Envisioning role of ammonia oxidizing bacteria in bioenergy production and its challenges: a review.

Ammonia oxidizing bacteria (AOB) play a key role in the biological oxidation of ammonia to nitrite and mark their significance in the biogeochemical nitrogen cycle. There has been significant development in harnessing the ammonia oxidizing potential of AOB in the past few decades. However, very little is known about the potential applications of AOB in the bioenergy sector. As alternate sources of energy represent a thrust area for environmental sustainability, the role of AOB in bioenergy production becomes a significant area of exploration. This review highlights the role of AOB in bioenergy production and emphasizes the understanding of the genetic make-up and key cellular biochemical reactions occurring in AOB, thereby leading to the exploration of its various functional aspects. Recent outcomes in novel ammonia/nitrite oxidation steps occurring in a model AOB - Nitrosomonas europaea propel us to explore several areas of environmental implementation. Here we present the significant role of AOB in microbial fuel cells (MFC) where Nitrosomonas sp. play both anodic and cathodic functions in the generation of bioelectricity. This review also presents the potential role of AOB in curbing fuel demand by producing alternative liquid fuel such as methanol and biodiesel. Herein, the multiple roles of AOB in bioenergy production namely: bioelectricity generation, bio-methanol, and biodiesel production have been presented.

Ecological Risk Assessment of Metals Contamination in the Sediments of Natural Urban Wetlands in Dry Tropical Climate.

The pollution load due to metal contamination in the sediments of urban wetlands (Dhanbad, India) due to illegal release of domestic and industrial wastewater was studied by using various geochemical indices, such as contamination factor (Cf), degree of contamination (Cd), modified degree of contamination (mCd), pollution load index (PLI) and geoaccumulation index (Igeo) for Cu, Co, Cd, Cr and Mn. Cluster analysis (CA) and Principal component analysis (PCA) of metals present in wetland sediments were carried out to assess their origin and relationship with each other. The Cf values for different metals in the wetlands under investigation indicated low to very high level of pollution (Cf ranged between 0.02 and 14.15) with highest Cf (14.15) for Cd. The wetland receiving both domestic and industrial wastewater had the highest values of Cd, mCd and PLI as 17.48, 3.49 and 1.03 respectively.

Genomic and transcriptomic analyses of the facultative methanotroph Methylocystis sp. strain SB2 grown on methane or ethanol.

A minority of methanotrophs are able to utilize multicarbon compounds as growth substrates in addition to methane. The pathways utilized by these microorganisms for assimilation of multicarbon compounds, however, have not been explicitly examined. Here, we report the draft genome of the facultative methanotroph Methylocystis sp. strain SB2 and perform a detailed transcriptomic analysis of cultures grown with either methane or ethanol. Evidence for use of the canonical methane oxidation pathway and the serine cycle for carbon assimilation from methane was obtained, as well as for operation of the complete tricarboxylic acid (TCA) cycle and the ethylmalonyl-coenzyme A (EMC) pathway. Experiments with Methylocystis sp. strain SB2 grown on methane revealed that genes responsible for the first step of methane oxidation, the conversion of methane to methanol, were expressed at a significantly higher level than those for downstream oxidative transformations, suggesting that this step may be rate limiting for growth of this strain with methane. Further, transcriptomic analyses of Methylocystis sp. strain SB2 grown with ethanol compared to methane revealed that on ethanol (i) expression of the pathway of methane oxidation and the serine cycle was significantly reduced, (ii) expression of the TCA cycle dramatically increased, and (iii) expression of the EMC pathway was similar. Based on these data, it appears that Methylocystis sp. strain SB2 converts ethanol to acetyl-coenzyme A, which is then funneled into the TCA cycle for energy generation or incorporated into biomass via the EMC pathway. This suggests that some methanotrophs have greater metabolic flexibility than previously thought and that operation of multiple pathways in these microorganisms is highly controlled and integrated.

Biogeochemical modelling to assess the effect of bioclogging on multiple electron acceptor-mediated petroleum hydrocarbon bioremediation in vadose zone.

