Monitoring of microbial diversity and activity during bioremediation of crude oil-contaminated soil with different treatments

The present study compared the microbial diversity and activity during the application of various bioremediation processes to crude oil-contaminated soil. Five different treatments, including natural attenuation (NA), biostimulation (BS), biosurfactant addition (BE), bioaugmentation (BA), and a combined treatment (CT) of biostimulation, biosurfactant addition, and bioaugmentation, were used to analyze the degradation rate and microbial communities. After 120 days, the level of remaining hydrocarbons after all the treatments was similar, however, the highest rate (k) of total petroleum hydrocarbon (TPH) degradatioN was observed with the CT treatment (P < 0.05). The total bacterial counts increased during the first 2 weeks with all the treatments, and then remained stable. The bacterial communities and alkane monooxygenase gene fragment, alkB, were compared by denaturing gradient gel electrophoresis (DGGE). The DGGE analyses of the BA and CT treatments, which included Nocardia sp. H17-1, revealed a simple dominant population structure, compared with the other treatments. The Shannon-Weaver diversity index (H') and Simpson dominance index (D), calculated from the DGGE profiles using 16S rDNA, showed considerable qualitative differences in the community structure before and after the bioremediation treatment as well as between treatment conditions.     

Effect of biostimulants on 2,4,6-trinitrotoluene (TNT) degradation and bacterial community composition in contaminated aquifer sediment enrichments

2,4,6-Trinitrotoluene (TNT) is a toxic and persistent explosive compound occurring as a contaminant at numerous sites worldwide. Knowledge of the microbial dynamics driving TNT biodegradation is limited, particularly in native aquifer sediments where it poses a threat to water resources. The purpose of this study was to quantify the effect of organic amendments on anaerobic TNT biodegradation rate and pathway in an enrichment culture obtained from historically contaminated aquifer sediment and to compare the bacterial community dynamics. TNT readily biodegraded in all microcosms, with the highest biodegradation rate obtained under the lactate amended condition followed by ethanol amended and naturally occurring organic matter (extracted from site sediment) amended conditions. Although a reductive pathway of TNT degradation was observed across all conditions, denaturing gradient gel electrophoresis (DGGE) analysis revealed distinct bacterial community compositions. In all microcosms, Gram-negative γ- or β-Proteobacteria and Gram-positive Negativicutes or Clostridia were observed. A Pseudomonas sp. in particular was observed to be stimulated under all conditions. According to non-metric multidimensional scaling analysis of DGGE profiles, the microcosm communities were most similar to heavily TNT-contaminated field site sediment, relative to moderately and uncontaminated sediments, suggesting that TNT contamination itself is a major driver of microbial community structure. Overall these results provide a new line of evidence of the key bacteria driving TNT degradation in aquifer sediments and their dynamics in response to organic carbon amendment, supporting this approach as a promising technology for stimulating in situ TNT bioremediation in the subsurface.     

Molinate biodegradation in soils: natural attenuation versus bioaugmentation

The aims of the present study were to assess the potential of natural attenuation or bioaugmentation to reduce soil molinate contamination in paddy field soils and the impact of these bioremediation strategies on the composition of soil indigenous microbiota. A molinate mineralizing culture (mixed culture DC) was used as inoculum in the bioaugmentation assays. Significantly higher removal of molinate was observed in bioaugmentation than in natural attenuation microcosms (63 and 39 %, respectively) after 42 days of incubation at 22 °C. In the bioaugmentation assays, the impact of Gulosibacter molinativorax ON4^T on molinate depletion was observed since the gene encoding the enzyme responsible for the initial molinate breakdown (harboured by that actinobacterium) was only detected in inoculated microcosms. Nevertheless, the exogenous mixed culture DC did not overgrow as the heterotrophic counts of the bioaugmentation microcosms were not significantly different from those of natural attenuation and controls. Moreover, the actinobacterial clone libraries generated from the bioaugmentation microcosms did not include any 16S rRNA gene sequences with significant similarity to that of G. molinativorax ON4^T. The multivariate analysis of the 16S rRNA DGGE patterns of the soil microcosm suggested that the activity of mixed culture DC did not affect the soil bacterial community structure since the DGGE patterns of the bioaugmentation microcosms clustered with those of natural attenuation and controls. Although both bioremediation approaches removed molinate without indigenous microbiota perturbation, the results suggested that bioaugmentation with mixed culture DC was more effective to treat soils contaminated with molinate.     

