Rapid measurement of bacterial contamination in water: A catalase responsive-electrochemical sensor.

The present study describes the development of a potentiometric sensor for microbial monitoring in water based on catalase activity. The sensor comprises a MnO2-modified electrode that responds linearly to hydrogen peroxide (H2O2) from 0.16 M to 3.26 M. The electrode potential drops when the H2O2 solution is spiked with catalase or catalase-producing microorganisms that decompose H2O2. The sensor is responsive to different bacteria and their catalase activities. The electrochemical sensor exhibits a lower limit of detection (LOD) for Escherichia coli at 11 CFU/ml, Citrobacter youngae at 12 CFU/ml, and Pseudomonas aeruginosa at 23 CFU/ml. The sensor shows high sensitivity at 3.49, 3.02, and 4.24 mV/cm2dec for E. coli, C. youngae, and P. aeruginosa, respectively. The abiotic sensing electrode can be used multiple times without changing the response potential (up to 100 readings) with a shelf-life of over six months. The response time is a few seconds, with a total test time of 5 min. Additionally, the sensor effectively tested actual samples (drinking and grey water), which makes it a quick and reliable sensing tool. Therefore, the study offers a promising water monitoring tool with high sensitivity, stability, good detection limit, and minimum interference from other water contaminants.

A Lab Prototype for Rapid Electrochemical Detection of Escherichia coli in Water Using Modified Screen-Printed Electrodes.

Recognizing the need for a hand-held device capable of quantitatively measuring the concentration of bacteria in water, this paper describes a label-free method for rapid detection of Escherichia coli (E. coli) in water via H2O2 decomposition using screen-printed electrodes (SPE) modified with nanostructured metal oxide layers. The study encompasses sensor preparation, bacteria culture, synthesis and characterization of nanostructures, and development of a readout circuitry for lab prototyping. During sensing measurements, the bacteria are first made to interact with H2O2 and subsequently, the H2O2 solution is exposed on the sensing surface. The electrochemical sensors are fabricated by modifying the working electrode of SPE with nanostructured metal oxide layers of MnO2 and TiO2 as these play a crucial role in the detection of E. coli in water. The sensing experiments of MnO2-modified SPE show a significant response to bacteria with a sensitivity of 0.82 mV.mL/log CFU and a limit of detection (LOD) of 1.8 CFU/mL, while the TiO2-modified SPE exhibits a linear response over a wide range of bacterial concentrations with a sensitivity of 1.12 mV·mL/log CFU and a limit of detection of 2.23 CFU/mL. Both sensors demonstrate a rapid response, stability, repeatability, and a recovery time of 70 ms. Additionally, selectivity with respect to other bacteria, wastewater components such as glucose, ammonium sulfate, and sodium carbonate, and testing with RO, DI, and tap water samples are conducted to evaluate the sensors' performance. A detailed sensing mechanism has been developed to comprehend the results, including chemical and biological reactions, metal oxide interfaces, morphology, and other surface studies of the sensing surface. A prototype comprising a sensor chip, an Arduino board, and other necessary circuit components is tested with various bacterial solutions. This enables its use for on-field rapid detection of bacteria in water using smaller volumes and a portable system.

The versatility of microbial fuel cells as tools for organic matter monitoring.

Water monitoring and remediation require robust, low-cost, and reliable test systems that can couple with prompt treatment interventions. Organic matter (BOD, COD), toxicants, heavy metals, and other pollutants in water need to be regularly inspected. Microbial fuel cells (MFCs) have already gained popularity as BOD biomonitoring systems as these don't need an external transducer or power source. Moreover, these systems are cost-effective, compact, biodegradable, reusable, portable, and applicable for on-site measurements. MFCs truly stands out as online BOD measurement devices as they provide wide detection range (0-25 g/L), low response time (2-4 min) and longer stability in continuous operations (2-5 years) in a cost-effective approach. This review examines the benefits, kinds, performance metrics, and signal optimization of the current state-of-the-art of the BOD measurement, with detailed focus on MFC-based BOD biomonitoring systems. This review covers the important technological breakthroughs in practical applications with associated bottlenecks to develop reliable sensing systems.

Co-culturing Chlorella vulgaris and Cystobasidium oligophagum JRC1 in the microbial fuel cell cathode for lipid biosynthesis.

