Recent progress in the characterization and application of exo-electrogenic microorganisms.

Exo-electrogenic microorganisms are characterized by their special metabolic capability of transferring metabolic electrons out of their cell, into insoluble external electron acceptors such as iron or manganese oxides and electrodes, or vice versa take up electron from electrodes. Their conventional application is primarily limited to microbial fuel cells for electrical power generation and microbial electrolysis cells for the production of value-added products such as biohydrogen, biomethane and hydrogen peroxide. The utility of exo-electrogenic organisms has expanded into many other applications in recent times. Such examples include microbial desalination cells, microbial electro-synthesis cells producing value-added chemicals such as bio-butanol and their applications in other carbon sequestration technologies. Additionally, electrochemically-active organisms are now beginning to be employed in biosensor applications for environmental monitoring. Additionally, the utility of biocathodes in bio-electrochemical systems is also a novel application in catalyzing the cathodic oxygen reduction reaction to enhance their electrochemical performance. Advances have also been made in the expansion and use of other organisms such as the usage of photosynthetic microorganisms for the fabrication of self-sustained bio-electrochemical systems. This review attempts to provide a comprehensive picture of the state-of the art of exo-electrogenic organisms and their novel utility in bioelectrochemical systems.

A novel growth and isolation medium for exoelectrogenic bacteria.

A microbiological isolation and growth medium that can effectively discriminate electrochemically active exoelectrogenic bacteria from other non-exoelectrogenic bacteria, is currently unavailable. In this study, we developed a novel chromogenic growth and isolation solid medium based on MnO2 that can selectively allow the growth of exoelectrogenic bacteria and change the medium colour in the process. Known exoelectrogenic bacteria such as Shewanella oneidensis MR1 and other such bacteria from functional microbial fuel cell (MFC) anodes were capable of growing and changing colour in the novel growth medium. On the contrary, non-exoelectrogenic bacteria such as Escherichia coli ATCC 25922 were incapable of growing and inducing a colour change in the novel medium. Further biochemical characterisation of these isolated exoelectrogenic bacteria by Raman micro-spectroscopy demonstrated that these bacteria over express cytochrome proteins that are vital in extracellular electron transfer events. This medium is a convenient method to isolate exoelectrogenic bacteria from complex environmental samples.

Resolving the individual contribution of key microbial populations to enhanced biological phosphorus removal with Raman-FISH.

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. Candidatus Accumulibacter conform to the classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under in situ conditions, and applied it to eight full-scale EBPR plants. Besides Ca. Accumulibacter, members of the genus Tetrasphaera were found to be important microbes for P accumulation, and in six plants they were the most important. As these Tetrasphaera cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations in situ in other ecosystems and might become a valuable tool for many environmental microbiologists.

Genomic and in Situ Analyses Reveal the Micropruina spp. as Abundant Fermentative Glycogen Accumulating Organisms in Enhanced Biological Phosphorus Removal Systems.

Enhanced biological phosphorus removal (EBPR) involves the cycling of biomass through carbon-rich (feast) and carbon-deficient (famine) conditions, promoting the activity of polyphosphate accumulating organisms (PAOs). However, several alternate metabolic strategies, without polyphosphate storage, are possessed by other organisms, which can compete with the PAO for carbon at the potential expense of EBPR efficiency. The most studied are the glycogen accumulating organisms (GAOs), which utilize aerobically stored glycogen to energize anaerobic substrate uptake and storage. In full-scale systems the Micropruina spp. are among the most abundant of the proposed GAO, yet little is known about their ecophysiology. In the current study, genomic and metabolomic studies were performed on Micropruina glycogenica str. Lg2T and compared to the in situ physiology of members of the genus in EBPR plants using state-of-the-art single cell techniques. The Micropruina spp. were observed to take up carbon, including sugars and amino acids, under anaerobic conditions, which were partly fermented to lactic acid, acetate, propionate, and ethanol, and partly stored as glycogen for potential aerobic use. Fermentation was not directly demonstrated for the abundant members of the genus in situ, but was strongly supported by the confirmation of anaerobic uptake of carbon and glycogen storage in the absence of detectable polyhydroxyalkanoates or polyphosphate reserves. This physiology is markedly different from the classical GAO model. The amount of carbon stored by fermentative organisms has potentially important implications for phosphorus removal - as they compete for substrates with the Tetrasphaera PAO and stored carbon is not made available to the "Candidatus Accumulibacter" PAO under anaerobic conditions. This study shows that the current models of the competition between PAO and GAO are too simplistic and may need to be revised to take into account the impact of potential carbon storage by fermentative organisms.

Culture-Independent Analyses Reveal Novel Anaerolineaceae as Abundant Primary Fermenters in Anaerobic Digesters Treating Waste Activated Sludge.

