Fourier transform infrared spectroscopy as a metabolite fingerprinting tool for monitoring the phenotypic changes in complex bacterial communities capable of degrading phenol

The coking process produces great volumes of wastewater contaminated with pollutants such as cyanides, sulfides and phenolics. Chemical and physical remediation of this wastewater removes the majority of these pollutants; however, these processes do not remove phenol and thiocyanate. The removal of these compounds has been effected during bioremediation with activated sludge containing a complex microbial community. In this investigation we acquired activated sludge from an industrial bioreactor capable of degrading phenol. The sludge was incubated in our laboratory and monitored for its ability to degrade phenol over a 48 h period. Multiple samples were taken across the time‐course and analysed by Fourier transform infrared (FT‐IR) spectroscopy. FT‐IR was used as a whole‐organism fingerprinting approach to monitor biochemical changes in the bacterial cells during the degradation of phenol. We also investigated the ability of the activated sludge to degrade phenol following extended periods (2–131 days) of storage in the absence of phenol. A reduction was observed in the ability of the microbial community to degrade phenol and this was accompanied by a detectable biochemical change in the FT‐IR fingerprint related to cellular phenotype of the microbial community. In the absence of phenol a decrease in thiocyanate vibrations was observed, reflecting the ability of these communities to degrade this substrate. Actively degrading communities showed an additional new band in their FT‐IR spectra that could be attributed to phenol degradation products from the ortho‐ and meta‐cleavage of the aromatic ring. This study demonstrates that FT‐IR spectroscopy when combined with chemometric analysis is a very powerful high throughput screening approach for assessing the metabolic capability of complex microbial communities.     

Effect of adaptation to phenol on biodegradation of monosubstituted phenols by aquatic microbial communities.

The adaptation of a mixed aquatic microbial community to phenol was examined in microcosms receiving phenol as a sole carbon source. Extended exposure (adaptation) to phenol resulted in adaptation of the microbial community to the structurally related aromatic compounds m-cresol, m-aminophenol, and p-chlorophenol. The increased biodegradation potential of the phenol-adapted microbial community was accompanied by a concurrent increase in the number of microorganisms able to degrade the three test compounds. Thus, adaptation to the three test chemicals was likely a growth-related result of extended exposure to phenol. The results indicate that adaptation to a single chemical may increase the assimilative capacity of an aquatic environment for other related chemicals even in the absence of adaptation-inducing levels of those materials.     

The influence of the invasive shrub, Lonicera maackii, on leaf decomposition and microbial community dynamics

Amur honeysuckle (Lonicera maackii) is an exotic invasive shrub that is rapidly expanding into forests of eastern North America. This species forms a dense forest understory, alters tree regeneration, negatively affects herb-layer biodiversity, and alters ecosystem function. In a second-growth forest in central Kentucky, we examined the timing and production of leaf litter and compared litter chemistry, decay rates, and microbial community colonization of Amur honeysuckle to that of two native trees, white ash (Fraxinus americana) and hickory (Carya spp.). The distribution of Amur honeysuckle was clumped, allowing us to compare differences in decomposition under and away from Amur honeysuckle shrubs. Amur honeysuckle leaf litter had significantly higher nitrogen, lower C:N, and lower lignin than the other species, and decomposition rates were greater than 5×?faster. Despite the much higher rate of Amur honeysuckle decomposition compared with the native species (p?<?0.0001), decomposition of all species was significantly slower (p?=?0.0489) in sites located under Amur honeysuckle shrubs. Nitrogen concentration increased through time in decomposing Amur honeysuckle litter; however, total mass of N rapidly declined. We found the initial microbial community on leaf litter of Amur honeysuckle was distinct from two native species and although all microbial communities changed through time, the microbial community of Amur honeysuckle remained distinct from native communities. In summary, a distinct microbial community that may originate on Amur honeysuckle leaves prior to senescence could explain the rapid decay rates.     

Microbial degradation of pollutants at high salt concentrations

Though our knowledge on microbial degradation of organic pollutants at high salt concentrations is still limited, the list of toxic compounds shown to be degraded or transformed in media of high salinity is growing. Compounds transformed aerobically include saturated and aromatic hydrocarbons (by certain archaeobacteria), certain aromatic compounds, organophosphorus compounds, and formaldehyde (by halotolerant eubacteria). Anaerobic microbial transformations of toxic compounds occurring at high salt concentrations include reduction of nitroaromatic compounds, and possibly transformation of chlorinated aromatic compounds.     

