Biodegradation of Maya crude oil fractions by bacterial strains and a defined mixed culture isolated from Cyperus laxus rhizosphere soil in a contaminated site

Ten bacterial strains were isolated by enrichment culture, using as carbon sources either aliphatics or an aromatic-polar mixture. Oxygen uptake rate was used as a criterion to determine culture transfer timing at each enrichment stage. Biodegradation of aliphatics (10,000 mg L(-1)) and an aromatic-polar mixture (5000 mg L(-1), 2:1) was evaluated for each of the bacterial strains and for a defined culture made up with a standardized mixture of the isolated strains. Degradation of total hydrocarbons (10,000 mg L(-1)) was also determined for the defined mixed culture. Five bacterial strains were able to degrade more than 50% of the aliphatic fraction. The most extensive biodegradation (74%) was obtained with strain Bs 9A, while strains Ps 2AP and UAM 10AP were able to degrade up to 15% of the aromatic-polar mixture. The defined mixed culture degraded 47% of the aliphatics and 6% of the aromatic-polar mixture. The defined mixed culture was able to degrade about 40% of the aliphatic fraction and 26% of the aromatic fraction when grown in the presence of total hydrocarbons, while these microorganisms did not consume the polar hydrocarbons fraction. The proposed strategy that combines enrichment culture together with oxygen uptake rate allowed the isolation of bacterial strains that are able to degrade specific hydrocarbons fractions at high consumption rates.     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

Biofilm-producing abilities of Salmonella strains isolated from Turkey

In the present study the biofilm-forming characteristics of 99 serotyped (DMC strains) and 41 genus level-identified (IS strains) Salmonella strains originating from Turkey were investigated. The strains were selected based on their ability to show the biofilm morphotype on Congo red agar plates. In addition, all strains were evaluated with regard to properties related to forming pellicle structures, physical differences of pellicles, any changes in the media associated with the formation of pellicles, and the presence of cellulose within the formed biofilm matrix as determined using 366 nm UV light. The Salmonella Typhimurium DMC4 strain was the best producer of biofilm grown on polystyrene microtiter plates (optical density at 595 nm: 3.418). In subsequent experiments industrial process conditions were used to investigate different morphotyped Salmonella strains’ biofilm-forming capability on stainless steel, a commonly preferred surface for the food industries, and on polystyrene surfaces. The effect of other important industrial conditions, such as temperature (5, 20, 37°C), pH (4.5, 5.5, 6.5, 7.4) and NaCl concentration (0.5, 1.5, 5.5, 10.5%) on the production of biofilm of the different morphotyped Salmonella strains (DMC4; red, dry and rough morphotyped S. Typhimurium, DMC12; brown, dry and rough morphotyped S. Infantis, DMC13; pink, dry and rough morphotyped S. subsp. Roughform) were also assessed. On the other hand, pH values exhibited variable effects on biofilm-forming features for different Salmonella strains on both polystyrene and stainless steel surfaces.     

Analysis of the bacterial strains using Biolog plates in the contaminated soil from Riyadh community

Routine manufacture, detonation and disposal of explosives in land and groundwater have resulted in complete pollution. Explosives are xenobiotic compounds, being toxic to biological systems, and their recalcitrance leads to persistence in the environment. The methods currently used for the remediation of explosive contaminated sites are expensive and can result in the formation of toxic products. The present study aimed to investigate the bacterial strains using the Biolog plates in the soil from the Riyadh community. The microbial strains were isolated using the spread plate technique and were identified using the Biolog method. In this study we have analyzed from bacterial families of soil samples, obtained from the different sites in 5 regions at Explosive Institute. Our results conclude that Biolog MicroPlates were developed for the rapid identification of bacterial isolates by sole-carbon source utilization and can be used for the identification of bacteria. Out of five communities, only four families of bacteria indicate that the microbial community lacks significant diversity in region one from the Riyadh community in Saudi Arabia. More studies are needed to be carried out in different regions to validate our results.     

Diversity ofFrankia strains isolated from a single alder stand

One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined at random. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates.     