Both Cycloclasticus spp. and Pseudomonas spp. as PAH-degrading bacteria in the Seine estuary (France)

Like other highly urbanized and industrialized estuaries, the Seine estuary (France) has, for decades, received high inputs of polycyclic aromatic hydrocarbons (PAHs). In order to estimate the bioremediation potentials and to identify the bacterial species involved in hydrocarbon degradation, we used microcosms containing seawater from the Seine estuary supplemented with either naphthalene, phenanthrene, fluorene or pyrene. In the microcosms enriched with naphthalene or phenanthrene, hydrocarbon biodegradation was significant within 9 weeks (43% or 46%, respectively), as shown by analyses in GC-MS. In similar microcosms incubated also with naphthalene or phenanthrene, analysis of the 16S rRNA gene sequences (DNA and cDNA) with denaturing gradient gel electrophoresis and clone libraries indicated that the PAH-degrading communities were dominated by Cycloclasticus spp., confirming their universal key role in degradation of low-molecular-weight PAHs in marine environments. However, in contrast to previous studies, we found that Pseudomonas spp. also degraded naphthalene and phenanthrene in seawater; this occurred only after 21 days, as was confirmed by real-time PCR. Although this genus has been abundantly described in the literature as a good PAH-degrading bacterial group in soil or in sediment, to our knowledge, this is the first evidence of a significant fitness in PAH degradation in seawater.     

Indigenous PAH-degrading bacteria from oil-polluted sediments in Caleta Cordova,Patagonia Argentina

Indigenous bacteria with the capability to degrade polycyclic aromatic hydrocarbons (PAH) were isolated from polluted sediment samples recovered from Caleta Cordova by using selective enrichment cultures supplemented with phenanthrene. Bacterial communities were evaluated by denaturing gradient gel electrophoresis (DGGE) in order to detect changes along enrichment culture and relationships with the representative strains subsequently isolated. Members of these communities included marine bacteria such as Lutibacter, Polaribacter, Arcobacter and Olleya, whose degradation pathway of PAH has not been studied yet. However, isolated bacteria obtained from this enrichment comprised the genus Pseudomonas, Marinobacter, Salinibacterium and Brevibacterium. The ability of isolates to grow and degrade naphthalene, phenanthrene and pyrene was demonstrated by detection of the residual substrate by HPLC. Archetypical naphthalene and catechol dioxygenase genes were found in two isolates belonging to genus Pseudomonas (Pseudomonas monteilii P26 and Pseudomonas xanthomarina N12), suggesting biodegradation potential in these sediments. The successful bacterial isolation with the ability to degrade PAH in pure culture suggest the possibility to study and further consider strategies like growth stimulation in situ, in order to increase the intrinsic bioremediation opportunities in the polluted Caleta Cordova harbor.     

Natural Attenuation, Biostimulation, and Bioaugmentation in 4-Chloroaniline-Contaminated Soil

Bioremediation treatments including natural attenuation (NA), biostimulation (BS), and bioaugmentation (BA) were performed and compared regarding the degradation of 4-chloroaniline (4CA) contaminating two types of agricultural soil collected from Nakornnayok (NN) and Chiangmai (CM) provinces, Thailand. Despite the different soil properties, both soil types exhibited intrinsic potential for biodegradation. 4CA degradation by NA in loam soil-NN was fairly effective (ca. 40%), while in sandy-clay loam soil-CM it occurred poorly (<10%). Compared to NA, BS with aniline and BA with 4CA-degrading Klebseilla sp. CA17 were comparatively more effective techniques, although the degradation occurred differently in each soil type. In soil-NN, the biodegradation of 4CA took place at a higher rate, achieving biodegradation of 70–75% within 4 weeks, than in soil-CM, i.e., up to 40–46% within 8 weeks. During each treatment, changes in soil microbial activity, numbers of 4CA-degrading micro-organisms, and dynamic modification of soil microbial community structure were also monitored. The results suggest that both BS and BA are feasible techniques for bioremediation of 4CA accumulated in soil, although the biodegrading efficiency in soil environment depends not only on site characteristics but also on the characteristics of either indigenous microbial population or the survival and stability of bioaugmented cultures.     

