Bacterial succession in a petroleum land treatment unit

Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C(10) to C(32) range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.     

Kinetic Modeling and Error Analysis for Decontamination of Different Petroleum Hydrocarbon Components in Biostimulation of Oily Soil Microcosm

A microcosm study was constructed to investigate the effect of complex co-substrate (corn steep liquor, CSL) addition on indigenous bacterial community, rate and extent of petroleum hydrocarbons (PH) degradation in an oily soil with total petroleum hydrocarbons (TPH) content of 63353 mg kg^?1. TPH degradation was found to be characterized by a rapid phase of degradation during the first three weeks where 76% removal of TPH occurred, followed by a slower degradation phase, where further 7% of the initial TPH was removed by the end of incubation period, 35 d. Branched alkanes are more resistant to microbial degradation than n-alkanes. Furthermore, the unresolved complex mixtures (UCM) of hydrocarbons are less degradable than n- and iso-alkanes. Pristane (Pr) was the most recalcitrant aliphatic compound studied in this work. These results in addition to the extensive bacterial growth observed (from 10⁷ to 10¹⁰ CFU g^?1 soil) give strong support that the addition of CSL resulted in increased degradation rates. The indigenous bacteria grew exponentially during the incubation period of 35 d with a growth rate of 0.26 d^?1. Kinetic modeling was performed to estimate the rates of biodegradation of each hydrocarbon type component in the studied system. Five different error functions were used in this study to evaluate the fitness of the model equation to the obtained experimental data. This showed that the degradation of ∑nC₂₀-nC_24, ∑nC₃₅-nC₄₂ and nC₁₈ can be better represented by a second order model, whereas the TPH, total resolvable peaks (TRP), nC₁₇, UCM, ∑nC₁₀-nC₁₄, ∑nC₁₅-nC_19, ∑nC₂₅-nC₂₉, ∑nC₃₀-nC_34, ∑nC_n, and ∑isoC_n and isoprenoids Pr and phytane (Ph) were similarly following the first order model.     

Remarkable impact of PAHs and TPHs on the richness and diversity of bacterial species in surface soils exposed to long-term hydrocarbon pollution

Nowadays, because of substantial use of petroleum-derived fuels the number and extension of hydrocarbon polluted terrestrial ecosystems is in growth worldwide. In remediation of aforementioned sites bioremediation still tends to be an innovative, environmentally attractive technology. Although huge amount of information is available concerning the hydrocarbon degradation potential of cultivable hydrocarbonoclastic bacteria little is known about the in situ long-term effects of petroleum derived compounds on the structure of soil microbiota. Therefore, in this study our aim was to determine the long-term impact of total petroleum hydrocarbons (TPHs), volatile petroleum hydrocarbons (VPHs), total alkyl benzenes (TABs) as well as of polycyclic aromatic hydrocarbons (PAHs) on the structure of bacterial communities of four different contaminated soil samples. Our results indicated that a very high amount of TPH affected positively the diversity of hydrocarbonoclastic bacteria. This finding was supported by the occurrence of representatives of the α-, β-, γ-Proteobacteria, Actinobacteria, Flavobacteriia and Bacilli classes. High concentration of VPHs and TABs contributed to the predominance of actinobacterial isolates. In PAH impacted samples the concentration of PAHs negatively correlated with the diversity of bacterial species. Heavily PAH polluted soil samples were mainly inhabited by the representatives of the β-, γ-Proteobacteria (overwhelming dominance of Pseudomonas sp.) and Actinobacteria.     

Bacterial Community Dynamics and Polycyclic Aromatic Hydrocarbon Degradation during Bioremediation of Heavily Creosote-Contaminated Soil

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the α-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the γ-Proteobacteria group (genus Xanthomonas), the α-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the γ-Proteobacteria group (genus Xathomonas), the β-Proteobacteria group (genera Alcaligenes and Achromobacter), and the α-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.     