Crude petroleum-oil biodegradation efficiency of Bacillus subtilis and Pseudomonas aeruginosa strains isolated from a petroleum-oil contaminated soil from North-East India

The efficiency of Bacillus subtilis DM-04 and Pseudomonas aeruginosa M and NM strains isolated from a petroleum contaminated soil sample from North-East India was compared for the biodegradation of crude petroleum-oil hydrocarbons in soil and shake flask study. These bacterial strains could utilize crude petroleum-oil hydrocarbons as sole source of carbon and energy. Bioaugmentation of TPH contaminated microcosm with P. aeruginosa M and NM consortia and B. subtilis strain showed a significant reduction of TPH levels in treated soil as compared to control soil at the end of experiment (120 d). P. aeruginosa strains were more efficient than B. subtilis strain in reducing the TPH content from the medium. The plate count technique indicated expressive growth and biosurfactant production by exogenously seeded bacteria in crude petroleum-oil rich soil. The results showed that B. subtilis DM-04 and P. aeruginosa M and NM strains could be effective for in situ bioremediation.     

Diversity of chlorophenol-degrading bacteria isolated from contaminated boreal groundwater

Chlorophenol-degrading bacteria from a long-term polluted groundwater aquifer were characterized. All isolates degraded 2,4,6-trichlorophenol and 2,3,4,6-tetrachlorophenol at concentrations detected in the contaminated groundwater (< 10 mg l^–1). Pentachlorophenol was degraded by three isolates when present alone. In two gram-positive isolates, 2,3,4,6-tetrachlorophenol was required as an inducer for the degradation of pentachlorophenol. The gram-positive isolates were sensitive to pentachlorophenol, with an IC₅₀ value of 5 mg/l. Isolates belonging to the Cytophaga/Flexibacter/Bacteroides phylum had IC₅₀ values of 25 and 63 mg/l. Isolates belonging to α-, β- and γ-Proteobacteria generally tolerated the highest pentachlorophenol concentrations (> 100 mg/l). Polychlorophenol-degrading capacity was found in strains of Nocardioides, Pseudomonas, Ralstonia, Flavobacterium, and Caulobacter previously not known to degrade polychlorophenols. In addition, six polychlorophenol-degrading sphingomonads were found. Received: 27 September 1998 / Accepted: 21 December 1998     

Nutrient-limited biodegradation of PAH in various soil strata at a creosote contaminated site

The effects of nutrient addition on the in situ biodegradation of polycyclic aromatic hydrocarbons in creosote contaminated soil were studied in soil columns taken from various soil strata at a wood preserving plant in Norway. Three samples were used: one from the topsoil (0–0.5 m), one from an organic rich layer (2–2.5 m) and one from the sandy aquifer (4.5–5 m). The addition of inorganic nitrogen and phosphorous stimulated the degradation of polycyclic aromatic hydrocarbons (PAHs) in the top soil and the aquifer sand. These two soils, which differed strongly in contamination levels, responded similarly to nutrient addition with the corresponding degradation of 4-ring PAHs. The ratio between available nitrogen (N) and phosphorous (P) might explain the degree of degradation observed for the 4-ring PAHs. However, the degree of degradation of 3-ring PAHs did not significantly increase after nutrient addition. An increase in the respiration rate, after nutrient addition, could only be observed in the topsoil. In the aquifer sand, 4-ring PAH degradation was not accompanied by an increase in the respiration rate or the number of heterotrophic micro-organisms. PAH degradation in the organic layer did not respond to nutrient addition. This was probably due to the low availability of the contaminants for micro-organisms, as a result of sorption to the soil organic matter. Our data illustrate the need for a better understanding of the role of nutrients in the degradation of high molecular weight hydrocarbons for the successful application of bioremediation at PAH contaminated sites.     

Exploring bacterial diversity from contaminated soil samples from river Yamuna

The Yamuna is the source of key water supply in the national capital region of India. Due to its immense importance, the pollution of Yamuna has become an imperative issue of study. Various initiatives have been taken by the Indian Government to decontaminate this river, but so far no possible outcome has been obtained. Therefore bioremediation may seem to be a promising approach. To assess the bioremediation potential of the microbes at river Yamuna, study of microbial diversity was carried out. Escherichia, Pseudomonas, Bacillus, Thermomicrobium, Azoarcus, Nitrosomonas and Shigella were the dominant genera present at the contaminated river coastal zone. The presence of Escherichia and Shigella indicated the sewage contamination in the river. On the other hand, the presence of Pseudomonas and Bacillus indicated the existence of indigenous bacterial communities capable of de-polluting the river, thus providing a promising approach to decontaminate Yamuna by natural means.     