This study investigated the effect of co-culturing the photobiont and mycobiont in the microbial fuel cell (MFC) cathode on biomass production, lipid generation, and power output. Chlorella vulgaris provides oxygen and nutrients for the yeast Cystobasidium oligophagum JRC1, while the latter offers CO2 and quench oxygen for higher algal growth. The MFC with co-culture enhanced the lipid output of biomass by 28.33%, and the total yield and productivity were 1.47 ± 0.18 g/l and 0.123 g/l/day, respectively. Moreover, with co-culture, the open circuit voltage of 685 ± 11 mV was two times higher than algae alone. The specific growth rate (day-1) at the cathode was 0.367 ± 0.04 in co-culture and 0.288 ± 0.05 with C. vulgaris only. The power density of the system was 5.37 ± 0.21 mW/m2 with 75.88 ± 1.89% of COD removal. The co-culture thus proved beneficial at the MFC cathode in terms of total energy output as 11.5 ± 0.035 kWh/m3, which was 1.4-fold higher than algae alone.

Integration of third generation biofuels with bio-electrochemical systems: Current status and future perspective.

Biofuels hold particular promise as these can replace fossil fuels. Algae, in particular, are envisioned as a sustainable source of third-generation biofuels. Algae also produce several low volume high-value products, which enhance their prospects of use in a biorefinery. Bio-electrochemical systems such as microbial fuel cell (MFC) can be used for algae cultivation and bioelectricity production. MFCs find applications in wastewater treatment, CO2 sequestration, heavy metal removal and bio-remediation. Oxidation of electron donor by microbial catalysts in the anodic chamber gives electrons (reducing the anode), CO2, and electrical energy. The electron acceptor at the cathode can be oxygen/NO3 -/NO2 -/metal ions. However, the need for a continuous supply of terminal electron acceptor in the cathode can be eliminated by growing algae in the cathodic chamber, as they produce enough oxygen through photosynthesis. On the other hand, conventional algae cultivation systems require periodic oxygen quenching, which involves further energy consumption and adds cost to the process. Therefore, the integration of algae cultivation and MFC technology can eliminate the need of oxygen quenching and external aeration in the MFC system and thus make the overall process sustainable and a net energy producer. In addition to this, the CO2 gas produced in the anodic chamber can promote the algal growth in the cathodic chamber. Hence, the energy and cost invested for CO2 transportation in an open pond system can be saved. In this context, the present review outlines the bottlenecks of first- and second-generation biofuels along with the conventional algae cultivation systems such as open ponds and photobioreactors. Furthermore, it discusses about the process sustainability and efficiency of integrating algae cultivation with MFC technology in detail.

Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase.

BACKGROUND: Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase. RESULTS: In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg(-1) protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac. CONCLUSION: Laccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L(-1) in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation.

Genetic diversity and population structure of critically endangered Dactylorhiza hatagirea (D. Don) Soo from North-Western Himalayas and implications for conservation.

Dactylorhiza hatagirea (D. Don) Soo is medicinally important herb, which is widely used in ayurveda, unani, and folk/traditional medicine system to cure diseases. Due to its immense ethno-botanical properties, the trade of D. hatagirea is estimated to be USD 1 billion/year in India. Unfortunately, due to overexploitation of the herb from the wild, has resulted in dwindling of its populations in their natural habitats, which has led to its critically endangered status. Molecular genetic studies are still scarce in D. hatagirea, therefore, in current study, genetic diversity and population structure analysis was carried out of 10 populations (48 individuals) collected from three cold desert regions (2527 m-3533 m amsl) of Himachal Pradesh. Mean observed heterozygosity (Ho) and expected heterozygosity (He) was recorded 0.185 and 0.158. The maximum values for Fst (fixation index) and Nm (gene flow) were recorded 0.945 at locus KSSR14 and 1.547 at locus KSSR 4 respectively. Mean genetic differentiation (Fst) coefficient was estimated to 0.542. Overall, low levels of genetic diversity was recorded in the populations of D. hatagirea, might be due to habitat specificity (alpine meadows ecosystem; humid laden undulating habitat), restricted distribution and high anthropogenic activities. However, two populations viz., Bathad and Rangrik were recorded with high diversity and largest number of private alleles, stipulates that these populations might have high evolutionary significance and response to selection. Dendrogram analysis revealed that the populations of D. hatagirea were clustered into four major clusters, which was supported by Bayesian based STRUCTURE predictions. Clustering pattern of majority individuals of different populations revealed consistency with their geographic origin. Outcomes of current study reveals the status of genetic diversity and population structure of endangered D. hatagirea, which can be futuristically utilised for appropriate planning of conservation strategies.