Bioremediation is an economically viable and sustainable clean-up strategy. Hydrodynamic, as well as transport characteristics of the porous medium, can evolve over the period as a result of biological clean-up activities. The present study proposes a 2-D numerical framework to simulate the effect of bioclogging on multiple electron acceptor-mediated petroleum hydrocarbon bioremediation in the vadose zone. For modelling, a spill of BTEX (benzene, toluene, ethylbenzene and xylene) is assumed near source zone. The developed model results are validated using three previously published datasets on flow, transport and biodegradation in the vadose zone. Simulations are performed for three types of soil, including clay, sand and loam. The analysis shows that sand has a maximum infiltration rate and clay has a minimum. Hydraulic conductivity and saturation profile peaks reach their minimal value at a shallower depth (around four times) when bioclogging is present compared to when it is absent. The migration depth and concentration of BTEX are observed to be restricted to a shallower depth in aquifers with the presence of microbial clogging. The outcome shows that electron acceptor consumption is more (around sevenfold for oxygen, fourfold for nitrate and threefold for sulphate) in the presence of bioclogging at the shallower zone. Zeroth order spatial moment and sensitivity analyses show that biological clogging, number of electron acceptors and inhibition constant substantially affect BTEX bioremediation in the vadose zone.

Methanobactin and MmoD work in concert to act as the 'copper-switch' in methanotrophs.

Biological oxidation of methane to methanol by aerobic bacteria is catalysed by two different enzymes, the cytoplasmic or soluble methane monooxygenase (sMMO) and the membrane-bound or particulate methane monooxygenase (pMMO). Expression of MMOs is controlled by a 'copper-switch', i.e. sMMO is only expressed at very low copper : biomass ratios, while pMMO expression increases as this ratio increases. Methanotrophs synthesize a chalkophore, methanobactin, for the binding and import of copper. Previous work suggested that methanobactin was formed from a polypeptide precursor. Here we report that deletion of the gene suspected to encode for this precursor, mbnA, in Methylosinus trichosporium OB3b, abolishes methanobactin production. Further, gene expression assays indicate that methanobactin, together with another polypeptide of previously unknown function, MmoD, play key roles in regulating expression of MMOs. Based on these data, we propose a general model explaining how expression of the MMO operons is regulated by copper, methanobactin and MmoD. The basis of the 'copper-switch' is MmoD, and methanobactin amplifies the magnitude of the switch. Bioinformatic analysis of bacterial genomes indicates that the production of methanobactin-like compounds is not confined to methanotrophs, suggesting that its use as a metal-binding agent and/or role in gene regulation may be widespread in nature.

Detoxification of mercury by methanobactin from Methylosinus trichosporium OB3b.

Many methanotrophs have been shown to synthesize methanobactin, a novel biogenic copper-chelating agent or chalkophore. Methanobactin binds copper via two heterocyclic rings with associated enethiol groups. The structure of methanobactin suggests that it can bind other metals, including mercury. Here we report that methanobactin from Methylosinus trichosporium OB3b does indeed bind mercury when added as HgCl2 and, in doing so, reduced toxicity associated with Hg(II) for both Alphaproteobacteria methanotrophs, including M. trichosporium OB3b, M. trichosporium OB3b ΔmbnA (a mutant defective in methanobactin production), and Methylocystis sp. strain SB2, and a Gammaproteobacteria methanotroph, Methylomicrobium album BG8. Mercury binding by methanobactin was evident in both the presence and absence of copper, despite the fact that methanobactin had a much higher affinity for copper due to the rapid and irreversible binding of mercury by methanobactin. The formation of a gray precipitate suggested that Hg(II), after being bound by methanobactin, was reduced to Hg(0) but was not volatilized. Rather, mercury remained associated with methanobactin and was also found associated with methanotrophic biomass. It thus appears that although the mercury-methanobactin complex was cell associated, mercury was not removed from methanobactin. The amount of biomass-associated mercury in the presence of methanobactin from M. trichosporium OB3b was greatest for M. trichosporium wild-type strain OB3b and the ΔmbnA mutant and least for M. album BG8, suggesting that methanotrophs may have selective methanobactin uptake systems that may be based on TonB-dependent transporters but that such uptake systems exhibit a degree of infidelity.

Priority pollutant degradation by the facultative methanotroph, Methylocystis strain SB2.