Sustainable remediation: electrochemically assisted microbial dechlorination of tetrachloroethene‐contaminated groundwater

Microbial electric systems (MESs) hold significant promise for the sustainable remediation of chlorinated solvents such as tetrachlorethene (perchloroethylene, PCE). Although the bio‐electrochemical potential of some specific bacterial species such as Dehalcoccoides and Geobacteraceae have been exploited, this ability in other undefined microorganisms has not been extensively assessed. Hence, the focus of this study was to investigate indigenous and potentially bio‐electrochemically active microorganisms in PCE‐contaminated groundwater. Lab‐scale MESs were fed with acetate and carbon electrode/PCE as electron donors and acceptors, respectively, under biostimulation (BS) and BS‐bioaugmentation (BS‐BA) regimes. Molecular analysis of the indigenous groundwater community identified mainly Spirochaetes, Firmicutes, Bacteroidetes, and γ and δ‐Proteobacteria. Environmental scanning electron photomicrographs of the anode surfaces showed extensive indigenous microbial colonization under both regimes. This colonization and BS resulted in 100% dechlorination in both treatments with complete dechlorination occurring 4 weeks earlier in BS‐BA samples and up to 11.5 μA of current being generated. The indigenous non‐Dehalococcoides community was found to contribute significantly to electron transfer with ～61% of the current generated due to their activities. This study therefore shows the potential of the indigenous non‐Dehalococcoides bacterial community in bio‐electrochemically reducing PCE that could prove to be a cost‐effective and sustainable bioremediation practice.     

The Impact of Sediment Characteristics on PCB-dechlorinating Cultures: Implications for Bioaugmentation

Polychlorinated Biphenyl (PCB)-dechlorinating cultures with complimentary activities, previously derived from estuarine Baltimore Harbor (B), marine Palos Verdes (P), and riverine Hudson River (H) sediments, were mixed and then inoculated into sterile sediments from the same sources. In the treatments containing sterile B sediment, the different inocula had limited impact on the bacterial community development and on dechlorination patterns, all of which were similar. In treatments containing sterile P or H sediment, however, different inocula resulted in significantly different PCB dechlorination patterns and bacterial communities. The B sediment appeared to support not only the most extensive and rapid dechlorination of the three sediments, but also supported a more diverse bacterial community. This was thought to be a result of nutritional richness, as it was high in organic carbon and micronutrients such as zinc and cobalt. Although mixing three PCB-dechlorinating cultures was able to produce a culture capable of enhanced PCB-dechlorinating activity as compared to single cultures, some activities were lost upon culture transfer. This indicates that care must be taken to establish robust PCB-dechlorinating cultures capable of extensive dechlorination prior to pursuing bioaugmentation. In addition, our results indicate that the concentration and availability of macro-and micronutrients could have a significant impact on the microbial community structure, and thus a thorough characterization of the sediment at contaminated sites is essential for implementing bioaugmentation for PCB bioremediation.     

Importance of Gram-positive naphthalene-degrading bacteria in oil-contaminated tropical marine sediments

AIMS: The aim of this study was to isolate, characterize and evaluate the importance of naphthalene-degrading bacterial strains from oil-contaminated tropical marine sediments. METHODS AND RESULTS: Three Gram-positive naphthalene-degrading bacteria were isolated from oil-contaminated tropical intertidal marine sediments by direct isolation or enrichment using naphthalene as the sole source of carbon and energy. Bacillus naphthovorans strain MN-003 can also grow on benzene, toluene, xylene and diesel fuel while Micrococcus sp. str. MN-006 can also grow on benzene. Staphylococcus sp. str. MN-005 can only degrade naphthalene and was not able to use the other aromatic hydrocarbons tested. Strain MN-003 possessed the highest maximal specific growth rate with naphthalene as sole carbon source. An enrichment culture fed with naphthalene as sole carbon source exhibited a significant increase in the relative abundances of the three isolates after 21 days of incubation. The three isolates constituted greater than 69% of the culturable naphthalene-degrading microbial community. Strain MN-003 outcompeted and dominated the other two isolates in competition studies involving batch cultures inoculated with equal cell densities of the three isolates and incubated with between 1 and 10 mg l-1 of naphthalene. CONCLUSIONS: Three Gram-positive naphthalene-degrading bacteria were successfully isolated from oil-contaminated tropical marine sediments. Gram-positive bacteria might play an important role in naphthalene degradation in the highly variable environment of oil-contaminated tropical intertidal marine sediments. Among the three isolates, strain MN-003 has the highest maximal specific growth rate when grown on naphthalene, and outgrew the other two isolates in competition experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: This research will aid in the development of bioremediation schemes for oil-contaminated marine environments. Strain MN-003 could potentially be exploited in such schemes.     