Distribution,abundance, and size structure of Amur kaluga <Emphasis Type="Italic">Acipenser dauricus</Emphasis> and Amur sturgeon <Emphasis Type="Italic">A. schrenckii</Emphasis> in the Lower Amur and Amur Estuary

Distribution, population structure, and abundance of Amur kaluga Acipenser dauricus and Amur sturgeon A. schrenckii in the Lower Amur and Amur Estuary have been considered based on the materials of two surveys performed in 2011. Both species occur along almost the entire length of the Lower Amur and in major part of the Amur Estuary. The density of their aggregations has significantly decreased in most regions of the river if compared to the 1960s. In the Amur Estuary, A. dauricus and A. schrenckii are most abundant in freshwater and light salted water bodies of its western part. In 2011, the abundance and biomass of A. dauricus and A. schrenckii in the Lower Amur and Amur Estuary was 345000 specimens and 7110 tons and 289000 specimens and 1946 tons, respectively.     

Enrichment and isolation of non-specific aromatic degraders from unique uncontaminated (plant and faecal material) sources and contaminated soils

Microbial analysis of contaminated soil and uncontaminated plant and faecal material resulted in the enrichment of a number of microbial communities capable of utilizing a range of environmental pollutants. Growth was observed on polycyclic aromatic hydrocarbons, polychlorinated biphenyls, heterocyclic aromatic compounds and organochlorine pesticides. However, none of the communities could grow on pentachlorophenol. Pure cultures were isolated from microbial communities using phenanthrene and pyrene as the sole carbon and energy source. Isolates were also obtained using DDT, DOH, DBH and PCPA when peptone was supplemented to the medium. Strain AJR39,504, isolated using DDT and peptone, could not be positively identified on the basis of substrate utilization tests. However, it most closely resembled Stenotrophomonas maltophilia (0.424 similarity) using the Microlog 3 database software. Isolate AJR39, 504 could also grow on polycyclic aromatic hydrocarbons, chlorinated- and nitro-aromatic compounds. In addition, the degradation of DDT (100 mg l(-1)) by isolate AJR39,504 resulted in a 35% decrease in DDT concentration after 28 days with a concomitant increase in DDD concentration.     

Identification of Genes and Pathways Related to Phenol Degradation in Metagenomic Libraries from Petroleum Refinery Wastewater

Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.     

Increasing concentrations of phenol progressively affect anaerobic digestion of cellulose and associated microbial communities

Performance stability is a key issue when managing anaerobic digesters. However it can be affected by external disturbances caused by micropollutants. In this study the influence of phenol on the methanization of cellulose was evaluated through batch toxicity assays. Special attention was given to the dynamics of microbial communities by means of automated ribosomal intergenic spacer analysis. We observed that, as phenol concentrations increased, the different steps of anaerobic cellulose digestion were unevenly and progressively affected, methanogenesis being the most sensitive: specific methanogenic activity was half-inhibited at 1.40 g/L of phenol, whereas hydrolysis of cellulose and its fermentation to VFA were observed at up to 2.00 g/L. Depending on the level of phenol, microbial communities resisted either through physiological or structural adaptation. Thus, performances at 0.50 g/L were maintained in spite of the microbial community’s shift. However, the communities’ ability to adapt was limited and performances decreased drastically beyond 2.00 g/L of phenol.     

Time-series metagenomic analysis reveals robustness of soil microbiome against chemical disturbance

Soil microbial communities have great potential for bioremediation of recalcitrant aromatic compounds. However, it is unclear which taxa and genes in the communities, and how they contribute to the bioremediation in the polluted soils. To get clues about this fundamental question here, time-course (up to 24 weeks) metagenomic analysis of microbial community in a closed soil microcosm artificially polluted with four aromatic compounds, including phenanthrene, was conducted to investigate the changes in the community structures and gene pools. The pollution led to drastic changes in the community structures and the gene sets for pollutant degradation. Complete degradation of phenanthrene was strongly suggested to occur by the syntrophic metabolism by Mycobacterium and the most proliferating genus, Burkholderia. The community structure at Week 24 (∼12 weeks after disappearance of the pollutants) returned to the structure similar to that before pollution. Our time-course metagenomic analysis of phage genes strongly suggested the involvement of the ‘kill-the-winner’ phenomenon (i.e. phage predation of Burkholderia cells) for the returning of the microbial community structure. The pollution resulted in a decrease in taxonomic diversity and a drastic increase in diversity of gene pools in the communities, showing the functional redundancy and robustness of the communities against chemical disturbance.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

