降解菌共14株并对其降解性能进行对比研究。结果表明,筛选得到的菌株分别属于9个菌属,其中2种菌源共有的菌属为Mycobacterium sp.、Ralstonia sp.和Shinella sp.。芘和PAHs的高效降解菌(CP16和CM32)均属于分支杆菌属(Mycobacterium),来源于焦化废水活性污泥;菌株CP16对芘(50mg/L)的7 d降解率为74.99%,CM32对PAHs(菲50 mg/L、荧蒽和芘各10 mg/L)的7 d降解率为100%。因此,以焦化废水活性污泥为菌源更有利于获得高效的多环芳烃降解菌。     

Oxidation of triphenylarsine to triphenylarsineoxide by Trichoderma harzianum and other fungi

Arsenic resistant strains of bacteria and fungi were isolated from soil contaminated by chemical warfare agents. Until now, no metabolic products of microbial attack against the phenyl residues of the model substrate triphenylarsine (TP) were found if it was incubated together with these strains in liquid culture assays. However, one of the isolated fungi, Trichoderma harzianum As 11, was found to oxidize TP to triphenylarsineoxide (TPO). The yeast Trichosporon mucoides SBUG 801 and the white-rot fungus Phanerochaete chrysosporium were also able to oxidize the As(III) in TP. In addition, P. chrysosporium transformed phenylarsineoxide (PAO) to phenylarsonic acid (PAA) under O2-atmosphere. By means of a respirometer system, the oxidation of TP by T. harzianum As 11 was confirmed by a significantly higher consumption of oxygen in the presence of these compounds. HPLC analysis of the oxidation products TPO and PAA in the medium of the assays provided evidence for the transfer reaction of As(III) to As(V) in organic bonds. The oxidation products TPO and PAA are more hydrophilic than TP and PAO. Therefore, it was concluded that particular fungi contribute to the mobilization of arsenic in soil contaminated by chemical warfare agents.     

代谢表面活性剂菌处理含油污泥的研究

试验采用异位生物修复技术堆肥法,对某炼厂油泥进行生物修复处理研究.用微生物代谢的表面活性剂对油泥进行预处理,洗脱油泥中部分油分后进行堆肥试验,投加从油田含油土壤中获得的以石油为唯一碳源、代谢高效生物表面活性剂的微生物C-2菌、F-2菌以及无机营养物和疏松剂(锯末),降解油泥中的石油污染物.经过外源微生物和内源微生物共同作用120 d,油泥中的石油烃总量由22 910 mg/kg下降到3 000 mg/kg以下.试验利用色谱-质谱联用方法分析了降解前后石油组分的变化.菌株经传统方法鉴定为蜡状芽孢杆菌、枯草芽孢杆菌.     

The fate of methoxychlor in soils and transformation by soil microorganisms

Methoxychlor was found to be sufficiently persistent in soil and its residues were present even 18 months after the soil treatment. Saprophytes, fungi and actinomyces were unaffected by varying concentrations of methoxychlor, azotobacter however was susceptable. Soil strains isolated did not utilize methoxychlor as a sole carbon source except for 9 cultures belonging to the genera Bacillus, Acinetobacter and Rhodococcus which carried out the complete dechlorination, demethylation and splitting of one of methoxychlor aromatic rings. Anaerobic conditions were more favorable for methoxychlor biodegradation by soil and pure microbial cultures.     

Microbial transformation and degradation of polychlorinated biphenyls

This paper reviews the potential of microorganisms to transform polychlorinated biphenyls (PCBs). In anaerobic environments, higher chlorinated biphenyls can undergo reductive dehalogenation. Meta- and para-chlorines in PCB congeners are more susceptible to dechlorination than ortho-chlorines. Anaerobes catalyzing PCB dechlorination have not been isolated in pure culture but there is strong evidence from enrichment cultures that some Dehalococcoides spp. and other microorganisms within the Chloroflexi phylum can grow by linking the oxidation of H(2) to the reductive dechlorination of PCBs. Lower chlorinated biphenyls can be co-metabolized aerobically. Some aerobes can also grow by utilizing PCB congeners containing only one or two chlorines as sole carbon/energy source. An example is the growth of Burkholderia cepacia by transformation of 4-chlorobiphenyl to chlorobenzoates. The latter compounds are susceptible to aerobic mineralization. Higher chlorinated biphenyls therefore are potentially fully biodegradable in a sequence of reductive dechlorination followed by aerobic mineralization of the lower chlorinated products.     