Utilization of bioremediation processes for the treatment of PAH-contaminated sediments

The widespread contamination of aquatic sediments by polycyclic aromatic hydrocarbons (PAHs) has created a need for cost-effective remediation processes. Many common PAHs are biodegradable, leading to studies investigating the potential of sediment bioremediation. This article reviews several factors that currently complicate the implementation of sediment bioremediation processes: the effect of complex mixtures of contaminants on the rate and extent of degradation observed, the bioavailability of PAHs in sorbed- and nonaqueous-phase, and methods being evaluated to enhance degradation/availability (surfactant-enhanced solubility, nutrient addition, and bioaugmentation). Received 13 November 1995/ Accepted in revised form 23 July 1996     

Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial community composition in deep-sea sediments of the South China Sea

The South China Sea, which is one of the largest marginal seas in the world, is predicted to have suitable accumulation conditions and exporting prospects for natural gas hydrate. The aim of this study was to explore the bacterial community composition of deep-sea sediments from such an ecosystem. DNA was extracted by five different methods and used as templates for PCR amplification of the V3 regions of the 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) was used to separate the amplified products and analyse the 16S rRNA gene diversity of sediment samples. The results of DGGE indicated that the bacterial community composition is influenced by DNA extraction methods. Sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belong to Proteobacteria, Bacteroidetes, gram-positive bacteria and Archaea. Integrating different DNA extraction procedures are needed to analyse the actual bacterial diversity from environment when the amplification of 16S rRNA gene and construction of representative clone library were adopted.     

Enrichment, isolation, and phylogenetic identification of polycyclic aromatic hydrocarbon-degrading bacteria from Elizabeth River sediments

The diversity of indigenous bacteria in sediments from several sites in the Elizabeth River (Virginia) able to degrade multiple polycyclic aromatic hydrocarbons (PAHs) was investigated by the use of classical selective enrichment and molecular analyses. Enrichment cultures containing naphthalene, phenanthrene, fluoranthene, or pyrene as a sole carbon and energy source were monitored by denaturing gradient gel electrophoresis (DGGE) to detect changes in the bacterial-community profile during enrichment and to determine whether the representative strains present were successfully cultured. The DGGE profiles of the final enrichments grown solely on naphthalene and pyrene showed no clear relationship with the site from which the inoculum was obtained. The enrichments grown solely on pyrene for two sample sites had >80% similarity, which suggests that common pyrene-degrading strains may be present in these sediments. The final enrichments grown on fluoranthene and phenanthrene remained diverse by site, suggesting that these strains may be influenced by environmental conditions. One hundred and one isolates were obtained, comprising representatives of the actinomycetes and alpha-, beta-, and gammaproteobacteria, including seven novel isolates with 16S rRNA gene sequences less than 98% similar to known strains. The ability to degrade multiple PAHs was demonstrated by mineralization of 14C-labeled substrate and growth in pure culture. This supports our hypothesis that a high diversity of bacterial strains with the ability to degrade multiple PAHs can be confirmed by the combined use of classical selective enrichment and molecular analyses. This large collection of diverse PAH-degrading strains provides a valuable resource for studies on mechanisms of PAH degradation and bioremediation.     