Methylocystis strain SB2, a facultative methanotroph capable of growth on multi-carbon compounds, was screened for its ability to degrade the priority pollutants 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA), and 1,1-dichloroethylene (1,1-DCE), as well as cis-dichloroethylene (cis-DCE) when grown on methane or ethanol. Methylocystis strain SB2 degraded 1,2-DCA and 1,1,2-TCA when grown on either substrate and cis-DCE when grown on methane. Growth of Methylocystis strain SB2 on methane was inhibited in the presence of all compounds, while only 1,1-DCE and cis-DCE inhibited growth on ethanol. No degradation of any chlorinated hydrocarbon was observed in ethanol-grown cultures when particulate methane monooxygenase (pMMO) activity was inhibited with the addition of acetylene, indicating that competition for binding to the pMMO between the chlorinated hydrocarbons and methane limited both methanotrophic growth and pollutant degradation when this strain was grown on methane. Characterization of Methylocystis strain SB2 found no evidence of a high-affinity form of pMMO for methane, nor could this strain utilize 1,2-DCA or its putative oxidative products 2-chloroethanol or chloroactetic acid as sole growth substrates, suggesting that this strain lacks appropriate dehydrogenases for the conversion of 1,2-DCA to glyoxylate. As ethanol: (1) can be used as an alternative growth substrate for promoting pollutant degradation by Methylocystis strain SB2 as the pMMO is not required for its growth on ethanol and (2) has been used to enhance the mobility of chlorinated hydrocarbons in situ, it is proposed that ethanol can be used to enhance both pollutant transport and biodegradation by Methylocystis strain SB2.

Biogeochemical modelling to assess benzene removal by biostimulation in aquifers containing natural reductants.

Biostimulation of aquifers contaminated with gasoline spills is vigorously affected by the biogeochemical environment existing there. In this study, biostimulation of benzene is simulated using a 2D coupled multispecies biogeochemical reactive transport (MBRT) model. The model is implemented at an oil spill site near a hypothetical aquifer containing natural reductants. Multiple electron acceptors are introduced to promote faster biodegradation rate. However, after reaction with natural reductants, it reduces the number of available electron acceptors, acidifies the subsurface environment, and inhibits bacterial growth. These mechanisms are assessed using seven coupled MBRT models sequentially. The finding of the present analysis reveals that biostimulation has caused a substantial drop in concentration of benzene and is efficient in reducing its penetration depth. The results also shows that the intervention of natural reductants in the biostimulation process is slightly diminished by pH adjustment of aquifers. When the pH level in aquifer changes from acidic pH 4 to neutral pH 7, it is observed that the biostimulation rate of benzene as well as microbial activity increases. Electron acceptors consumption is more at neutral pH. Overall, it can be inferred from zeroth-order spatial moment and sensitivity analyses that retardation factor, inhibition constant, pH, and dispersivity in vertical direction significantly affect benzene biostimulation in aquifers.

Identification, characterization, and lipid profiling of microalgae Scenedesmus sp. NC1, isolated from coal mine effluent with potential for biofuel production.

An autoflocculating microalgal strain was isolated from coal mine effluent wastewater which was named as Scenedesmus sp. NC1 after morphological and molecularly characterization. Further analysis of internal transcribed spacer 2 (ITS2) and compensatory base changes (CBCs) showed it does not belong to the clade comprising Scenedesmus sensu stricto. In stationary phase of growth, Scenedesmus sp. NC1 exhibited excellent autoflocculation efficiency (> 88 %) within 150 min of setting. Temperature, pH, and inorganic metals exhibited minor influence on the autoflocculation activity of Scenedesmus sp. NC1. The fatty acid profiling of Scenedesmus sp.NC1 showed that palmitic acid (C16:0), oleic acid (C18:1), and stearic acid (18:0) accounted for more than 68 % of total fatty acids. Moreover, Scenedesmus sp. NC1 demonstrated significant bioflocculation potential over non-flocculating freshwater microalgae, Chlorella sp. NCQ and Micractinium sp. NCS2. Hence, Scenedesmus sp. NC1 could be effective for economical harvesting of other non-flocculating microalgae for productions of biodiesel and other metabolites.

Optimization of phosphate recovery as struvite from synthetic distillery wastewater using a chemical equilibrium model.

This study investigates the feasibility of recovery of phosphorus via struvite precipitation from a synthetic anaerobically treated distillery spent wash by optimizing the process using a chemical equilibrium model, namely Visual MINTEQ. Process parameters such as Mg2+, [Formula: see text], and [Formula: see text] ion concentrations and pH were used as inputs into the model. Increasing the molar ratio of [Formula: see text] from 0.8:1 to 1.6:1 at pH 9 led to an increase in phosphate recovery from 88.2 to 99.5%. The model and experimental results were in good agreement in terms of phosphate recovery, indicating that the Visual MINTEQ model can be used to pre-determine the process parameters for struvite synthesis. Increasing the concentration of calcium ion adversely affected the synthesis and purity of struvite, whereas the presence of melanoidins had no significant impact. This study demonstrates that phosphorus recovery through struvite precipitation is a sustainable approach to reclaim phosphorus from high-strength industrial wastewater.

Arsenite biotransformation by Rhodococcus sp.: Characterization, optimization using response surface methodology and mechanistic studies.

