Looking through the FOG: microbiome characterization and lipolytic bacteria isolation from a fatberg site.

Sewer systems are complex physical, chemical and microbial ecosystems where fats, oils and grease (FOG) present a major problem for sewer management. Their accumulation can lead to blockages ('Fatbergs'), sewer overflows and disruption of downstream wastewater treatment. Further advancements of biological FOG treatments need to be tailored to degrade the FOG, and operate successfully within the sewer environment. In this study we developed a pipeline for isolation of lipolytic strains directly from two FOG blockage sites in the UK, and isolated a range of highly lipolytic bacteria. We selected the five most lipolytic strains using Rhodamine B agar plates and pNP-Fatty acid substrates, with two Serratia spp., two Klebsiella spp. and an environmental Acinetobacter strain that all have the capacity to grow on FOG-based carbon sources. Their genome sequences identified the genetic capacity for fatty acid harvesting (lipases), catabolism and utilization (Fad genes). Furthermore, we performed a preliminary molecular characterization of the microbial community at these sites, showing a diverse community of environmental bacteria at each site, but which did include evidence of sequences related to our isolates. This study provides proof of concept to isolation strategies targeting Fatberg sites to yield candidate strains with bioremediation potential for FOG in the wastewater network. Our work sets the foundation for development of novel bioadditions tailored to the environment with non-pathogenic Acinetobacter identified as a candidate for this purpose.

Enumeration of sulphate-reducing bacteria for assessing potential for hydrogen sulphide production in urban drainage systems.

Urban drainage structures have increasing demands which can lead to increasing hydrogen sulphide related problems forming in places where they have not previously been prevalent. This puts pressure on the methods currently used to monitor and diagnose these problems and more sophisticated methods may be needed for identifying the origin of the problems. Molecular microbiological techniques, such as quantitative polymerase chain reaction, offer a potential alternative for identifying and quantifying bacteria likely to be causing the production of hydrogen sulphide, information that, when combined with an appropriate sampling programme, can then be used to identify the potentially most effective remediation technique. The application of these methods in urban drainage systems is, however, not always simple, but good results can be achieved. In this study bacteria producing hydrogen sulphide were quantified in three small combined sewer overflow storage tanks. Bacterial counts were compared between wastewater, biofilms and sediments. Similar numbers were found in the wastewater and biofilms, with the numbers in the sediments being lower. If remediation methods for hydrogen sulphide are deemed necessary in the tanks, methods that target both the wastewater and the biofilms should therefore be considered.

Biodesalination: a case study for applications of photosynthetic bacteria in water treatment.

Shortage of freshwater is a serious problem in many regions worldwide, and is expected to become even more urgent over the next decades as a result of increased demand for food production and adverse effects of climate change. Vast water resources in the oceans can only be tapped into if sustainable, energy-efficient technologies for desalination are developed. Energization of desalination by sunlight through photosynthetic organisms offers a potential opportunity to exploit biological processes for this purpose. Cyanobacterial cultures in particular can generate a large biomass in brackish and seawater, thereby forming a low-salt reservoir within the saline water. The latter could be used as an ion exchanger through manipulation of transport proteins in the cell membrane. In this article, we use the example of biodesalination as a vehicle to review the availability of tools and methods for the exploitation of cyanobacteria in water biotechnology. Issues discussed relate to strain selection, environmental factors, genetic manipulation, ion transport, cell-water separation, process design, safety, and public acceptance.

Development of wastewater treatment system based on cascade dielectric barrier discharge plasma atomizers.

A novel design for a cascade dielectric barrier discharge (DBD) atomizer was applied for treating samples of water containing biological and organic contaminants. Several experimental investigations were conducted on artificial samples and real sample (digested sludge collected from a wastewater treatment plant, WWTP). The artificial water samples were prepared by using different concentrations of E. coli for biological samples, whereas acetic acid was used to prepare the organic samples. The biological samples were subjected to the plasma effect for different treatment periods, and the growth curves of E. coli were generated for 24 h after treatment. Moreover, the viable cells were counted after each treatment period and the change in E. coli morphology was monitored. The results showed that a significant reduction in the viable cell number, by 3 orders of magnitude, occurred for an artificial biological sample after only 5-min treatment. The treatment of organic samples for 10 min showed a significant reduction in the concentration of acetic acid by 50%. In consequence, treatment of real wastewater sample for 10 min resulted in more than 70% reduction in BOD5 and 30% reduction in COD.