Rhamnolipid production using Shewanella seohaensis BS18 and evaluation of its efficiency along with phytoremediation and bioaugmentation for bioremediation of hydrocarbon contaminated soils

Abstract

This study reports the combined use of a rhamnolipid type biosurfactant (BS) along with phytoremediation and bioaugmentation (BA) for bioremediation of hydrocarbon-contaminated soils. Bacterial isolates obtained from hydrocarbon contaminated soil were screened for rhamnolipid production and isolate BS18, identified as Shewanella seohaensis, was selected for bioremediation experiments. Growth of BS18 in mineral salt medium (MSM) with diesel oil as the carbon source showed a maximum biomass of 8.2?g L^?1, rhamnolipid production of 2.2?mg g^?1 cell dry weight, surface tension reduction of 28.6?mN/m and emulsification potential (EI₂₄%) of 65.6. Characterization of rhamnolipid based on Fourier transmittance infrared (FTIR) analysis confirmed the presence of OH, CH₂/CH₃, C=O, and COO stretching vibrations, respectively, which are distinctive features of rhamnolipid type BSs. In bioremediation experiments, the lowest hydrocarbon concentration of 2.1?mg g^?1 of soil for non-sterilized soil and 4.3?mg g^?1 of soil for sterilized soil was recorded in the combined application of rhamnolipid, phytoremediation, and BA. This treatment also yielded the highest hydrocarbon degrading bacterial population (6.4 Log Cfu g^?1 of soil), highest plant biomass (8.3?g dry weight plant^?1), and the highest hydrocarbon uptake (512.3?mg Kg^?1 of plant).     

Prokaryotic metabolic activity and community structure in Antarctic continental shelf sediments

The prokaryote community activity and structural characteristics within marine sediment sampled across a continental shelf area located off eastern Antarctica (66 degrees S, 143 degrees E; depth range, 709 to 964 m) were studied. Correlations were found between microbial biomass and aminopeptidase and chitinase rates, which were used as proxies for microbial activity. Biomass and activity were maximal within the 0- to 3-cm depth range and declined rapidly with sediment depths below 5 cm. Most-probable-number counting using a dilute carbohydrate-containing medium recovered 1.7 to 3.8% of the sediment total bacterial count, with mostly facultatively anaerobic psychrophiles cultured. The median optimal growth temperature for the sediment isolates was 15 degrees C. Many of the isolates identified belonged to genera characteristic of deep-sea habitats, although most appear to be novel species. Phospholipid fatty acid (PLFA) and isoprenoid glycerol dialkyl glycerol tetraether analyses indicated that the samples contained lipid components typical of marine sediments, with profiles varying little between samples at the same depth; however, significant differences in PLFA profiles were found between depths of 0 to 1 cm and 13 to 15 cm, reflecting the presence of a different microbial community. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified bacterial 16S rRNA genes revealed that between samples and across sediment core depths of 1 to 4 cm, the community structure appeared homogenous; however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. Sequencing of DGGE bands and rRNA probe hybridization analysis revealed that the major community members belonged to delta proteobacteria, putative sulfide oxidizers of the gamma proteobacteria, Flavobacteria, Planctomycetales, and Archaea. rRNA hybridization analyses also indicated that these groups were present at similar levels in the top layer across the shelf region.     

Studies on Biodegradation of Kerosene in Soil under Different Bioremediation Strategies

The effectiveness of bioremediation is often a function of the microbial population and how they can be enriched and maintained in an environment. Strategies for inexpensive in situ bioremediation of soil contaminated with petroleum hydrocarbons include stimulation of the indigenous microorganisms by introduction of nutrients (biostimulation) and/or through inoculation of an enriched mixed microbial culture into soil (bioaugmentation). To demonstrate the potential use of bioremediation in soil contaminated with kerosene, a laboratory study with the objective of evaluating and comparing the effects of bioattenuation, biostimulation, bioaugmentation, and combined biostimulation and bioaugmentation was performed. The present study dealt with the biodegradation of kerosene in soil under different bioremediation treatment strategies: bioattenuation, biostimulation, bioaugmentation, and combined biostimulation and bioaugmentation, respectively. Each treatment strategy contained 10% (w/w) kerosene in soil as a sole source of carbon and energy. After 5 weeks of remediation, the results revealed that bioattenuation, bioaugmentation, biostimulation, and combined biostimulation and bioaugmentation exhibited 44.1%, 67.8%, 83.1%, and 87.3% kerosene degradation, respectively. Also, the total hydrocarbon-degrading bacteria (THDB) count in all the treatments increased with time up till the second week after which it decreased. The highest bacterial growth was observed for combined biostimulation and bioaugmentation treatment strategy. A first-order kinetic model equation was fitted to the biodegradation data to further evaluate the rate of biodegradation and the results showed that the specific degradation rate constant (k) value was comparatively higher for combined biostimulation and bioaugmentation treatment strategy than the values for other treatments. Therefore, value of the kinetic parameter showed that the degree of effectiveness of these bioremediation strategies in the clean up of soil contaminated with kerosene is in the following order: bioattenuation < bioaugmentation < biostimulation < combined biostimulation and bioaugmentation. Conclusively, the present work has defined combined biostimulation and bioaugmentation treatment strategy requirements for kerosene oil degradation and thus opened an avenue for its remediation from contaminated soil.     