Diversity of bacterial communities inhabiting soil and groundwater of arsenic contaminated areas in West Bengal,India

Soil and water contaminated with arsenic (As) through natural or anthropogenic inputs are commonly considered as native source of tolerant bacterial strains. The present study was successful in characterizing 12 hyper-tolerant bacteria, satisfying maximum tolerable concentration (MTC) for arsenate (As⁵⁺) ≥ 300 mM and arsenite (As³⁺) ≥ 30 mM, isolated from As affected North 24 Parganas and South 24 Parganas districts of West Bengal, India. Most of the bacteria showing higher level of tolerance to As⁵⁺ and As³⁺ were found as gram-positive and bacilli in shape. Positive responses to different biochemical tests indicated that some of these bacteria could be potent sources of various biotechnologically important enzymes. Some of the hyper-tolerant bacteria could reduce As⁵⁺ to As³⁺ while all others could oxidise As³⁺ to As⁵⁺. Phylogenetic analysis revealed that those hyper-tolerant bacterial strains were distributed among three phyla such as Actinobacteria, Firmicutes, and γ-Proteobacteria. The Firmicutes were well represented in this study with more than half of the hyper-tolerant strains corresponding to members of this group. Moreover, majority of the isolates except SR10 belonging to this phylum were affiliated to different species of the genus Bacillus and showed different tolerance capability to As³⁺ and As⁵⁺. We present the first report of the genus Paenibacillus as being involved in arsenite oxidation with hyper-tolerance property to As. Four isolates named as SDe5, SDe12, SDe13, and SDe15 belonging to genera Bacillus and Rhodococcus exhibited highest tolerance to As and therefore represented as good candidates for bioremediation processes of native polluted soil and ground water.     

Diversity of hydrocarbon-degrading Klebsiella strains isolated from hydrocarbon-contaminated estuaries

Aims:  To investigate the diversity and the catabolic capacity of oil-degrading Klebsiella strains isolated from hydrocarbon-contaminated sediments in Santos–São Vicente estuary systems in Brazil.
Methods and Results:  Klebsiella strains obtained from the estuary were characterized using 16S rRNA gene sequencing and BOX-PCR patterns, testing their catabolic capacity to degrade toluene, xylene, naphthalene and nonane, and identifying the catabolic genes present in the oil-degrading strains. Results show that Klebsiella strains were widespread in the estuary. Twenty-one isolates from the Klebsiella genus were obtained; 14 had unique BOX patterns and were further investigated. Among four distinct catabolic genes tested ( todC 1, ndoB , xylE and alkB 1), only the todC 1 gene could be amplified in two Klebsiella strains. The biodegradation assay showed that most of the strains had the ability to degrade all of the tested hydrocarbons; however, the strains displayed different efficiencies.
Conclusions:  The oil-degrading Klebsiella isolates obtained from the estuary were closely related to Klebsiella pneumoniae and Klebsiella ornithinolytica . The isolates demonstrated a substantial degree of catabolic plasticity for hydrocarbon degradation.
Significance and Impact of the Study:  The results of this study show that several strains from the Klebsiella genus are able to degrade diverse hydrocarbon compounds. These findings indicate that Klebsiella spp. can be an important part of the oil-degrading microbial community in estuarine areas exposed to sewage.     

Biological degradation of selected hydrocarbons in an old PAH/creosote contaminated soil from a gas work site

An old PAH/creosote contaminated soil (total approximately 300 microg PAH/g soil) from a former gas work site in Stockholm, Sweden, has been treated at 20 degrees C with the addition of various nutrients and inoculated with bacteria (isolated from the soil) to enhance the degradation of selected hydrocarbons. Microcosm studies showed that the soil consisted of two contaminant fractions: one available, easily degraded fraction and a strongly sorbed, recalcitrant one. The bioavailable fraction, monitored by headspace solid phase microextraction, contained aromatics with up to three rings, and these were degraded within 20 days down to non-detectable levels (ng PAH/g soil) by both the indigenous bacteria and the externally inoculated samples. The nutrient additives were: a minimal medium (Bushnell-Haas), nitrate, nitrite, potting soil (Anglamark, Sweden), sterile water and aeration with Bushnell-Haas medium. After 30 days treatment most of the sorbed fractions were still present in the soil. Stirring or mechanical mixing of the soil slurries had the greatest effect on degradation, indicating that the substances were too strongly sorbed for the microorganisms. When stirring the choice of nutrient seemed less important. For the non-stirred samples the addition of nitrate with the bacterial inoculum showed the best degradation, compared to the other non-stirred samples. At the end of the experiments, accumulations of metabolites/degradation products, such as 9H-fluorenone, 4-hydroxy-9H-fluorenone, 9,10-phenanthrenedione and 4H-cyclopenta[def]phenanthrenone were detected. The metabolite 4-hydroxy-9H-fluorenone increased by several orders of magnitude during the biological treatments. Microbial activity in the soil was measured by oxygen consumption and carbon dioxide production.     