Phylogenetic Analysis of Microbial Communities in Different Regions of the Gastrointestinal Tract in Panaque nigrolineatus,a Wood-Eating Fish

The Neotropical detritivorous catfish Panaque nigrolineatus imbibes large quantities of wood as part of its diet. Due to the interest in cellulose, hemi-cellulose and lignin degradation pathways, this organism provides an interesting model system for the detection of novel microbial catabolism. In this study, we characterize the microbial community present in different regions of the alimentary tract of P. nigrolineatus fed a mixed diet of date palm and palm wood in laboratory aquaria. Analysis was performed on 16S rRNA gene clone libraries derived from anterior and posterior regions of the alimentary tract and the auxiliary lobe (AL), an uncharacterized organ that is vascularly attached to the midgut. Sequence analysis and phylogenetic reconstruction revealed distinct microbial communities in each tissue region. The foregut community shared many phylotypes in common with aquarium tank water and included Legionella and Hyphomicrobium spp. As the analysis moved further into the gastrointestinal tract, phylotypes with high levels of 16S rRNA sequence similarity to nitrogen-fixing Rhizobium and Agrobacterium spp. and Clostridium xylanovorans and Clostridium saccharolyticum, dominated midgut and AL communities. However, the hindgut was dominated almost exclusively by phylotypes with the highest 16S rRNA sequence similarity to the Cytophaga-Flavobacterium-Bacteroides phylum. Species richness was highest in the foregut (Chao₁ = 26.72), decreased distally through the midgut (Chao₁ = 25.38) and hindgut (Chao₁ = 20.60), with the lowest diversity detected in the AL (Chao₁ = 18.04), indicating the presence of a specialized microbial community. Using 16S rRNA gene phylogeny, we report that the P. nigrolineatus gastrointestinal tract possesses a microbial community closely related to microorganisms capable of cellulose degradation and nitrogen fixation. Further studies are underway to determine the role of this resident microbial community in Panaque nigrolineatus.     

Prepared Bed Land Treatment of Soils Containing Diesel and Crude Oil Hydrocarbons

An ex situ, field-scale, prepared bed land treatment unit (LTU) was used to bio-remediate soils containing petroleum hydrocarbons. Two soils were treated in side-by-side units to compare performance: (1) a clayey silt containing crude oil hydrocarbons from releases 30 to 40 years ago and (2) a silty sand containing diesel fuel hydrocarbons from a leak about three years prior to the bioremediation. The effectiveness of the bioremediation in the LTU was evaluated over a period of 18 months. The results indicated that: (1) prepared bed bioremediation reduced the hydrocarbon concentration, mobility, and relative toxicity in the soil with the diesel fuel, and (2) chemical bioavailability appeared to limit bioremediation of the soil containing the crude oil hydrocarbons. Although the soils containing the crude oil hydrocarbons contained an average of 10,000?mg TPH/kg dry soil, these soils had limited hydrocarbon availability, nontoxic conditions, and low potential for chemical migration. For the soils containing the diesel fuel, active prepared bed bioremediation of about 15 weeks was adequate to reach an environmentally acceptable endpoint. At that time, there was little further TPH loss, no Microtox^TM toxicity, and limited hydrocarbon mobility.     

Investigating the Diversity of Pseudomonas spp. in Soil Using Culture Dependent and Independent Techniques

Less than 1 % of bacterial populations present in environmental samples are culturable, meaning that cultivation will lead to an underestimation of total cell counts and total diversity. However, it is less clear whether this is also true for specific well-defined groups of bacteria for which selective culture media is available. In this study, we use culture dependent and independent techniques to describe whether isolation of Pseudomonas spp. on selective nutrient-poor NAA 1:100 agar-medium can reflect the full diversity, found by pyrosequencing, of the total soil Pseudomonas community in an urban waste field trial experiment. Approximately 3,600 bacterial colonies were isolated using nutrient-poor NAA 1:100 medium from soils treated with different fertilizers; (i) high N-level sewage sludge (SA), (ii) high N-level cattle manure (CMA), and (iii) unfertilized control soil (U). Based on Pseudomonas specific quantitative-PCR and Pseudomonas CFU counts, less than 4 % of Pseudomonas spp. were culturable using NAA 1:100 medium. The Pseudomonas selectivity and specificity of the culture medium were evaluated by 454 pyrosequencing of 16S rRNA gene amplicons generated using Bacteria- and Pseudomonas-specific primers. Pyrosequencing results showed that most isolates were Pseudomonas and that the culturable fraction of Pseudomonas spp. reflects most clusters of the total Pseudomonas diversity in soil. This indicates that NAA 1:100 medium is highly selective for Pseudomonas species, and reveals the ability of NAA 1:100 medium to culture mostly the dominant Pseudomonas species in soil.     