Prokaryotic Metabolic Activity and Community Structure in Antarctic Continental Shelf Sediments

The prokaryote community activity and structural characteristics within marine sediment sampled across a continental shelf area located off eastern Antarctica (66°S, 143°E; depth range, 709 to 964 m) were studied. Correlations were found between microbial biomass and aminopeptidase and chitinase rates, which were used as proxies for microbial activity. Biomass and activity were maximal within the 0- to 3-cm depth range and declined rapidly with sediment depths below 5 cm. Most-probable-number counting using a dilute carbohydrate-containing medium recovered 1.7 to 3.8% of the sediment total bacterial count, with mostly facultatively anaerobic psychrophiles cultured. The median optimal growth temperature for the sediment isolates was 15°C. Many of the isolates identified belonged to genera characteristic of deep-sea habitats, although most appear to be novel species. Phospholipid fatty acid (PLFA) and isoprenoid glycerol dialkyl glycerol tetraether analyses indicated that the samples contained lipid components typical of marine sediments, with profiles varying little between samples at the same depth; however, significant differences in PLFA profiles were found between depths of 0 to 1 cm and 13 to 15 cm, reflecting the presence of a different microbial community. Denaturing gradient gel electrophoresis (DGGE) analysis of amplified bacterial 16S rRNA genes revealed that between samples and across sediment core depths of 1 to 4 cm, the community structure appeared homogenous; however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. Sequencing of DGGE bands and rRNA probe hybridization analysis revealed that the major community members belonged to delta proteobacteria, putative sulfide oxidizers of the gamma proteobacteria, Flavobacteria, Planctomycetales, and Archaea. rRNA hybridization analyses also indicated that these groups were present at similar levels in the top layer across the shelf region.     

Microbial diversity and anaerobic hydrocarbon degradation potential in an oil-contaminated mangrove sediment

ABSTRACT: BACKGROUND: Mangrove forests are coastal wetlands that provide vital ecosystem services and serve as barriers against natural disasters like tsunamis, hurricanes and tropical storms. Mangroves harbour a large diversity of organisms, including microorganisms with important roles in nutrient cycling and availability. Due to tidal influence, mangroves are sites where crude oil from spills farther away can accumulate. The relationship between mangrove bacterial diversity and oil degradation in mangrove sediments remains poorly understood. RESULTS: Mangrove sediment was sampled from 0--5, 15--20 and 35--40 cm depth intervals from the Surui River mangrove (Rio de Janeiro, Brazil), which has a history of oil contamination. DGGE fingerprinting for bamA, dsr and 16S rRNA encoding fragment genes, and qPCR analysis using dsr and 16S rRNA gene fragment revealed differences with sediment depth. CONCLUSIONS: Analysis of bacterial 16S rRNA gene diversity revealed changes with depth. DGGE for bamA and dsr genes shows that the anaerobic hydrocarbon-degrading community profile also changed between 5 and 15 cm depth, and is similar in the two deeper sediments, indicating that below 15 cm the anaerobic hydrocarbon-degrading community appears to be well established and homogeneous in this mangrove sediment. qPCR analysis revealed differences with sediment depth, with general bacterial abundance in the top layer (0--5 cm) being greater than in both deeper sediment layers (15--20 and 35--40 cm), which were similar to each other.     