Comparative study of in vitro expansion of bone marrow-derived mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BMD-MSCs) are of great interest for tissue engineering, but require expansion before they can be used for therapeutic applications. We compared three different culture techniques for their potential for large scale expansion of rat BMD-MSCs, i.e. monolayer cultures, stirred suspension cultures and pour-off cultures, and found that pour-off cultures supported the biggest expansion in BMD-MSCs as measured by the fibroblastic-colony forming unit assay (CFU-f). BMD-MSCs expanded in stirred suspension cultures stopped proliferating altogether and, although monolayer cultures allowed for expansion of BMD-MSCs, they favoured a differentiated phenotype over uncommitted MSCs. Only BMD-MSCs expanded in pour-off cultures were able to differentiate into both osteoblastic and adipocytic lineages and maintain CFU-f numbers. These data suggest that pour-off cultures are a viable method of BMD-MSC expansion.

Influence of fermentation conditions on the surface properties and adhesion of Lactobacillus rhamnosus GG.

BACKGROUND: The surface properties of probiotic bacteria influence to a large extent their interactions within the gut ecosystem. There is limited amount of information on the effect of the production process on the surface properties of probiotic lactobacilli in relation to the mechanisms of their adhesion to the gastrointestinal mucosa. The aim of this work was to investigate the effect of the fermentation pH and temperature on the surface properties and adhesion ability to Caco-2 cells of the probiotic strain Lactobacillus rhamnosus GG. RESULTS: The cells were grown at pH 5, 5.5, 6 (temperature 37°C) and at pH 6.5 (temperature 25°C, 30°C and 37°C), and their surfaces analysed by X-ray photoelectron spectrometry (XPS), Fourier transform infrared spectroscopy (FT-IR) and gel-based proteomics. The results indicated that for all the fermentation conditions, with the exception of pH 5, a higher nitrogen to carbon ratio and a lower phosphate content was observed at the surface of the bacteria, which resulted in a lower surface hydrophobicity and reduced adhesion levels to Caco-2 cells as compared to the control fermentation (pH 6.5, 37°C). A number of adhesive proteins, which have been suggested in previous published works to take part in the adhesion of bacteria to the human gastrointestinal tract, were identified by proteomic analysis, with no significant differences between samples however. CONCLUSIONS: The temperature and the pH of the fermentation influenced the surface composition, hydrophobicity and the levels of adhesion of L. rhamnosus GG to Caco-2 cells. It was deduced from the data that a protein rich surface reduced the adhesion ability of the cells.

An insight into iTRAQ: where do we stand now?

The iTRAQ (isobaric tags for relative and absolute quantification) technique is widely employed in proteomic workflows requiring relative quantification. Here, we review the iTRAQ literature; in particular, we focus on iTRAQ usage in relation to other commonly used quantitative techniques e.g. stable isotope labelling in culture (SILAC), label-free methods and selected reaction monitoring (SRM). As a result, we identify several issues arising with respect to iTRAQ. Perhaps frustratingly, iTRAQ's attractiveness has been undermined by a number of technical and analytical limitations: it may not be truly quantitative, as the changes in abundance reported will generally be underestimated. We discuss weaknesses and strengths of iTRAQ as a methodology for relative quantification in the light of this and other technical issues. We focus on technical developments targeted at iTRAQ accuracy and precision, use of 4-plex over 8-plex reagents and application of iTRAQ to post-translational modification (PTM) workflows. We also discuss iTRAQ in relation to label-free approaches, to which iTRAQ is losing ground.

Using a multi-faceted approach to determine the changes in bacterial cell surface properties influenced by a biofilm lifestyle.

Biofilm formation is a developmental process in which initial reversible adhesion is governed by physico-chemical forces, whilst irreversible adhesion is mediated by biological changes within a cell, such as the production of extracellular polymeric substances. Using two bacteria, E. coli MG1655 and B. cereus ATCC 10987, this study establishes that the surface of the bacterial cell also undergoes specific modifications, which result in biofilm formation and maintenance. Using various surface characterisation techniques and proteomics, an increase in the surface exposed proteins on E. coli cells during biofilm formation was demonstrated, along with an increase in hydrophobicity and a decrease in surface charge. For B. cereus, an increase in the surface polysaccharides during biofilm formation was found as well as a decrease in hydrophobicity and surface charge. This work therefore shows that surface modifications during biofilm formation occur and understanding these specific changes may lead to the formulation of effective biofilm control strategies in the future.