Phytoremediation Effect and Growth Responses of Cynodon spp. and Agropyron desertorum in a Petroleum-Contaminated Soil

In order to compare the petroleum tolerance and phytoremediation ability of a native grass, Agropyron desertorum (desert Wheatgrass) with Cynodon spp. (Bermuda grass) in a petroleum hydrocarbon-contaminated soil, a 7-month greenhouse experiment was performed. There were 4 soil treatments with 0% (uncontaminated soil), 2%, 4%, and 12% (w_oil/w_soil) petroleum concentration. Parameters including shoot and root fresh weight and dry weight, root penetration depth and root density depth, soil respiration, and total petroleum hydrocarbons (TPH) degradation were measured during and after experiments. The results showed an increase in shoot fresh weight of A. desertorum in soil polluted with 2% petroleum sludge compared to the uncontaminated soil, whereas the growth of Bermuda grass significantly decreased in corresponding treatment. Root growth of A. desertorum was decreased in 2% and 4% petroleum sludge, whereas it was increased in Bermuda grass species. Overall, root fresh weight of Bermuda grass was higher than that of A. desertorum in all treatments. Significant increase in microorganisms' activity was observed in the presence of petroleum sludge and plants in soil compared with uncontaminated soil without plants, and the highest soil respiration (37.6 mg C-CO₂/kg soil day) has been observed in the rhizosphere of Bermuda grass in treatment with 12% petroleum sludge. Plants had a significant role in the degradation of soil contaminants as TPH degradation in planted soils was significantly higher than that in unplanted soil (TPH degradation (%) was 30.4 and 38.9 in A. desertorum and Bermuda grass, respectively, whereas it was just 13.3 in unplanted soil). The rhizosphere of Bermuda grass had significantly less residual TPHs compared to A. desertorum. The results indicated that both Cynodon spp. and A. desertorum had a peculiar tolerance to petroleum pollution. Therefore, as Bermuda grass has already been suggested to be a typical and efficient species for phytoremediating petroleum-contaminated sites, A. desertorum may also prove to be a suitable native alternative.     

Bacterial community dynamics and polycyclic aromatic hydrocarbon degradation during bioremediation of heavily creosote-contaminated soil

Bacterial community dynamics and biodegradation processes were examined in a highly creosote-contaminated soil undergoing a range of laboratory-based bioremediation treatments. The dynamics of the eubacterial community, the number of heterotrophs and polycyclic aromatic hydrocarbon (PAH) degraders, and the total petroleum hydrocarbon (TPH) and PAH concentrations were monitored during the bioremediation process. TPH and PAHs were significantly degraded in all treatments (72 to 79% and 83 to 87%, respectively), and the biodegradation values were higher when nutrients were not added, especially for benzo(a)anthracene and chrysene. The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment. All treatments resulted in a high first-order degradation rate during the first 45 days, which was markedly reduced after 90 days. A sharp increase in the size of the heterotrophic and PAH-degrading microbial populations was observed, which coincided with the highest rates of TPH and PAH biodegradation. At the end of the incubation period, PAH degraders were more prevalent in samples to which nutrients had not been added. Denaturing gradient gel electrophoresis analysis and principal-component analysis confirmed that there was a remarkable shift in the composition of the bacterial community due to both the biodegradation process and the addition of nutrients. At early stages of biodegradation, the alpha-Proteobacteria group (genera Sphingomonas and Azospirillum) was the dominant group in all treatments. At later stages, the gamma-Proteobacteria group (genus Xanthomonas), the alpha-Proteobacteria group (genus Sphingomonas), and the Cytophaga-Flexibacter-Bacteroides group (Bacteroidetes) were the dominant groups in the nonnutrient treatment, while the gamma-Proteobacteria group (genus Xathomonas), the beta-Proteobacteria group (genera Alcaligenes and Achromobacter), and the alpha-Proteobacteria group (genus Sphingomonas) were the dominant groups in the nutrient treatment. This study shows that specific bacterial phylotypes are associated both with different phases of PAH degradation and with nutrient addition in a preadapted PAH-contaminated soil. Our findings also suggest that there are complex interactions between bacterial species and medium conditions that influence the biodegradation capacity of the microbial communities involved in bioremediation processes.     

