Ian M. Head

Accurate determination of microbial diversity from 454 pyrosequencing data.

We present an algorithm, PyroNoise, that clusters the flowgrams of 454 pyrosequencing reads using a distance measure that models sequencing noise. This infers the true sequences in a collection of amplicons. We pyrosequenced a known mixture of microbial 16S rDNA sequences extracted from a lake and found that without noise reduction the number of operational taxonomic units is overestimated but using PyroNoise it can be accurately calculated.

Microbial Evolution, Diversity, and Ecology: A Decade of Ribosomal RNA Analysis of Uncultivated Microorganisms

A bstractThe application of molecular biological methods to study the diversity and ecology of microorganisms in natural environments has been practiced since the mid-1980s. Since that time many new insights into the composition of uncultivated microbial communities have been gained. Whole groups of organisms that are only known from molecular sequences are now believed to be quantitatively significant in many environments. Molecular methods have also allowed characterization of many long-recognized but poorly understood organisms. These organisms have eluded laboratory cultivation and, hence, have remained enigmatic. This review provides an outline of the main methods used in molecular microbial ecology, and their limitations. Some discoveries, made through the application of molecular biological methods, are highlighted, with reference to morphologically distinctive, uncultivated bacteria; an important biotechnological process (wastewater treatment); and symbiotic relationships between Bacteria, Archaea and Eukarya.

Marine microorganisms make a meal of oil

Hundreds of millions of litres of petroleum enter the environment from both natural and anthropogenic sources every year. The input from natural marine oil seeps alone would be enough to cover all of the worlds oceans in a layer of oil 20 molecules thick. That the globe is not swamped with oil is testament to the efficiency and versatility of the networks of microorganisms that degrade hydrocarbons, some of which have recently begun to reveal the secrets of when and how they exploit hydrocarbons as a source of carbon and energy.

Crude-oil biodegradation via methanogenesis in subsurface petroleum reservoirs

Biodegradation of crude oil in subsurface petroleum reservoirs has adversely affected the majority of the world’s oil, making recovery and refining of that oil more costly. The prevalent occurrence of biodegradation in shallow subsurface petroleum reservoirs has been attributed to aerobic bacterial hydrocarbon degradation stimulated by surface recharge of oxygen-bearing meteoric waters. This hypothesis is empirically supported by the likelihood of encountering biodegraded oils at higher levels of degradation in reservoirs near the surface. More recent findings, however, suggest that anaerobic degradation processes dominate subsurface sedimentary environments, despite slow reaction kinetics and uncertainty as to the actual degradation pathways occurring in oil reservoirs. Here we use laboratory experiments in microcosms monitoring the hydrocarbon composition of degraded oils and generated gases, together with the carbon isotopic compositions of gas and oil samples taken at wellheads and a Rayleigh isotope fractionation box model, to elucidate the probable mechanisms of hydrocarbon degradation in reservoirs. We find that crude-oil hydrocarbon degradation under methanogenic conditions in the laboratory mimics the characteristic sequential removal of compound classes seen in reservoir-degraded petroleum. The initial preferential removal of n-alkanes generates close to stoichiometric amounts of methane, principally by hydrogenotrophic methanogenesis. Our data imply a common methanogenic biodegradation mechanism in subsurface degraded oil reservoirs, resulting in consistent patterns of hydrocarbon alteration, and the common association of dry gas with severely degraded oils observed worldwide. Energy recovery from oilfields in the form of methane, based on accelerating natural methanogenic biodegradation, may offer a route to economic production of difficult-to-recover energy from oilfields.

Robust Hydrocarbon Degradation and Dynamics of Bacterial Communities during Nutrient-Enhanced Oil Spill Bioremediation