Proteomic analysis of the impact of static culturing on the expansion of rat bone marrow mesenchymal stem cells.

The clinical potential of mesenchymal stem cells (MSC) in tissue engineering and regenerative medicine is due to their self-renewal, proliferation and multi-lineage differentiation potential. Clinical use requires large cell numbers; which can, theoretically, be generated by ex vivo expansion of plastic adherent, MSC subpopulation, of bone marrow cells (BMC). Effects of serial culture on MSC phenotype were investigated using non-gel based quantitative proteomic methodology for static monolayer cultures of rat BMC. In total, 382 proteins were relatively quantified (≥ 2 peptides). Nine proteins were up-regulated and seven down-regulated at passage 4 relative to passage 2 (p ≤ 0.05). We propose that serial culture impacts on MSC expansion (observed decline in colony forming potential and colony size) is through a combination of osteogenic differentiation and ageing/senescence and propose six novel protein biomarkers as candidates for quality control purposes in bioprocessing.

Quantitative protein expression and cell surface characteristics of Escherichia coli MG1655 biofilms.

Cell surface physicochemical characterization techniques were combined with quantitative changes in protein expression, to investigate the biological and biophysical changes of Escherichia coli MG1655 cells when grown as a biofilm (BIO). The overall surface charge of BIO cells was found to be less negative, highlighting the need for a lower electrophoretic mobility for attachment to occur. Comparison of the chemical functional groups on the cell surface showed similar profiles, with the absorbance intensity higher for proteins and carbohydrates in the BIO cells. Quantitative proteomic analysis demonstrated that 3 proteins were significantly increased, and 9 proteins significantly decreased in abundance, in cells grown as a BIO compared to their planktonic counterparts, with 7 of these total 12 proteins unique to this study. Proteins showing significant increased or decreased abundance include proteins involved in acid resistance, DNA protection and binding and ABC transporters. Further predictive analysis of the metabolic pathways showed an increased abundance of the amino acid metabolism and tricarboxylic acid (TCA) cycle, with a decrease in expression within the pentose phosphate and glycolysis pathways. It is therefore hypothesized that cells grown as a BIO are still energetically viable potentially using amino acids as an indirect carbon backbone source into the TCA cycle.

Macromolecular fingerprinting of sulfolobus species in biofilm: a transcriptomic and proteomic approach combined with spectroscopic analysis.

Microorganisms in nature often live in surface-associated sessile communities, encased in a self-produced matrix, referred to as biofilms. Biofilms have been well studied in bacteria but in a limited way for archaea. We have recently characterized biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus, and S. tokodaii. These strains form different communities ranging from simple carpet structures in S. solfataricus to high density tower-like structures in S. acidocaldarius under static condition. Here, we combine spectroscopic, proteomic, and transcriptomic analyses to describe physiological and regulatory features associated with biofilms. Spectroscopic analysis reveals that in comparison to planktonic life-style, biofilm life-style has distinctive influence on the physiology of each Sulfolobus spp. Proteomic and transcriptomic data show that biofilm-forming life-style is strain specific (eg ca. 15% of the S. acidocaldarius genes were differently expressed, S. solfataricus and S. tokodaii had ~3.4 and ~1%, respectively). The -omic data showed that regulated ORFs were widely distributed in basic cellular functions, including surface modifications. Several regulated genes are common to biofilm-forming cells in all three species. One of the most striking common response genes include putative Lrs14-like transcriptional regulators, indicating their possible roles as a key regulatory factor in biofilm development.

Mechanisms of Bacillus cereus biofilm formation: an investigation of the physicochemical characteristics of cell surfaces and extracellular proteins.

Microbial biofilms contribute to biofouling in a wide range of processes from medical implants to processed food. The extracellular polymeric substances (EPS) are implicated in imparting biofilms with structural stability and resistance to cleaning products. Still, very little is known about the structural role of the EPS in Gram-positive systems. Here, we have compared the cell surface and EPS of surface-attached (biofilm) and free-floating (planktonic) cells of Bacillus cereus, an organism routinely isolated from within biofilms on different surfaces. Our results indicate that the surface properties of cells change during biofilm formation and that the EPS proteins function as non-specific adhesions during biofilm formation. The physicochemical traits of the cell surface and the EPS proteins give us an insight into the forces that drive biofilm formation and maintenance in B. cereus.