A large population of the world is under increased health risk due to consumption of arsenic contaminated groundwater. The present study investigates the arsenic resistance and arsenic biotransforming ability in three bacterial species, namely Bacillus arsenicus, Rhodococcus sp. and Alcaligenes faecalis for employing them in potential groundwater bioremediation programmes. The tolerance to pH levels for the 3 organisms are 6-9 for A. faecalis, 5-10 for Rhodococcus and 5-9 for B. arsenicus. The arsenic bio-oxidation capacity was qualitatively confirmed by using the silver nitrate method and all three bacteria were able to convert arsenite to arsenate. The arsenite tolerance capacity (MIC values) were found to be 3 mM, 7 mM and 12 mM for B. arsenicus, A. faecalis and Rhodococcus sp. respectively. The changes in cellular morphology of these strains under various arsenic stress conditions were studied using advanced cell imaging techniques such as scanning electron microscopy and Atomic Force Microscopy. Rhodococcus sp. emerged as a potential candidate for bioremediation application. A response surface methodology was employed to optimize key parameters affecting arsenic removal (pH, Iron (II) soluble, concentration of humic acid and initial arsenic concentration) and at optimized conditions, experimental runs demonstrated 48.34% removal of As (III) (initial concentration = 500 µg/L) in a duration of 6 h, with complete removal after 48 h. Evidences from this work indicate that arsenic removal occurs through bioaccumulation, biotransformation and biosorption. The present study makes the first attempt to investigate the arsenic removal capability of Rhodococcus sp. in synthetic groundwater by employing bacterial whole cell assays. This study also sheds light on the arsenic tolerance and detoxification mechanisms employed by these bacteria, knowledge of which could be crucial in the successful implementation of in-situ bioremediation programmes.

A novel non-starch based cationic polymer as flocculant for harvesting microalgae.

This work intends towards the preparation of different grades of cationic locust bean gum biopolymer (CLBG) through the incorporation of 2,3-epoxypropyltrimethylammonium chloride (GTMAC) on to the pristine locust bean gum (LBG) biopolymer. Among them the best grade was further selected, characterized and their flocculation efficacy was evaluated towards harvesting of three different indigenous isolated green microalgae viz. Chlorella sp. NCQ, Micractinium sp. NCS2 and Scenedesmus sp. CBIIT(ISM). Flocculation efficiency of 96.68%, 96.64%, and 97.42% were obtained for Chlorella sp. NCQ, Micractinium sp. NCS2 and Scenedesmus sp. CBIIT(ISM) at an optimum dosage of 55, 40, and 30 ppm respectively. Thus CLBG was proven to be an efficient flocculant towards harvesting of green microalgae than its natural form.

Recent developments in synthetic biology and metabolic engineering in microalgae towards biofuel production.

In the wake of the uprising global energy crisis, microalgae have emerged as an alternate feedstock for biofuel production. In addition, microalgae bear immense potential as bio-cell factories in terms of producing key chemicals, recombinant proteins, enzymes, lipid, hydrogen and alcohol. Abstraction of such high-value products (algal biorefinery approach) facilitates to make microalgae-based renewable energy an economically viable option. Synthetic biology is an emerging field that harmoniously blends science and engineering to help design and construct novel biological systems, with an aim to achieve rationally formulated objectives. However, resources and tools used for such nuclear manipulation, construction of synthetic gene network and genome-scale reconstruction of microalgae are limited. Herein, we present recent developments in the upcoming field of microalgae employed as a model system for synthetic biology applications and highlight the importance of genome-scale reconstruction models and kinetic models, to maximize the metabolic output by understanding the intricacies of algal growth. This review also examines the role played by microalgae as biorefineries, microalgal culture conditions and various operating parameters that need to be optimized to yield biofuel that can be economically competitive with fossil fuels.

Genetic identification of arsenate reductase and arsenite oxidase in redox transformations carried out by arsenic metabolising prokaryotes - A comprehensive review.

Arsenic (As) contamination in water is a cause of major concern to human population worldwide, especially in Bangladesh and West Bengal, India. Arsenite (As(III)) and arsenate (As(V)) are the two common forms in which arsenic exists in soil and groundwater, the former being more mobile and toxic. A large number of arsenic metabolising microorganisms play a crucial role in microbial transformation of arsenic between its different states, thus playing a key role in remediation of arsenic contaminated water. This review focuses on advances in biochemical, molecular and genomic developments in the field of arsenic metabolising bacteria - covering recent developments in the understanding of structure of arsenate reductase and arsenite oxidase enzymes, their gene and operon structures and their mechanism of action. The genetic and molecular studies of these microbes and their proteins may lead to evolution of successful strategies for effective implementation of bioremediation programs.