Anaerobic digestion for biogas production is reliant on the tightly coupled synergistic activities of complex microbial consortia. Members of the uncultured A6 phylotype, within the phylum Chloroflexi, are among the most abundant genus-level-taxa of mesophilic anaerobic digester systems treating primary and surplus sludge from wastewater treatment plants, yet are known only by their 16S rRNA gene sequence. This study applied metagenomics to obtain a complete circular genome (2.57 Mbp) from a representative of the A6 taxon. Preliminary annotation of the genome indicates these organisms to be anaerobic chemoorganoheterotrophs with a fermentative metabolism. Given their observed abundance, they are likely important primary fermenters in digester systems. Application of fluorescence in situ hybridisation probes designed in this study revealed their morphology to be short filaments present within the flocs. The A6 were sometimes co-located with the filamentous Archaea Methanosaeta spp. suggesting potential undetermined synergistic relationships. Based on its genome sequence and morphology we propose the species name Brevefilum fermentans gen. nov. sp. nov.

Treatment of colour industry wastewaters with concomitant bioelectricity production in a sequential stacked mono-chamber microbial fuel cells-aerobic system.

The scalability of any microbial fuel cell (MFC)-based system is of vital importance if it is to be utilized for potential field applications. In this study, an integrated MFC-aerobic bioreactor system was investigated for its scalability with the purpose of treating a simulated dye wastewater and industrial wastewaters originated from textile dyebaths and leather tanning. The influent containing real wastewater was fed into the reactor in continuous mode at ambient temperature. Three MFC units were integrated to act in unison as a single module for wastewater treatment and a continuously stirred aerobic bioreactor operating downstream to the MFC module was installed in order to ensure more complete degradation of colouring agents found in the wastewater. Total colour removal in the final effluent exceeded 90% in all experiments where both synthetic (AO-7 containing) and real wastewater were used as the influent feed. The chemical oxygen demand reduction also exceeded 80% in all experiments under the same conditions. The MFC modules connected in parallel configuration allowed obtaining higher current densities than that can be obtained from a single MFC unit. The maximum current density of the MFC stack reached 1150 mA m(-2) when connected in a parallel configuration. The outcome of this work implies that suitably up-scaled MFC-aerobic integrated bioprocesses could be used for colour industry wastewater treatment under industrially relevant conditions with possible prospects of bioelectricity generation.

Complete degradation of the azo dye Acid Orange-7 and bioelectricity generation in an integrated microbial fuel cell, aerobic two-stage bioreactor system in continuous flow mode at ambient temperature.

In this study, the commercially used model azo dye Acid Orange-7 (AO-7) was fully degraded into less toxic intermediates using an integrated microbial fuel cell (MFC) and aerobic bioreactor system. The integrated bioreactor system was operated at ambient temperature and continuous-flow mode. AO-7 loading rate was varied during experiments from 70gm(-3)day(-1) to 210gm(-3)day(-1). Colour and soluble COD removal rates reached>90% under all AO-7 loading rates. The MFC treatment stage prompted AO-7 to undergo reductive degradation into its constituent aromatic amines. HPLC-MS analysis of metabolite extracts from the aerobic stage of the bioreactor system indicated further oxidative degradation of the resulting aromatic amines into simpler compounds. Bioluminescence based Vibrio fischeri ecotoxicity testing demonstrated that aerobic stage effluent exhibited toxicity reductions of approximately fivefold and ten-fold respectively compared to the dye wastewater influent and MFC-stage effluent.

Simultaneous co-metabolic decolourisation of azo dye mixtures and bio-electricity generation under thermophillic (50 °C) and saline conditions by an adapted anaerobic mixed culture in microbial fuel cells.

In this study, azo dye adapted mixed microbial consortium was used to effectively remove colour from azo dye mixtures and to simultaneously generate bio-electricity using microbial fuel cells (MFCs). Operating temperature (20-50 °C) and salinity (0.5-2.5%w/v) were varied during experiments. Reactor operation at 50 °C improved dye decolourisation and COD removal kinetic constants by approximately 2-fold compared to the kinetic constants at 30 °C. Decolourisation and COD removal kinetic constants remained high (0.28 h(-1) and 0.064 h(-1) respectively) at moderate salinity (1%w/v) but deteriorated approximately 4-fold when the salinity was raised to 2.5% (w/v). Molecular phylogenetic analysis of microbial cultures used in the study indicated that both un-acclimated and dye acclimated cultures from MFCs were predominantly comprised of Firmicutes bacteria. This study demonstrates the possibility of using adapted microbial consortia in MFCs for achieving efficient bio-decolourisation of complex azo dye mixtures and concomitant bio-electricity generation under industrially relevant conditions.