Performance evaluation of a photosynthetic microbial fuel cell (PMFC) using Chlamydomonas reinhardtii at cathode.

This study reports the use of Chlamydomonas reinhardtiiat the cathode in a photosynthetic microbial fuel cell (PMFC). The PMFC produced power and current density of 15.21 W m-3 and 39 A m-3, respectively. The specific growth rate of algae culture at the cathode was 0.284 day-1. The system achieved COD removal at 73.30% with a Coulombic efficiency of 9.068%. The usability of algae biomass was assessed in terms of its total phenol content (157.69 mg GAE/g algae DW), antioxidant activity (IC50 = 67.07 µg/ml), total Chlorophyll (18.95 mg/g), total Carotenoids (2.40 mg/g), and antibacterial properties against known pathogens. Overall, the study's findings suggested thatC. reinhardtiisupports high power output from a PMFC and is highly resourceful in terms of value-added products.

Effect of cathodic culture on wastewater treatment and power generation in a photosynthetic sediment microbial fuel cell (SMFC): Canna indica v/s Chlorella vulgaris.

The aim of this work was to compare the performance of the two types of photosynthetic microbial fuel cells (MFCs) fed with real wastewater- one having plant Canna indica (PMFC) and the other having alga Chlorella vulgaris (AMFC) at the cathode. The chemical oxygen demand (COD), phosphate, and nitrate removal stood at 57.16% 88.81%, 59.82% for PMFC and 65.27%, 95.59%, 66.61% for the AMFC. While AMFC was slightly superior in water treatment, the power output was 6 times higher in PMFC (22.76 mW m-2) than the AMFC (3.64 mW m-2). The biomass growth was good in both systems, with biomass productivity of 0.031 Kg m-3 day-1 in AMFC and a leaf area index of 0.006 in PMFC. The study's findings suggest that PMFCs are equally good or even better than AMFCs when the goal is simultaneous water treatment and power generation.

Performance evaluation of algae assisted microbial fuel cell under outdoor conditions.

This study reports for the first time an operation of an outdoor algae assisted Microbial Fuel Cell (MFC). The MFC (10 L) comprised of low-cost materials like rock phosphate blended clayware & low-density polyethylene bags as anodic & cathodic chamber respectively. Algae biomass after lipid extraction at 2 g/l served as electron donor at the anode. Chlorella vulgaris at cathode provided oxygen as electron acceptor and served as lipid source. The MFCs performed well in all aspects namely energy recovery, algae productivity, and cost of operation. The 5% RP-MFCs gave 0.307 kg/m3d algal productivity, 0.09 kg/m3d lipid productivity, and 11.5318 kWh/m3 of net energy recovery (NER). Rock phosphate served as a slow and constant source of phosphorus supporting algae growth. Proteobacteria (45.14%) were the dominant phyla while Alicyliphilus (5.46%) and Dechloromonas (4.74%) were the dominant genera at the anode. The estimated cost of the system was $11.225 only.

Assessing oil accumulation in the oleaginous yeast Cystobasidium oligophagum JRC1 using dairy waste cheese whey as a substrate.

This study assesses the potential for the lipid production by the oleaginous yeast Cystobasidium oligophagum JRC1 using dairy industry waste cheese whey as a substrate. Cheese whey was used either untreated (UCW) or deproteinized (DCW) at different concentrations (25-100%) to serve as the carbon and energy source. Both UCW and DCW supported high biomass and lipid productivities. The biomass productivity of 0.076 ± 0.0004 and 0.124 ± 0.0021 g/L h, lipid productivity of 0.0335 ± 0.0004 and 0.0272 ± 0.0008 g/L h, and the lipid content of 44.12 ± 0.84 and 21.79 ± 1.00% were achieved for 100% DCW and UCW, respectively. The soluble chemical oxygen demand (sCOD) removal rate was 8.049 ± 0.198 and 10.61 ± 0.0165 g/L day (84.91 ± 0.155 and 86.82 ± 0.067% removal) for 100% DCW and UCW, respectively. Fatty acid methyl ester (FAME) composition obtained using GC-FID studies revealed the presence of C16 and C18 fatty acid in the lipid extract and the biodiesel properties were found to be in accordance with ASTM and EN standards. The study presents a method for the valorization of cheese whey waste into a feasible feedstock for biodiesel.