ABSTRACT Degradation of oil on beaches is, in general, limited by the supply of inorganic nutrients. In order to obtain a more systematic understanding of the effects of nutrient addition on oil spill bioremediation, beach sediment microcosms contaminated with oil were treated with different levels of inorganic nutrients. Oil biodegradation was assessed respirometrically and on the basis of changes in oil composition. Bacterial communities were compared by numerical analysis of denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA genes and cloning and sequencing of PCR-amplified 16S rRNA genes. Nutrient amendment over a wide range of concentrations significantly improved oil degradation, confirming that N and P limited degradation over the concentration range tested. However, the extent and rate of oil degradation were similar for all microcosms, indicating that, in this experiment, it was the addition of inorganic nutrients rather than the precise amount that was most important operationally. Very different microbial communities were selected in all of the microcosms. Similarities between DGGE profiles of replicate samples from a single microcosm were high (95% ± 5%), but similarities between DGGE profiles from replicate microcosms receiving the same level of inorganic nutrients (68% ± 5%) were not significantly higher than those between microcosms subjected to different nutrient amendments (63% ± 7%). Therefore, it is apparent that the different communities selected cannot be attributed to the level of inorganic nutrients present in different microcosms. Bioremediation treatments dramatically reduced the diversity of the bacterial community. The decrease in diversity could be accounted for by a strong selection for bacteria belonging to the alkane-degrading Alcanivorax/Fundibacter group. On the basis of Shannon-Weaver indices, rapid recovery of the bacterial community diversity to preoiling levels of diversity occurred. However, although the overall diversity was similar, there were considerable qualitative differences in the community structure before and after the bioremediation treatments.

The controls on the composition of biodegraded oils in the deep subsurface—part 1: biodegradation rates in petroleum reservoirs

Abstract Biodegradation rates in oilfields have been assessed conservatively using whole oil-column minimum rate estimates, diffusion-controlled oil column compositional gradient modelling and mixed oil kinetic models. Biodegradation rate constants (first order) are around 10−6–10−7 yr−1 for hydrocarbons in the degradation zones these corresponding well with zero order field-wide minimum rate estimates of about 10−8 kg hydrocarbons/kg oil/year for the whole oil column. With biodegradation induction times of around 1–2 Ma to perturb an entire oil column for light oil reservoirs and 10–20 Ma for heavy oil reservoir degradation the results indicate that where we see continuous gradients in the oil columns, degradation must have been occurring episodically for many millions of years. To remove the n-alkanes from an oil (i.e. about 10% of an oil) around ca 15 Ma is needed for a heavy oil (ca 5 Ma for a light N. Sea oil). The timescales of oilfield degradation and filling are thus very similar and consequently the degree of biodegradation will be substantially controlled by oilfield charge history. Assessment of mixed degraded/non-degraded oil occurrence provides an independent confirmation that these rates are realistic and that timescales of degradation and field charging are similar. The maximum effective rate constant of degradation, ultimately controlled by the limiting effect of diffusion of alkanes to the oil water contact (OWC) (ca 10−4 yr−1 for a 130 m thick oil column first order rate constant) is well above the estimated rate constants indicating oil biodegradation rate is not limited by electron donor supply (i.e. hydrocarbons) but by supply of nutrients or oxidants. This suggests that diffusive transport of nutrients and electron acceptors in the aquifer to the site of biodegradation may be adequate to maintain the low rate biosphere.

Bacterial community dynamics and hydrocarbon degradation during a field-scale evaluation of bioremediation on a mudflat beach contaminated with buried oil

ABSTRACT A field-scale experiment with a complete randomized block design was performed to study the degradation of buried oil on a shoreline over a period of almost 1 year. The following four treatments were examined in three replicate blocks: two levels of fertilizer treatment of oil-treated plots, one receiving a weekly application of liquid fertilizer and the other treated with a slow-release fertilizer; and two controls, one not treated with oil and the other treated with oil but not with fertilizer. Oil degradation was monitored by measuring carbon dioxide evolution and by chemical analysis of the oil. Buried oil was degraded to a significantly greater extent in fertilized plots, but no differences in oil chemistry were observed between the two different fertilizer treatments, although carbon dioxide production was significantly higher in the oil-treated plots that were treated with slow-release fertilizer during the first 14 days of the experiment. Bacterial communities present in the beach sediments were profiled by denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA gene fragments and 16S rRNA amplified by reverse transcriptase PCR. Similarities between the DGGE profiles were calculated, and similarity matrices were subjected to statistical analysis. These analyses showed that although significant hydrocarbon degradation occurred both in plots treated with oil alone and in the plots treated with oil and liquid fertilizer, the bacterial community structure in these plots was, in general, not significantly different from that in the control plots that were not treated with oil and did not change over time. In contrast, the bacterial community structure in the plots treated with oil and slow-release fertilizer changed rapidly, and there were significant differences over time, as well as between blocks and even within plots. The differences were probably related to the higher concentrations of nutrients measured in interstitial water from the plots treated with slow-release fertilizer. Bacteria with 16S rRNA sequences closely related (>99.7% identity) to Alcanivorax borkumensis and Pseudomonas stutzeri sequences dominated during the initial phase of oil degradation in the plots treated with slow-release fertilizer. Field data were compared to the results of previous laboratory microcosm experiments, which revealed significant differences.