Biodegradation of lindane by a native bacterial consortium isolated from contaminated river sediment

The aerobic biodegradation of lindane (γ-hexachlorocyclohexane) by a consortium of acclimated bacteria from sediment at a polluted site on the Suquia River, Cordoba, Argentina, is reported. The bacteria were acclimated for 30 days under aerobic conditions, using a minimal culture medium containing lindane (0.034 mM) as sole carbon source. Growth of the bacterial consortium decreased at a lindane concentration of 1.03 mM and was totally inhibited at 2.41 mM. The consortium showed initial lindane degradation rates of 4.92×10⁻³, 11.0×10⁻³ and 34.8×10⁻³ mM h⁻¹ when exposed to lindane concentrations of 0.069, 0.137 and 0.412 mM, respectively. Chloride concentration increased during aerobic biodegradation, indicating lindane mineralization. A metabolite identified as γ-2,3,4,5,6-pentachlorocyclohexene appeared during the first 24 h of biodegradation. Four different bacteria, identified as Sphingobacterium spiritivorum, Ochrobactrum anthropi, Bosea thiooxidans and Sphingomonas paucimobilis, were isolated. Pure strains of B. thiooxidans and S. paucimobilis degraded lindane after 3 days of aerobic incubation. This is the first report of lindane biodegradation by B. thiooxidans.     

Evaluation of chemical pretreatment of contaminated soil for improved PAH bioremediation

The efficiency of several chemical treatments as potential enhancers of the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil was evaluated by analyzing the mineralization of ¹⁴C-labeled phenanthrene, pyrene, and benzo(a)pyrene. The effect of nonionic surfactants with Fenton oxidation and combinations of surfactants with the Fenton oxidation was evaluated in a microtiter plate assay. The surfactants selected for the study were Tween 80, Brij 35, Tergitol NP-10, and Triton X-100. The addition of Fentons reagent significantly enhanced the mineralization of pyrene at the two concentrations studied: 2.8 M H₂O₂ with 0.1 M FeSO₄ and 0.7 M H₂O₂ with 0.025 M FeSO₄. Phenanthrene mineralization was also positively induced by the Fenton treatments. However, none of the treatments had a significant effect on benzo(a)pyrene mineralization. Surfactant additions at concentrations of 20% and 80% of the aqueous critical micelle concentration did not significantly affect the mineralization rates. When surfactant addition was combined with the Fenton oxidation, reduced mineralization rates were obtained when compared with mineralization after Fentons treatment alone. The results indicate that the addition of Fentons reagent may enhance the mineralization of PAHs in contaminated soil, whereas the addition of surfactants has no significant beneficial effect. The efficiency of the Fenton oxidation may decrease when surfactants are added simultaneously with Fentons reagent to contaminated soil.     

安徽省土壤绿僵菌分离株的分子鉴定

目的进一步验证ITS序列的系统发育分析可为绿僵菌属种的鉴定提供重要的参考依据。方法对分离自安徽土壤的13株绿僵菌菌株的内转录间隔区（ITS）片段进行PCR扩增和序列测定,采用Blast方法将测序结果在GenBank中进行同源搜索,依据邻接法构建获得与其相关菌株的ITS序列系统发育树。结果供试菌株分别位于系统发育树的3个分支上,分支I包括8个菌株和金龟子绿僵菌小孢变种,1个菌株和金龟子绿僵菌鳞鳃金龟变种形成分支III,另外4个菌株和黄绿绿僵菌棉蚜变种聚为分支X。结论结合同源比较的数据,将这8个、4个和1个绿僵菌菌株分别鉴定为金龟子绿僵菌小孢变种、黄绿绿僵菌棉蚜变种和金龟子绿僵菌鳞鳃金龟变种。     

Real-time reverse transcription PCR analysis of expression of atrazine catabolism genes in two bacterial strains isolated from soil