"Biofilmology": a multidisciplinary review of the study of microbial biofilms.

The observation of biofilm formation is not a new phenomenon. The prevalence and significance of biofilm and aggregate formation in various processes have encouraged extensive research in this field for more than 40 years. In this review, we highlight techniques from different disciplines that have been used to successfully describe the extracellular, surface and intracellular elements that are predominant in understanding biofilm formation. To reduce the complexities involved in studying biofilms, researchers in the past have generally taken a parts-based, disciplinary specific approach to understand the different components of biofilms in isolation from one another. Recently, a few studies have looked into combining the different techniques to achieve a more holistic understanding of biofilms, yet this approach is still in its infancy. In order to attain a global understanding of the processes involved in the formation of biofilms and to formulate effective biofilm control strategies, researchers in the next decade should recognise that the study of biofilms, i.e. biofilmology, has evolved into a discipline in its own right and that mutual cooperation between the various disciplines towards a multidisciplinary research vision is vital in this field.

Effect of temperature on the substrate utilization profiles of microbial communities in different sewer sediments.

Sewer systems represent an essential component of modern society. They have a major impact on our quality of life by preventing serious illnesses caused by waterborne diseases, by protecting the environment, and by enabling economic and social development through reducing flood risk. In the UK, systems are normally large and complex and, because of the long lifespan of these assets, their performance and hence their management are influenced by long-term environmental and urban changes. Recent work has focussed on the long-term changes in the hydraulic performance of these systems in response to climate change, e.g. rainfall and economic development. One climate-related driver that has received little attention is temperature, which may in itself have a complex dependence on factors such as rainfall. This study uses Biolog EcoPlates to investigate the effect of different temperatures (4 degrees C, 24 degrees C and 30 degrees C) on the carbon substrate utilization profiles of bacterial communities within sewer sediment deposits. Distinct differences in the metabolic profiles across the different temperatures were observed. Increasing temperature resulted in a shift in biological activity with an increase in the number of different carbon sources that can be utilized. Certain carboxylic and amino acids, however, did not support growth, regardless of temperature. Distinct differences in carbon utilization profiles were also found within sewers that have similar inputs. Therefore, this study has demonstrated that the carbon utilization profile for microbial communities found within sewer sediment deposits is dependent on both temperature and spatial variations.

A new coupon design for simultaneous analysis of in situ microbial biofilm formation and community structure in drinking water distribution systems.

This study presents a new coupon sampling device that can be inserted directly into the pipes within water distribution systems (WDS), maintaining representative near wall pipe flow conditions and enabling simultaneous microscopy and DNA-based analysis of biofilms formed in situ. To evaluate this sampling device, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate changes in biofilms on replicate coupons within a non-sterile pilot-scale WDS. FISH analysis demonstrated increases in bacterial biofilm coverage of the coupon surface over time, while the DGGE analysis showed the development of increasingly complex biofilm communities, with time-specific clustering of these communities. This coupon design offers improvements over existing biofilm sampling devices in that it enables simultaneous quantitative and qualitative compositional characterization of biofilm assemblages formed within a WDS, while importantly maintaining fully representative near wall pipe flow conditions. Hence, it provides a practical approach that can be used to capture the interactions between biofilm formation and changing abiotic conditions, boundary shear stress, and turbulent driven exchange within WDS.

Publisher Correction: Deciphering the unique cellulose degradation mechanism of the ruminal bacterium Fibrobacter succinogenes S85.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Deciphering the unique cellulose degradation mechanism of the ruminal bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes S85, isolated from the rumen of herbivores, is capable of robust lignocellulose degradation. However, the mechanism by which it achieves this is not fully elucidated. In this study, we have undertaken the most comprehensive quantitative proteomic analysis, to date, of the changes in the cell envelope protein profile of F. succinogenes S85 in response to growth on cellulose. Our results indicate that the cell envelope proteome undergoes extensive rearrangements to accommodate the cellulolytic degradation machinery, as well as associated proteins involved in adhesion to cellulose and transport and metabolism of cellulolytic products. Molecular features of the lignocellulolytic enzymes suggest that the Type IX secretion system is involved in the translocation of these enzymes to the cell envelope. Finally, we demonstrate, for the first time, that cyclic-di-GMP may play a role in mediating catabolite repression, thereby facilitating the expression of proteins involved in the adhesion to lignocellulose and subsequent lignocellulose degradation and utilisation. Understanding the fundamental aspects of lignocellulose degradation in F. succinogenes will aid the development of advanced lignocellulosic biofuels.