Thaumarchaeotes abundant in refinery nitrifying sludges express amoA but are not obligate autotrophic ammonia oxidizers

Nitrification is a core process in the global nitrogen cycle that is essential for the functioning of many ecosystems. The discovery of autotrophic ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota has changed our perception of the microbiology of nitrification, in particular since their numerical dominance over ammonia-oxidizing bacteria (AOB) in many environments has been revealed. These and other data have led to a widely held assumption that all amoA-encoding members of the Thaumarchaeota (AEA) are autotrophic nitrifiers. In this study, 52 municipal and industrial wastewater treatment plants were screened for the presence of AEA and AOB. Thaumarchaeota carrying amoA were detected in high abundance only in four industrial plants. In one plant, thaumarchaeotes closely related to soil group I.1b outnumbered AOB up to 10,000-fold, and their numbers, which can only be explained by active growth in this continuous culture system, were two to three orders of magnitude higher than could be sustained by autotrophic ammonia oxidation. Consistently, 14CO2 fixation could only be detected in AOB but not in AEA in actively nitrifying sludge from this plant via FISH combined with microautoradiography. Furthermore, in situ transcription of archaeal amoA, and very weak in situ labeling of crenarchaeol after addition of 13CO2, was independent of the addition of ammonium. These data demonstrate that some amoA-carrying group I.1b Thaumarchaeota are not obligate chemolithoautotrophs.

Microbial landscapes: new paths to biofilm research.

It is the best of times for biofilm research. Systems biology approaches are providing new insights into the genetic regulation of microbial functions, and sophisticated modelling techniques are enabling the prediction of microbial community structures. Yet it is also clear that there is a need for ecological theory to contribute to our understanding of biofilms. Here, we suggest a concept for biofilm research that is spatially explicit and solidly rooted in ecological theory, which might serve as a universal approach to the study of the numerous facets of biofilms.

Composition and diversity of ammonia‐oxidising bacterial communities in wastewater treatment reactors of different design treating identical wastewater

Autotrophic ammonia-oxidising bacteria (AOB) are a crucial component of the microbial communities of nitrifying wastewater treatment systems. Nitrification is known to occur in reactors of different configuration, but whether AOB communities are different in reactors of different design is unknown. We compared the diversity and community structure of the betaproteobacterial AOB in two full-scale treatment reactors - a biological aerated filter (BAF) and a trickling filter - receiving the same wastewater. Polymerase chain reaction (PCR) of 16S ribosomal RNA (rRNA) gene fragments with AOB-selective primers was combined with denaturing gradient gel electrophoresis (DGGE) to allow comparative analysis of the dominant AOB populations. The phylogenetic affiliation of the dominant AOB was determined by cloning and sequencing PCR-amplified 16S rRNA gene fragments. DGGE profiles were compared using a probability-based similarity index (Raup and Crick). The use of a probability-based index of similarity allowed us to evaluate if the differences and similarities observed in AOB community structure in different samples were statistically significant or could be accounted for by chance matching of bands in DGGE profiles, which would suggest random colonisation of the reactors by different AOB. The community structure of AOB was different in different sections of each of the reactors and differences were also noted between the reactors. All AOB-like sequences identified, grouped within the genus Nitrosomonas. A greater diversity of AOB was detected in the trickling filters than in the BAF though all samples analysed appeared to be dominated by AOB most closely related to Nitrosococcus mobilis. Numerical analysis of DGGE profiles indicated that the AOB communities in depth profiles from the filter beds were selected in a non-random manner.