Effect of hydrogen on soil bacterial community structure in two soils as determined by terminal restriction fragment length polymorphism

The metabolism of hydrogen evolved from HUP^? legume nodules can alter bacterial community structures in the rhizosphere. Our earlier experiments demonstrated increased hydrogen uptake and appearance of white spots within bacterial colonies in H₂-treated soil. We were also able to isolate hydrogen-oxidizing bacteria from soil samples exposed to hydrogen, but not from samples exposed to air. To further understand the effect of hydrogen metabolism on soil microbial communities, in this study 16S rRNA terminal restriction fragment (TRF) profiles of different soil samples exposed to hydrogen gas under laboratory, greenhouse, and field conditions were analyzed. Relationships between soil bacterial community structures from hydrogen-treated soil samples and controls, illustrated by UPGMA (unpaired group mathematical averages) dendrograms, indicated a significant contribution of hydrogen metabolism to the variation in bacterial community. The intensity variation of TRF peaks includes both hydrogen-utilizing bacteria, whose growth were stimulated by hydrogen exposure, and other bacterial species whose growth was inhibited. Comparison of TRF profiles between laboratory and greenhouse samples showed that T-RFLP is a useful technique in the detection of root-related effects on soil bacterial community structure.     

Efficiency of Opuntia ficus in the phytoremediation of a soil contaminated with used motor oil and lead,compared to that of Lolium perenne and Aloe barbadensis

Industrial pollutants such as heavy metals and hydrocarbons in soils represent a serious concern due to their persistence and negative effects on the environment, affecting cellular processes in living organisms and even causing mutations and cancer. The main objectives of this work were to evaluate the efficiency of Opuntia ficus in the phytoremediation of a soil polluted with used motor oil. Two other species, one with different and one with similar characteristics, relatively, were used for comparison purposes: Lolium perenne and Aloe barbadensis. The effect of the plants on lead solubility and bioaccumulation, the biomass production of each specie and the microbial counts and bacterial identification for each experiment was studied. Total petroleum hydrocarbons (TPH) were measured every 5 weeks throughout the 20-week phytoremediation experiment. At the end of the experiment soluble Pb, Pb extracted by the plant species, microbiological counts, total biomass and bacterial species in soil were analyzed. Even though Lolium perenne showed the highest TPH removal (47%), Opuntia ficus produced the highest biomass and similar removal (46%). Since Opuntia ficus requires low amounts of water and grows fast, it would be a suitable option in the remediation of soils polluted with hydrocarbons and/or heavy metals.     

Metagenomic analysis of microbial communities across a transect from low to highly hydrocarbon-contaminated soils in King George Island,Maritime Antarctica

Soil samples from a transect from low to highly hydrocarbon-contaminated soils were collected around the Brazilian Antarctic Station Comandante Ferraz (EACF), located at King George Island, Antarctica. Quantitative PCR (qPCR) analysis of bacterial 16S rRNA genes, 16S rRNA gene (iTag), and shotgun metagenomic sequencing were used to characterize microbial community structure and the potential for petroleum degradation by indigenous microbes. Hydrocarbon contamination did not affect bacterial abundance in EACF soils (bacterial 16S rRNA gene qPCR). However, analysis of 16S rRNA gene sequences revealed a successive change in the microbial community along the pollution gradient. Microbial richness and diversity decreased with the increase of hydrocarbon concentration in EACF soils. The abundance of Cytophaga, Methyloversatilis, Polaromonas, and Williamsia was positively correlated (p-value = <.05) with the concentration of total petroleum hydrocarbons (TPH) and/or polycyclic aromatic hydrocarbons (PAH). Annotation of metagenomic data revealed that the most abundant hydrocarbon degradation pathway in EACF soils was related to alkyl derivative-PAH degradation (mainly methylnaphthalenes) via the CYP450 enzyme family. The abundance of genes related to nitrogen fixation increased in EACF soils as the concentration of hydrocarbons increased. The results obtained here are valuable for the future of bioremediation of petroleum hydrocarbon-contaminated soils in polar environments.     

Bacterial community structure analysis of a hot spring soil by next generation sequencing of ribosomal RNA

In the present study the whole bacterial community structure of Tapovan hot spring soil located in the state of Uttarakhand, India was analysed through next generation sequencing. The hot spring soil is slightly alkaline in nature with abundance of sulphur. The spring soil was rich in various metallic and non-metallic elements required for bacterial survival. The community was found to comprise of 14 bacterial phyla with representation of members belonging to Firmicutes, Proteobacteria, Thermi, Bacteroidetes, Aquificae, Actinobacteria, chloroflexi, TM7, Fusobacteria etc. At the genus level Bacillus, Pseudomonas, Symbiobacterium, Thermus, Geobacillus, Anoxybacillus were found in abundance as compared to other genera like Flavobacterium, Ureibacillus, Clostridium, Meiothermus, Acinetobacter, Desulfotomaculum and Rheinheimera.     