Importance of the Sedimentary Matrix for Anaerobic Oil Degradation by Marine Bacterial Assemblages

The microbial conversion of petroleum hydrocarbons is increasingly employed in bioremediation efforts. In marine sediments, oxygen levels are characteristically depleted, so anaerobic degradation coupled to sulfate reduction dominates. Prior studies have noted that anaerobic degradation is much reduced in the absence of sediments. In this study, a simple centrifugation protocol was used to extract sediment porewaters to obtain a sediment-free bacterial assemblage capable of anaerobic hydrocarbon degradation. In addition, the factors in sediment that were important to degradation rates were determined. Experiments were designed to differentiate among the effects of increased surface area associated with individual grains in sediments, differing levels of organic constituents in sediments and water, and disparate microbiota within the sedimentary matrix and those free living. Anaerobic alkane degradation, sulfate levels and bacterial community structure were monitored over 90 days in five treatments consisting of Bonny Light crude oil added to (1) intact sediment, (2) sediment-free supernate from centrifuged sediment, (3) supernate plus autoclaved sediment, (4) supernate plus organic-free (combusted) sediment, and (5) a control, with autoclaved supernate plus autoclaved sediment. Lack of surface area associated with sediment grains had little effect on degradation. Separation of porewaters from the sedimentary matrix resulted in loss of bacterial biomass, although this had only a temporary negative effect on degradation rates. Reduction of organic matter due to sediment removal had the largest effect, resulting initially in lower degradation rates. However, sulfate depletion in low organic treatments was also reduced so that long-term loss of alkanes was enhanced.     

Evaluation of different bioremediation protocols to enhance decomposition of organic polymers in harbour sediments

The response of the microbial community (in term of abundance and enzymatic activity) was investigated to test the effect of different bioremediation protocols to naturally enhance decomposition of organic polymers in harbour sediments (Genoa Harbour, Italy, N–W Mediterranean). Bioremediation techniques tested were bioaugmentation (5 different microorganisms inocula), biostimulation (air supply), and natural attenuation. The coupling bioaugmentation/biostimulation was also tested. After 60days, following the bioaugmentation protocol, bacterial densities correlated to the quantities of inocula amended to the boxes, suggesting that allochthonous community was able to survive and multiply. However, while bioaugmentation alone seems not to be able to carry out significative degradation, its coupling with air insufflations produced the best response: here bacterial densities increased, especially in the water (from 2.3×10⁷ to 3.50×10⁸cells ml^–1), average cell size and enzymatic activities increased, and sedimentary organic matter was significantly depleted (PRT 5-folds reduction, CHO 1.5-folds reduction). The strong coupling observed between the sediment and water compartments together with the greatest microbial response observed in this latter suggest that the sediment–water interface may constitute a key compartment for the occurring of biodegradation processes in organic-rich sediments.     

Evidence for indigenous Streptomyces populations in a marine environment determined with a 16S rRNA probe.

A 16S rRNA genus-specific probe was used to determine whether Streptomyces populations are an indigenous component of marine sediment bacterial communities. Previous debates have suggested that marine Streptomyces isolates are derived not from resident populations but from spores of terrestrial species which have been physically transported to marine ecosystems but remain dormant until isolation. Rigorously controlled hybridization of rRNA extracted from coastal marsh sediments with the genus-specific probe indicated that Streptomyces rRNA accounted for 2 to 5% of the sediment community rRNA and that spores are not the source of the hybridization signal. Streptomyces populations must therefore be at least the 26th most abundant genus-level source of bacterial rRNA. the relative amounts of rRNAs from Streptomyces spp. and members of the Bacteria (69 to 79%) and Archaea (4 to 7%) domains were highly consistent in these marine sediments throughout an annual cycle, indicating that the species composition of sediment bacterial communities may be more stable than recent studies suggest for marine planktonic bacterial communities. Laboratory studies designed to investigate the possible functional roles of Streptomyces populations in coastal sediments demonstrated that population levels of this genus changed relatively rapidly (within a time frame of 6 weeks) in response to manipulation of substrate availability. Amendments of intact sediment cores with two compounds (vanillic acid and succinic acid) consistently resulted in Streptomyces populations contributing an increased percentage of rRNA (6 to 15%) to the total bacterial rRNA pool.