The level of expression of highly conserved, plasmid-borne, and widely dispersed atrazine catabolic genes (atz) was studied by RT-qPCR in two telluric atrazine-degrading microbes. RT-qPCR assays, based on the use of real-time PCR, were developed in order to quantify atzABCDEF mRNAs in Pseudomonas sp. ADP and atzABC mRNAs in Chelatobacter heintzii. atz gene expression was expressed as mRNA copy number per 10(6) 16S rRNA. In Pseudomonas sp. ADP, atz genes were basally expressed. It confirmed atrazine-degrading kinetics indicating that catabolic activity starts immediately after adding the herbicide. atz gene expression increased transitorily in response to atrazine treatment. This increase was only observed while low amount of atrazine remained in the medium. In C. heintzii, only atzA was basally expressed. atzA and atzB expression levels were similarly and significantly increased in response to atrazine treatment. atzC was not expressed even in the presence of high amounts of atrazine. This study showed that atz genes are basally expressed and up-regulated in response to atrazine treatment. atz gene expression patterns are different in Pseudomonas ADP and C. heintzii suggesting that the host may influence the expression of plasmid-borne atrazine-catabolic potential.     

Diversity of biosurfactant producing microorganisms isolated from soils contaminated with diesel oil

Biosurfactant production is a desirable property of hydrocarbon-degrading microorganisms (HDM). We characterized biosurfactant producing microbial populations from a Long Beach soil, California (USA) and a Hong Kong soil (China), contaminated with diesel oil. A total of 33 hydrocarbon-utilizing microorganisms were isolated from the soils. Twelve isolates and three defined consortia were tested for biosurfactant production and emulsification activity. The highest reduction of surface tension was achieved with a consortium of L1, L2 and L3 isolates from a Long Beach soil (41.4mN m(-1)). Isolate L1 (Acinetobacter junii) displayed the highest reduction of surface tension (46.5 mN m(-1)). The emulsifying capacity evaluated by the E24 emulsification index was highest in the culture of isolate L5 (74%). No substantial emulsification was achieved with the cell-free extracts, indicating that the emulsifying activity was not extracellular. Based on surface tension and the E24 index results, isolates F1, F2, F3, F4, L1, L2, L3 and L4 were identified by 16S rRNA gene sequencing as Bacillus cereus, Bacillus sphaericus, B. fusiformis, Acinetobacter junii, a non-cultured bacterium, Pseudomonas sp. and B. pumilus, respectively. Cluster analyses of 16S rRNA gene sequences of the bacterial isolates revealed 70% similarity amongst hydrocarbon-degrading bacterial community present in both soils. Five isolates (isolates F1, F2, F3, F4 and L4) belong to the Firmicutes order, two isolates (L1 and L3) belong to the Proteobacteria order and one isolate (L2) is an Actinomyces sp. Simpson's index (1 - D) and the Shannon-Weaver index (H) revealed more diversity of HDM in the Hong Kong soil, while evenness (E) and the equitability (J) data indicated that there was not a dominant population. Bacterial isolates displaying substantial potential for production of biosurfactants can be applied in the bioremediation of soils contaminated with petroleum hydrocarbons.     

Studies on strains of Microsporon gypseum isolated from soil

Summary 180 samples collected from different types of soils were examined for presence of dermatophytes.17 strains ofM. gypseum isolated from these samples were studied for their morphology, perfect stage formation and animal pathogenicity. Two isolates could be induced to produce perfect stage,Nannizia incurvata and only one isolate was pathogenic to guinea pig.     

Characterization of bacterial composition and diversity in a long-term petroleum contaminated soil and isolation of high-efficiency alkane-degrading strains using an improved medium

Bacterial community and diversity in a long-term petroleum-contaminated soil of an oilfield were characterized using 16S rRNA gene-based Illumina MiSeq high-throughput sequencing. Results indicated that Proteobacteria (49.11%) and Actinobacteria (24.24%) were the most dominant phyla, and the most abundant genera were Pseudoxanthomonas (8.47%), Luteimonas (3.64%), Alkanindiges (9.76%), Acinetobacter (5.26%) and Agromyces (8.56%) in the soil. Meanwhile a series of cultivations were carried out for isolation of alkane degraders from petroleum-contaminated soil with gellan gum and agar as gelling agents. And the isolates were classified by their 16S rRNA genes. Nine of the isolates including Enterobacter, Pseudomonas,Acinetobacter, Rhizobium, Bacillus, Sphingomonas, Paenibacillus, Variovorax and Rhodococcus showed strong biodegradability of alkane mixture (C9–C30) in a wide range of chain-length, which could be potentially applied in enhancement of bioremediation.