The fate of methoxychlor in soils and transformation by soil microorganisms

Abstract

Methoxychlor was found to be sufficiently persistant in soil and its residues were present even 18 months after the soil treatment. Saprophytes, fungi and actinomyces were unaffected by varying concentrations of methoxychlor, azotobacter however was susceptable. Soil strains isolated did not utilize methoxychlor as a sole carbon source except for 9 cultures belonging to the genera Bacillus, Acineto‐bacter and Rhodococcus which carried out the complete dechlorination, demethylation and splitting of one of methoxychlor aromatic rings. Anaerobic conditions were more favorable for methoxychlor biodegradation by soil and pure microbial cultures.     

Crude oil and hydrocarbon-degrading strains of Rhodococcus rhodochrous isolated from soil and marine environments in Kuwait

Soil and marine samples collected from different localities in Kuwait were screened for microorganisms capable of oil degradation. Both fungi and bacteria were isolated. The fungal flora consisted of Aspergillus terreus, A. sulphureus, Mucor globosus, Fusarium sp. and Penicillum citrinum. Mucor globosus was the most active oil degrading fungus isolated. Bacterial isolates included Bacillus spp. Enterobacteriaceae, Pseudomonas spp., Nocardia spp., Streptomyces spp.,and Rhodococcus spp. Among these Rhodococcus strains were the most efficient in oil degradation and, relatively speaking, the most abundant. Bacterial and fungal isolates differed in their ability to degrade crude oil, with Rhodococcus isolates being more active that fungin in n-alkane biodegradation, particularly in the case of R. rhodochrous. In addition to medium chain n-alkanes, fungi utilized one or more of the aromatic hydrocarbons studied, while bacteria failed to do so. R. rhodochorous KUCC 8801 was shown by GLC and post-growth studies to be more efficient in oil degradation than isolates known to be active oil degraders.     

PLA对土壤微生物生长的影响及PLA的生物降解性

从不同土壤环境中筛选出的4类不同土壤微生物91株,分别标记为细菌、固氮菌、分解纤维素菌和放线菌。然后通过将定量的聚乳酸（PLA）分别加入对应液态培养基中,恒温（30℃）摇床培养,连续测定这4类不同的土壤微生物对PLA的降解性能以及该种聚合物对各种微生物生长的影响。结果表明,虽然在自然界中PLA的降解率比较高,但不同类型的土壤微生物对PLA的降解性能却存在明显个体差异。在测试期内,放线菌对于PLA的总降解率最高;而细菌中RB-4的日降解率最高,达到3%左右;纤维分解菌HX-8及固氮菌RG-28的日降解率能达到2.4%。PLA降解产物对于细菌及纤维分解菌的抑制性普遍较强。     

The use of glyphosate as the sole source of phosphorus or carbon for the selection of soil-borne fungal strains capable to degrade this herbicide

The herbicide glyphosate was used as a selection agent for isolation of fungal strains capable to degrade phosphorus-to-carbon bond from standard sandy-clay soil. The studies have shown that the herbicide used in Martin medium as a sole source of phosphorus br carbon caused the decrease of the fungal population and substantially changed strain composition, thus selecting those which are able to degrade glyphosate.     

The bacterial metabolism of 4,4′-dichlorobiphenyl,and its suppression by alternative carbon sources

The metabolism of 4,4′-dichlorobiphenyl by mixed cultures of bacteria, isolated from activated sludge, was studied in shake cultures and in soil, both in presence and absence of alternative carbon sources. When 4,4′-dichlorobiphenyl was used as sole carbon source, 4-chlorobenzoic acid and 4,4′-dichloro-2,3-biphenyldiol could be isolated from the culture medium. Polar metabolites however, could not be detected in soil and in media in which alternative carbon sources such as glucose, glycerol, peptone, yeast-extract, humic acid or activated sludge were present. No hydroxylated or carboxylic acid derivatives could be isolated when 2,4′, 5-tri-, 2,2′,5,5′,-textra-, 2,2′,3,4,5′-penta-, 2,2′,3,4,5,5′-hexa- and decachlorobiphenyl were used as the sole carbon sources for incubation with bacteria in shake culture.     