Bacterial degradation of airborne phenol in the phyllosphere

Despite the vast surface area of terrestrial plant leaves and the large microbial communities they support, little is known of the ability of leaf-associated microorganisms to access and degrade airborne pollutants. Here, we examined bacterial acquisition and degradation of phenol on leaves by an introduced phenol degrader and by natural phyllosphere communities. Whole-cell gfp-based Pseudomonas fluorescens bioreporter cells detected phenol on leaves that had previously been transiently exposed to gaseous phenol, indicating that leaves accumulated phenol; moreover, they accumulated it in sites that were accessible to epiphytic bacteria and to concentrations that were at least 10-fold higher than those in the air. After inoculated leaves were exposed to gaseous 14C-phenol, leaves harbouring the phenol-degrading Pseudomonas sp. strain CF600 released eight times more 14CO2 than did leaves harbouring a non-degrading mutant, demonstrating that CF600 actively mineralized phenol on leaves. We evaluated phenol degradation by natural microbial communities on green ash leaves that were collected from a field site rich in airborne organic pollutants. We found that significantly more phenol was mineralized by these leaves when the communities were present than by these leaves following surface sterilization. Thus, phenol-degrading organisms were present in these natural communities and were metabolically capable of phenol degradation. Collectively, these results provide the first direct evidence that bacteria on leaves can degrade an organic pollutant from the air, and indicate that bacteria on leaves could potentially contribute to the natural attenuation of organic air pollutants.     

Amended diagnosis,validity and relationships of the genus Acrolichanus Ward, 1917 (Digenea: Allocreadiidae) based on the 28S rRNA gene,and observations on its lineage diversity

Systematic Parasitology - Adult specimens of the allocreadiid trematode Acrolichanus auriculatus (Wedl, 1858) were collected from Acipenser schrenckii Brandt from the River Amur and Amur Estuary,...     

Microbial degradation of phenol and phenolic derivatives

Phenol and its derivatives are one of the largest groups of environmental pollutants due to their presence in many industrial effluents and broad application as antibacterial and antifungal agents. A number of microbial species possess enzyme systems that are applicable for the decomposition of various aliphatic and aromatic toxic compounds. Intensive efforts to screen species with high‐degradation activity are needed to study their capabilities of degrading phenol and phenolic derivatives. Most of the current research has been directed at the isolation and study of microbial species of potential ecological significance. In this review, some of the best achievements in degrading phenolic compounds by bacteria and yeasts are presented, which draws attention to the high efficiency of strains of Pseudomonas, Candida tropicalis, Trichosporon cutaneum, etc. The unique ability of fungi to maintain their degradation potential under conditions unfavorable for other microorganisms is outstanding. Mathematical models of the microbial biodegradation dynamics of single and mixed aromatic compounds, which direct to the benefit of the processes studied in optimization of modern environmental biotechnology are also presented.     

Methanogenic destruction of (amino)aromatic compounds by anaerobic microbial communities

Destruction of a number of aromatic substrates by anaerobic microbial communities was studied. Active methanogenic microbial communities decomposing aminoaromatic acids and azo dyes into CH4 and CO2 were isolated. Products of primary conversion were found to be 2-hydroxybenzyl and benzyl alcohols gradually transforming into benzoate. It was shown that isolated microbial communities are capable of converting the initial substrates--benzyl alcohol, benzoate, salicylic acid, and golden yellow azo dye--into biogas without a lag-phase but with different velocities. Aromatic and linear intermediates of biodestruction of aromatic amines by obtained enrichment cultures were determined for the first time. Selective effect of aromatic substrates on a microbial community that was expressed in decrease in diversity and gradual change of dominant morphotypes was revealed.     

Effects of Phenol Feeding Pattern on Microbial Community Structure and Cometabolism of Trichloroethylene

Cometabolism of trichloroethylene (TCE) by phenol-fed enrichments was evaluated in four reactors with distinct phenol feeding patterns. The reactors were inoculated from the same source, operated at the same average dilution rate, and received the same mass of phenol over time. Only the timing of phenol addition differed. Reactor C received phenol continuously; reactor SC5 received phenol semicontinuously--alternating between 5 h of feed and 3 h without feed; reactor SC2 alternated between 2 h of feed and 6 h without feed; and reactor P received a single pulse every 24 h. The structure of the enrichments and their capacity for TCE transformation were analyzed. In long-term operation, reactors C and SC5 were dominated by fungi, had higher levels of predators, were more susceptible to biomass fluctuations, and exhibited reduced capacity for TCE transformation. Reactors P and SC2 were characterized by lower levels of fungi, higher bacterial biomass, higher concentrations of TCE-degrading organisms, and higher rates of TCE transformation. After 200 days of operation, rates of TCE transformation increased 10-fold in reactor P, resulting in TCE transformation rates that were 20 to 100 times higher than the rates of the other reactor communities. The cause of this shift is unknown. Isolates capable of the highest rates of TCE transformation were obtained from reactor P. We conclude that cometabolic activity depends upon microbial community structure and that the community structure can be manipulated by altering the growth substrate feeding pattern.     