Biodegradation of diesel oil by an Arabian Sea sediment culture isolated from the vicinity of an oil field.

Laboratory scale batch studies were performed to test the diesel oil biodegradation ability of ES1 cultures isolated from Arabian Sea sediments obtained from the vicinity of an oil field. This culture could utilize diesel as the sole source of carbon and energy. Under aerobic conditions, 39% loss of diesel oil was observed over 8 days where 80% of the loss was due to aliphatic constituents. Under anoxic nitrate reducing conditions the rate and extent of degradation was significantly lower, i.e., 18% over 50 days. Salt acclimatized cultures could tolerate salinities up to 3.5% and demonstrated optimal performance at a salinity of 0.5%. The optimum N/P ratio for these cultures was found to be in the range of 2:1-5:1. Addition of two trace elemental substance formulations exhibited a significant inhibitory effect on culture growth. This culture has good potential for decontamination of oil-contaminated marine and subsurface environments.

Treatment of waste metalworking fluid by a hybrid ozone-biological process.

In metal machining processes, the regulation of heat generation and lubrication at the contact point are achieved by application of a fluid referred to as metalworking fluid (MWF). MWFs inevitably become operationally exhausted with age and intensive use, which leads to compromised properties, thereby necessitating their safe disposal. Disposal of this waste through a biological route is an increasingly attractive option, since it is effective with relatively low energy demands. However, successful biological treatment is challenging since MWFs are chemically complex, and include biocides specifically to retard microbial deterioration whilst the fluids are operational. In this study remediation of the recalcitrant component of a semi-synthetic MWF by a novel hybrid ozone-bacteriological treatment, was investigated. The hybrid treatment proved to be effective and reduced the chemical oxygen demand by 72% (26.9% and 44.9% reduction after ozonation and biological oxidation respectively). Furthermore, a near-complete degradation of three non-biodegradable compounds (viz. benzotriazole, monoethanolamine, triethanolamine), commonly added as biocides and corrosion inhibitors in MWF formulations, under ozonation was observed.

A novel hybrid nano zerovalent iron initiated oxidation--biological degradation approach for remediation of recalcitrant waste metalworking fluids.

Disposal of operationally exhausted metal working fluids (MWF) through a biological route is an attractive option, since it is effective with relatively low energy demands. However, it is enormously challenging since these fluids are chemically complex, including the addition of toxic biocides which are added specifically to retard bio-deterioration whilst the fluids are operational. Nano-sized elemental iron represents a new generation of environmental remediation technologies. Laboratory scale batch studies were performed to test the degradation ability of a semi-synthetic metalworking fluid (MWF) wastewater (which was found to be resistant to initial bacterial treatment in specifically established bioreactors) by employing a novel hybrid approach. The approach was to combine the synergistic effects of nano zerovalent iron (nZVI) induced oxidation, followed by biodegradation, specifically for the remediation of recalcitrant components of MWF effluent. Addition of nZVI particles to oxygenated wastewater resulted in oxidation of organic contaminants present. Our studies confirmed 78% reduction in chemical oxygen demand (COD) by nZVI oxidation at pH 3.0 and 67% reduction in neutral pH (7.5), and 85% concurrent reduction in toxicity. Importantly, this low toxicity made the nZVI treated effluent more amenable for a second stage biological oxidation step. An overall COD reduction of 95.5% was achieved by the novel combined treatment described, demonstrating that nZVI oxidation can be exploited for enhancing the biodegradability of a recalcitrant wastewater in treatment processes.

Harmonisation of chemical and biological process in development of a hybrid technology for treatment of recalcitrant metalworking fluid.

Disposal of operationally exhausted metalworking fluids (MWFs) is enormously challenging. In this study the feasibility of employing a sequential Fenton-biological oxidation for the treatment of recalcitrant components of MWF wastewater was investigated. A statistical experimental design was employed to address Fenton reagent (H2O2, Fe²âº) dose optimisation which ensured minimal concentrations of the reagents, thus making the treatment environmentally less toxic to subsequent biological steps and economically viable. This was achieved by employing a five-level-two-variable central composite experimental design. The results demonstrated that Fenton pre-treatment of the MWF effluent greatly improved biodegradability index (BOD5)/COD increased from 0.160 to 0.538) with a synchronous lowering in the toxicity of the wastewater, making the recalcitrant component more amenable to subsequent biological treatment. An overall decrease of 92% and 86% in chemical oxygen demand (COD) and total organic carbon (TOC), respectively, was achieved by the two-step treatment method developed.