Microbial conversion of fungicide vinclozolin

An ecological safety study of using vinclozolin in field and laboratory experiments showed that the effect of the preparation led to a decrease in the abundance of actinomycetes and mycelial fungi and an enhancement of nitrification. The residual amounts of vinclozolin in soil after 12 months were 6-12% of the dose introduced. The persistent chlorinated derivatives of the toxicant were found. Microbial strains pertaining to the genera Pseudomonas and Bacillus were isolated that utilized vinclozolin as the sole source of carbon and energy.     

Characterization of chlordecone-tolerant fungal populations isolated from long-term polluted tropical volcanic soil in the French West Indies

The insecticide chlordecone is a contaminant found in most of the banana plantations in the French West Indies. This study aims to search for fungal populations able to grow on it. An Andosol heavily contaminated with chlordecone, perfused for 1 year in a soil–charcoal system, was used to conduct enrichment cultures. A total of 103 fungal strains able to grow on chlordecone-mineral salt medium were isolated, purified, and deposited in the MIAE collection (Microorganismes d'Intérêt Agro-Environnemental, UMR Agroécologie, Institut National de la Recherche Agronomique, Dijon, France). Internal transcribed spacer sequencing revealed that all isolated strains belonged to the Ascomycota phylum and gathered in 11 genera: Metacordyceps, Cordyceps, Pochonia, Acremonium, Fusarium, Paecilomyces, Ophiocordyceps, Purpureocillium, Bionectria, Penicillium, and Aspergillus. Among predominant species, only one isolate, Fusarium oxysporum MIAE01197, was able to grow in a liquid culture medium that contained chlordecone as sole carbon source. Chlordecone increased F. oxysporum MIAE01197 growth rate, attesting for its tolerance to this organochlorine. Moreover, F. oxysporum MIAE01197 exhibited a higher EC₅₀ value than the reference strain F. oxysporum MIAE00047. This further suggests its adaptation to chlordecone tolerance up to 29.2 mg l^?1. Gas chromatography–mass spectrometry (GC-MS) analysis revealed that 40 % of chlordecone was dissipated in F. oxysporum MIAE01197 suspension culture. No chlordecone metabolite was detected by GC-MS. However, weak amount of ¹⁴CO₂ evolved from ¹⁴C₁₀-chlordecone and ¹⁴C₁₀-metabolites were observed. Sorption of ¹⁴C₁₀-chlordecone onto fungal biomass followed a linear relationship (r ²?=?0.99) suggesting that it may also account for chlordecone dissipation in F. oxysporum MIAE01197 culture.     

Interactions between glyphosate and autochthonous soil fungi surviving in aqueous solution of glyphosate

The survival of autochthonous fungi in soil treated with 1mM aqueous solution of glyphosate was investigated. Significant differences in the total number of fungi in the studied objects were observed, and additionally significant qualitative changes were encountered. The dominating group of fungi belonged to genus Fusarium: Fusarium solani H30, Fusarium solani H50 and Fusarium oxysporum H80. Interactions between the isolated strains of fungi and varying concentrations of glyphosate were determined. The studied strains possessed high tolerance against the applied doses of glyphosate (0.5-2.0 mM). In the presence of glyphosate (as a sole source of phosphorus) applied in concentrations of 1.0-1.5 mM the increase in dry mass of the tested fungi was highly significant. In the presence of glyphosate the phenotypic changes of studied strains were observed as was shown as the presence of colorants being indicators of such changes. Thus, their color and intensity depended on the age, pH and species present in the culture. The degradation of glyphosate by studied fungi was determined by means of TLC. Two types of compounds were formed. One of them (Rf=0.21-0.35) contained free amino group but was not either glycine nor AMPA. Survival of Fusarium in soil environment is potentially dangerous.     

Bacterial release of arsenic ions and organoarsenic compounds from soil contaminated by chemical warfare agents

The objective of this paper was to investigate possible participation of microorganisms in the release of soluble arsenical compounds from organoarsenic warfare agents in contaminated soil. A number of bacterial strains were isolated with high resistance against As3+ and As5+ ions which are able to degrade the water insoluble compounds triphenylarsine (TP) and triphenylarsineoxide (TPO). These strains belong to different genera of bacteria. Release of arsenic ions and soluble organoarsenic compounds from soil by the activity of autochthonic soil bacteria and a mixture of the isolated pure cultures was demonstrated by percolation experiments with undisturbed soil samples (core drills) from the contaminated site. This release increased after addition of nutrients (mineral nitrogen and phosphorus, sodium acetate and ethanol) and is nearly independent of the percolation temperature (5 degrees C and 22 degrees C). These results show that bacteria play an important role in the release of arsenical compounds from organoarsenic warfare agent contaminated soil. This release is limited by shortage of water and, above all, of nutrients for the microorganisms in the sandy forest soil. These results are important both for the management and security and possibly for bioremediation of military waste sites containing similar contaminations. Furthermore, this is the first report on bacterial degradation of organoarsenic warfare compounds.     