Application of enhanced assimilable organic carbon method across operational drinking water systems.

Assimilable organic carbon (AOC) is known to correlate with microbial growth, which can consequently degrade drinking water quality. Despite this, there is no standardised AOC test that can be applied to drinking water distribution systems (DWDS). Herein we report the development of a quick, robust AOC that incorporates known strains Pseudomonas fluorescens strain P-17 and Spirillum strain NOX, a higher inoculum volume and enumeration using flow cytometry to generate a quicker (total test time reduced from 14 to 8 days), robust method. We apply the developed AOC test to twenty drinking water treatment works (WTW) to validate the method reproducibility and resolution across a wide range of AOC concentrations. Subsequently, AOC was quantified at 32 sample points, over four DWDS, for a year in order to identify sinks and sources of AOC in operative networks. Application of the developed AOC protocol provided a previously unavailable insight and novel evidence of pipes and service reservoirs exhibiting different AOC and regrowth behaviour. Observed correlations between AOC and microbial growth highlight the importance of monitoring AOC as an integral part of managing drinking water quality at the consumers tap.

A cross-species quantitative proteomic study of salt adaptation in a halotolerant environmental isolate using 15N metabolic labelling.

We examine differential protein expression in Euhalothece sp. BAA001, an extremely halotolerant and unsequenced cyanobacterium, under adaptation to low (0% w/v), medium (3% w/v), high (6% w/v) and very high (9% w/v) salt concentrations using cross-species protein identification tools. We combine stable isotope labelling with 1-D SDS-PAGE, and MASCOT protein identification software with MS-driven BLAST searches, to produce an accurate method for protein identification and quantitation. The use of metabolic labelling to improve the confidence in identification of proteins in cross-species proteomics is demonstrated. Three hundred and eighty-three unique proteins were identified, and 72 were deemed to be differentially expressed (average CV for quantitations was 0.10 +/- 0.08), belonging to 24 functional groups. Responses to low salt as well as high salt are discussed in terms of adaptation and evidence shows that Euhalothece cells display 'stress' responses in nonsaline conditions as well as higher salt environments.

Characterization of the extracellular polymeric substances produced by Escherichia coli using infrared spectroscopic, proteomic, and aggregation studies.

The aim of this study was twofold: first, to characterize the free extracellular polymeric substances (EPS) and bound EPS produced by Escherichia coli during different growth phases in different media, and then to investigate the role of the free EPS in promoting aggregation. EPS was extracted from a population of E. coli MG1655 cells grown in different media composition (Luria-Bertani (LB) and Luria-Bertani with the addition of 0.5 w/v% glucose at the beginning of the growth phase (LBG)) and at different growth phases (6 and 24 h). The extracted EPS was characterized using Fourier transform infrared spectroscopy and further identified using one-dimensional gel-based electrophoresis and tandem mass spectrometry. E. coli MG1655 was found to produce significantly lower amounts of bound EPS compared to free EPS under all conditions. The protein content of free EPS increased as the cells progressed from the exponential to stationary phase when grown in LB or LBG, while the carbohydrate content only increased across the growth phases for cells grown in LBG. FTIR revealed a variation in the different functional groups such as amines, carboxyl, and phosphoryl groups for free EPS extracted at the different growth conditions. Over 500 proteins were identified in the free EPS, with 40 proteins common in all growth conditions. Proteins with functionality related to amino acid and carbohydrate metabolism, as well as cell wall and membrane biogenesis were among the highest proteins identified in the free EPS extracted from E. coli MG1655 under all growth and media conditions. The role of bound and free EPS was investigated using a standardized aggregation assay. Bound EPS did not contribute to aggregation of E. coli MG1655. The readdition of free EPS to E. coli MG1655 resulted in aggregation of the cells in all growth conditions. Free EPS extracted from the 24 h E. coli MG1655 cultures grown in LB had the greatest effect on aggregation of cells grow in LBG, with a 30% increase in aggregation observed.