Diversity and Structure of Bacterial Communities in Arctic versus Antarctic Pack Ice

A comprehensive assessment of bacterial diversity and community composition in arctic and antarctic pack ice was conducted through cultivation and cultivation-independent molecular techniques. We sequenced 16S rRNA genes from 115 and 87 pure cultures of bacteria isolated from arctic and antarctic pack ice, respectively. Most of the 33 arctic phylotypes were >97% identical to previously described antarctic species or to our own antarctic isolates. At both poles, the α- and γ-proteobacteria and the Cytophaga-Flavobacterium group were the dominant taxonomic bacterial groups identified by cultivation as well as by molecular methods. The analysis of 16S rRNA gene clone libraries from multiple arctic and antarctic pack ice samples revealed a high incidence of closely overlapping 16S rRNA gene clone and isolate sequences. Simultaneous analysis of environmental samples with fluorescence in situ hybridization (FISH) showed that ~95% of 4′,6′-diamidino-2-phenylindole (DAPI)-stained cells hybridized with the general bacterial probe EUB338. More than 90% of those were further assignable. Approximately 50 and 36% were identified as γ-proteobacteria in arctic and antarctic samples,respectively. Approximately 25% were identified as α-proteobacteria, and 25% were identified as belonging to the Cytophaga-Flavobacterium group. For the quantification of specific members of the sea ice community, new oligonucleotide probes were developed which target the genera Octadecabacter, Glaciecola, Psychrobacter, Marinobacter, Shewanella, and Polaribacter. High FISH detection rates of these groups as well as high viable counts corroborated the overlap of clone and isolate sequences. A terrestrial influence on the arctic pack ice community was suggested by the presence of limnic phylotypes.     

Bacterial community dynamics during <Emphasis Type="Italic">in-situ</Emphasis> bioremediation of petroleum waste sludge in landfarming sites

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75–100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.     

Microbial community dynamics and evaluation of bioremediation strategies in oil-impacted salt marsh sediment microcosms

Microbial community dynamics in wetlands microcosms emended with commercial products (surfactant, a biological agent, and nutrients) designed to enhance bioremediation was followed for 3 months. The effectiveness of enhanced degradation was assessed by determining residual concentrations of individual petroleum hydrocarbons by GC/MS. The size and composition of the sediment microbial community was assessed using a variety of indices, including bacterial plate counts, MPNs, and DNA hybridizations with domain- and group-specific oligonucleotide probes. The addition of inorganic nutrients was the most effective treatment for the enhancement of oil degradation, resulting in marked degradation of petroleum alkanes and a lesser extent of degradation of aromatic oil constituents. The enhanced degradation was associated with increases in the amount of extractable microbial DNA and Streptomyces in the sediment, although not with increased viable counts (plate counts, MPN). Bacteria introduced with one of the proprietary products were still detected in the microcosms after 3 months, but were not a major quantitative constituent of the community. The biological product enhanced oil degradation relative to the control, but to a lesser extent than the nutrient additions alone. In contrast, application of the surfactant to the oil-impacted sediment decreased oil degradation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 72–79. Received 18 March 2001/ Accepted in revised form 09 June 2001     

Biodegradation of petroleum sludge and petroleum polluted soil by a bacterial consortium: a laboratory study

This article presents a study of the efficiency and degradation pattern of samples of petroleum sludge and polluted sandy soil from an oil refinery. A bacterial consortium, consisting of strains from the genera Pseudomonas, Achromobacter, Bacillus and Micromonospora, was isolated from a petroleum sludge sample and characterized. The addition of nitrogen and phosphorus nutrients and a chemical surfactant to both the samples and bioaugmentation to the soil sample were applied under laboratory conditions. The extent of biodegradation was monitored by the gravimetric method and analysis of the residual oil by gas chromatography. Over a 12-week experiment, the achieved degree of TPH (total petroleum hydrocarbon) degradation amounted to 82–88% in the petroleum sludge and 86–91% in the polluted soil. Gas chromatography–mass spectrometry was utilized to determine the biodegradability and degradation rates of n-alkanes, isoprenoids, steranes, diasteranes and terpanes. Complete degradation of the n-alkanes and isoprenoids fractions occurred in both the samples. In addition, the intensities of the peaks corresponding to tricyclic terpenes and homohopanes were decreased, while significant changes were also observed in the distribution of diasteranes and steranes.     