Enhanced degradation of fluorene in soil slurry by Absidia cylindrospora and maltosyl-cyclodextrin

This study investigates the fungal biodegradation of fluorene, a polycyclic aromatic hydrocarbon, in liquid medium and soil slurry. Fungal strains and cyclodextrins were used in order to degrade fluorene and optimize fluorene bioavailability and degradation in soil slurries. After a procedure of selection in solid and liquid media, maltosyl-cyclodextrin, a branched cyclodextrin was chosen. 47 fungal strains isolated from a contaminated site were tested for biodegradation. Results showed the greater efficiency of "adapted" fungi isolated from contaminated soil vs reference strains belonging to the collection of the laboratory. These assays allowed us to select the most efficient strain, Absidia cylindrospora, which was used in a bioaugmentation process. Bioaugmentation tests were performed in an artificially contaminated non-sterile soil. In the presence of A. cylindrospora, more than 90% of the fluorene was degraded within 288 h, while 576 h were necessary in the absence of fungal bioremediation. It also appeared that biodegradation was enhanced by amendment with previously selected maltosyl-cyclodextrin. The results of this study indicate that A. cylindrospora and maltosyl-cyclodextrin could be used successfully in fluorene bioremediation systems.     

芘高效降解菌ZQ_5的培养条件及无机元素对其降解效率的影响

以从我国最大的石油污水灌区之一——沈抚灌区污染土壤分离到的以芘为惟一碳源、能源生长的高效降解菌株ZQ5为实验材料,通过对菌株ZQ5培养条件的优化,以及采用摇瓶振荡培养方法测定菌株ZQ5对不同浓度芘的降解率,表明：菌株ZQ5在30℃振荡培养16 d后对150 mg/L芘的降解率为90.31%。通过模拟稻田施用N、P和K肥等的土壤环境,探索了无机营养元素对降解菌ZQ5降解能力的影响,发现土壤中混合加入N、P和K无机营养元素的降解率能达到82%以上,比单加某种营养元素对降解菌ZQ5的降解效果好。本研究结果可以指导稻田PAHs的原位生物修复。     

Effects of trifluralin on soil microbial populations and the nitrogen fixation activities

Effects of trifluralin on soil microbial populations and the nitrogen fixation activity of nitrogen-fixing bacteria Azotobacter chroococcum and Bradyrhizobium japonicum and the decomposition of trifluralin by soil microorganisms were studied. Trifluralin at lower concentrations from 0.5 mg microg(-1) dry soil to lower than 10.0 mg microg(-1) dry soil appeared to stimulate the growth of soil bacteria, actinomycetes, mould, and the pure cultures of Br. japonicum and A. chroococcum. Not only the colony amounts of these two species of nitrogen-fixing bacteria increased, grown on agar medium containing lower concentrations of trifluralin, but also these colonies also enlarged in size and appeared obviously in shorter formation time. However, trifluralin at higher concentrations would inhibit the development of microbial colonies both in amount and size. Trifluralin inhibited the activity of acetylene reduction of A. chroococcum when it was added at the same time of inoculation with A. chroococcum, but it showed a noteworthy stimulation to nitrogen fixation of A.chroococcum when it was put into culture after the cells of the nitrogen-fixing bacterium had grown well. The observation that soil microorganisms could use trifluralin as sole carbon and nitrogen resources for their growth, indicated that microorganisms could decompose trifluralin well.     

Effect of some technical and formulated insecticides on microbial activities in soil

Abstract

The influence of 11 formulated and technical insecticides at 10 μg/g soil on growth and activities of microorganisms was determined. The populations of bacteria and fungi initially decreased with some pesticide treatments but recovered rapidly to levels similar to or greater than those of controls after three weeks. Both formulated and technical chlordane, chlorpyrifos and cypermethrin stimulated fungal growth. No inhibition on nitrification after two wks and sulfur oxidation after three wks was observed in treatments with either grade of insecticide. The effect of different treatments on respiration was equal to or greater than that of control sample. Less effect was observed with technical insecticides than the formulated ones on microbial populations and activities in the soil.