Influence of various phenolic compounds on phenol hydroxylase activity of a Trichosporon cutaneum strain

The phenol-degrading strain Trichosporon cutaneum R57 utilizes various aromatic and aliphatic compounds as a sole carbon and energy source. The intracellular activities of phenol hydroxylase [EC 1.14.13.7] of a Trichosporon cutaneum R57 strain grown on phenol (0.5 g/l) were measured. Different toxic phenol derivatives (cresols, nitrophenols and hydroxyphenols) were used as substrates in the reaction mixture for determination of the enzyme activity. The data obtained showed that the investigated enzyme was capable to hydroxylate all applied aromatic substrates. The measured activities of phenol hydroxylase varied significantly depending on the aromatic compounds used as substrates. The rate of phenol hydroxylase activity with phenol as a substrate (1.0 U/mg total cell protein) was accepted as 100%.     

Methanogenic transformation of aromatic hydrocarbons and phenols in groundwater aquifers

Fermentative and methanogenic bacteria have been found repeatedly as important members of microbial flora in anoxic zones of the subsurface—in pristine as well as in contaminated groundwater aquifers. These bacteria, which together with obligate proton reducers form complex methanogenic communities, are significant as decomposers of organic matter under conditions of exogenous electron acceptor depletion. Their metabolic activity has been demonstrated in laboratory microcosms derived from aquifer material, and also in the subsurface in situ. Methanogenic communities have been shown to transform numerous organic pollutants, or even to completely degrade these compounds with the production of carbon dioxide and methane. Depending on the chemical structure of the pollutant, such a compound can be used as an electron donor and a carbon/energy source for fermentative microorganisms (which is typically the case with highly reduced compounds); alternatively, a highly oxidized pollutant can be used as a potential electron acceptor or electron sink. This review addresses fermentative/methanogenic degradation of chlorinated and nonchlorinated aromatic hydrocarbons and phenols by subsurface microorganisms; for comparison, it briefly relates also other types of anaerobic transformations (under sulfate‐reducing, iron‐reducing, and denitrifying conditions). Furthermore, it outlines transformation pathways, those that are proposed as well as those that are already partially proved, for aromatic hydrocarbons and phenols under fermentative/methanogenic conditions; finally, it discusses the relevance of these processes to bioremediation of contaminated groundwater aquifers.     

Phytoplankton of the Amur River Estuary (Sea of Okhotsk) during the summer periods of 2005–2007

The qualitative composition of planktonic microalgae in the Amur River Estuary (in the Sea of Okhotsk) was studied in July 2005, June 2006, and June 2007. The biomass and abundance of phytoplankton were studied for the first time. The average cell density of the microalgae ranged from 194700 to 855100 cells/liter, being higher in 2006 than in 2005 and 2007. The average biomass of the community varied from 1.06 g/m³ in 2007 to 3.17 g/m³ in 2006. Analysis of the similarity of the species composition and cell density of the phytoplankton revealed two groups of stations: the first includes stations in the northern and central parts of the Amur Estuary; and the second includes stations in the southern and central parts. The first group was characterized by the predominance of freshwater diatoms and a high abundances of green and blue-green algae and the second group was characterized by the predominance of marine microalgae. The high cell density and biomass of phytoplankton were due to the increased nutrient input via the river runoff of the Amur.     

Toward Understanding the Dynamics of Microbial Communities in an Estuarine System

Community assembly theories such as species sorting theory provide a framework for understanding the structures and dynamics of local communities. The effect of theoretical mechanisms can vary with the scales of observation and effects of specific environmental factors. Based on 16S rRNA gene tag pyrosequencing, different structures and temporal succession patterns were discovered between the surface sediments and bottom water microbial communities in the Pearl River Estuary (PRE). The microbial communities in the surface sediment samples were more diverse than those in the bottom water samples, and several genera were specific for the water or sediment communities. Moreover, water temperature was identified as the main variable driving community dynamics and the microbial communities in the sediment showed a greater temporal change. We speculate that nutrient-based species sorting and bacterial plasticity to the temperature contribute to the variations observed between sediment and water communities in the PRE. This study provides a more comprehensive understanding of the microbial community structures in a highly dynamic estuarine system and sheds light on the applicability of ecological theoretical mechanisms.     

Microbial transformation of propenylbenzenes for natural flavour production

Propenylbenzenes are common aromatic compounds that are often used as starting compounds for the production of various flavours. Recently, microbial transformation has emerged as an important approach for producing natural flavours in high quantities. Because the biotransformation processes are environmentally friendly and the products are considered 'natural', flavour production using this method is attracting more and more attention. This paper reviews recent advances in the application of microbial metabolism to propenylbenzenes and in subsequent flavour production. Vanillin, a valuable aromatic compound, is used as a model to show recent progress in high-value natural flavour production. Future research should focus on metabolic-mechanism characterisation and on optimisation of biotransformation to improve the yields of target products for scale-up and industrial use.