Biodegradation of Petroleum Tar by Pseudomonas Spp. From Oil Field of Assam,India

ABSTRACT

Petroleum tar produced during the processing of crude oil is one of the earth's major pollutants. The potential of certain soil bacteria in the biodegradation of petroleum tar was assessed to develop an active indigenous bacterial consortium for bioremediation of petroleum tar–polluted sites of Assam, India. In vitro enrichment cultures of five Pseudomonas spp. were found to metabolize petroleum tar. The Fourier transform infrared (FTIR) analyses of the enrichment cultures revealed the presence of the functional groups, viz., –OH, –CHO, C?O, and –COOH, which provided evidence for the biodegradation of petroleum tar. Further, gas chromatography–flame ionization detection (GC-FID) analyses revealed complete degradation of low-molecular-weight hydrocarbons, and the subsequent appearance of some additional peaks reflected the formation of intermediate metabolites during the degradation of petroleum tar. A mixed culture with 0.1% Tween 80 as a surfactant exhibited almost complete degradation in contrast to the degradation by the mixed culture without Tween 80. This confirmed the effect of a surfactant for acceleration of the biodegradation process of petroleum tar.     

Dynamics of Bacterial Community Composition and Activity during a Mesocosm Diatom Bloom

Bacterial community composition, enzymatic activities, and carbon dynamics were examined during diatom blooms in four 200-liter laboratory seawater mesocosms. The objective was to determine whether the dramatic shifts in growth rates and ectoenzyme activities, which are commonly observed during the course of phytoplankton blooms and their subsequent demise, could result from shifts in bacterial community composition. Nutrient enrichment of metazoan-free seawater resulted in diatom blooms dominated by a Thalassiosira sp., which peaked 9 days after enrichment (≈24 μg of chlorophyll a liter⁻¹). At this time bacterial abundance abruptly decreased from 2.8 × 10⁶ to 0.75 × 10⁶ ml⁻¹, and an analysis of bacterial community composition, by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments, revealed the disappearance of three dominant phylotypes. Increased viral and flagellate abundances suggested that both lysis and grazing could have played a role in the observed phylotype-specific mortality. Subsequently, new phylotypes appeared and bacterial production, abundance, and enzyme activities shifted from being predominantly associated with the <1.0-μm size fraction towards the >1.0-μm size fraction, indicating a pronounced microbial colonization of particles. Sequencing of DGGE bands suggested that the observed rapid and extensive colonization of particulate matter was mainly by specialized α-Proteobacteria- and Cytophagales-related phylotypes. These particle-associated bacteria had high growth rates as well as high cell-specific aminopeptidase, β-glucosidase, and lipase activities. Rate measurements as well as bacterial population dynamics were almost identical among the mesocosms indicating that the observed bacterial community dynamics were systematic and repeatable responses to the manipulated conditions.     

Comparison of Diversities and Compositions of Bacterial Populations Inhabiting Natural Forest Soils

The diversity and composition of soil bacterial communities were compared among six Austrian natural forests, including oak-hornbeam, spruce-fir-beech, and Austrian pine forests, using terminal restriction fragment length polymorphism (T-RFLP, or TRF) analysis and sequence analysis of 16S rRNA genes. The forests studied differ greatly in soil chemical characteristics, microbial biomass, and nutrient turnover rates. The aim of this study was to relate these differences to the composition of the bacterial communities inhabiting the individual forest soils. Both TRF profiling and clone sequence analysis revealed that the bacterial communities in soils under Austrian pine forests, representing azonal forest types, were distinct from those in soils under zonal oak-hornbeam and spruce-fir-beech forests, which were more similar in community composition. Clones derived from an Austrian pine forest soil were mostly affiliated with high-G+C gram-positive bacteria (49%), followed by members of the α-Proteobacteria (20%) and the Holophaga/Acidobacterium group (12%). Clones in libraries from oak-hornbeam and spruce-fir-beech forest soils were mainly related to the Holophaga/Acidobacterium group (28 and 35%), followed by members of the Verrucomicrobia (24%) and the α-Proteobacteria (27%), respectively. The soil bacterial communities in forests with distinct vegetational and soil chemical properties appeared to be well differentiated based on 16S rRNA gene phylogeny. In particular, the outstanding position of the Austrian pine forests, which are determined by specific soil conditions, was reflected in the bacterial community composition.