Microbial community modulates electrochemical performance and denitrification rate in a biocathodic autotrophic and heterotrophic denitrifying microbial fuel cell.

A comparison of autotrophic (AD) and heterotrophic (HD) cathodic denitrification in a Microbial Fuel Cell (MFC) was made in this study. Denitrifying microbial consortia were developed from cow manure and soil and acclimatized under AD and HD conditions. The AD MFC supported the power output of 4.45 W m-3 while removing nitrate nitrogen (NO3--N) at the rate of 0.118 kg NO3--N m-3 d-1. Significant power output (3.02 W m-3) and nitrate removal rate (2.06 kg NO3--N m-3 d-1) were achieved in HD MFC. Further, 16S rDNA based community analysis revealed higher diversity in HDMFC. The genus Thauera and Pseudomonas were predominant in ADMFC while genus Klebsiella and Alkaliphilus were abundant in HDMFC. The abundance of the denitrifying genes namely narG, nirS, and nosZ were assessed with the help of quantitative PCR and presence of all the genes in both the conditions ensured the necessary molecular requirements for complete denitrification.

Microbial fuel cell powered by lipid extracted algae: A promising system for algal lipids and power generation.

In this study, a promising microbial fuel cell (MFC) system has been developed, wherein algae is cultivated in the cathode chamber, algae biomass is harvested and lipids are extracted. The lipid extracted algal (LEA) biomass was then used asan electron donor substrate. The performance of MFCs fed with LEA biomass was compared with that of fruit waste fed MFCs (FP-MFCs), wherein LEA-fed MFC was superior in all aspects. Power density of 2.7Wm-3 was obtained by LEA-fed MFCs which is 145% and 260% higher than FP MFC and control MFC respectively. The volumetric algae productivity of 0.028kgm-3day-1 in cathode chamber was achieved. The system was able to generate 0.0136kWhKg-1CODday-1 of electric energy and 0.0782kWhm-3day-1 of algal oil energy. The proposed system is a net energy producer which does not rely heavily on the external supply of electron donor substrates.

Halophilic starch degrading bacteria isolated from Sambhar Lake, India, as potential anode catalyst in microbial fuel cell: A promising process for saline water treatment.

In this study, Microbial Fuel Cell (MFC) capable of treating saline starch water was developed. Sodium chloride (NaCl) concentrations ranging from 500 mM to 3000 mM were tested at the anode. Nitrate was used as an electron acceptor at the biocathode. The halophilic bacteria were isolated from Sambhar Lake, India. Results indicated successful removal of starch (1.83 kg/m3-d) and nitrate (0.13 kg/m3-d NO3--N) with concomitant power output of 207.05 mW/m2 at 1000 mM NaCl concentration. An increase in power density from 71.06 mW/m2 to 207.05 mW/m2 (2.92 folds) was observed when NaCl concentration was increased from 500 mM to 1000 mM. A decline in power density was observed when the salt concentrations >1000 mM were used. Concentration of 3000 mM supported power output as well as the highest starch degradation (3.2 kg/m3-d) and amylase activity of 2.26 IU/ml. The halophilic exoelectrogens were isolated and identified. The present study demonstrates the utility of MFC for degrading starch in saline water.

Isolation, identification and characterization of Cystobasidium oligophagum JRC1: A cellulase and lipase producing oleaginous yeast.

Oleaginous yeast closely related to Cystobasidium oligophagum was isolated from soil rich in cellulosic waste. The yeast was isolated based on its ability to accumulate intracellular lipid, grow on carboxymethylcellulose (CMC) and produce lipase. It could accumulate up to 39.44% lipid in a glucose medium (12.45±0.97g/l biomass production). It was able to grow and accumulate lipids (36.46%) in the medium containing CMC as the sole carbon source. The specific enzyme activities obtained for endoglucanase, exoglucanase, and ß-glucosidase were 2.27, 1.26, and 0.98IU/mg respectively. The specific enzyme activities obtained for intracellular and extracellular lipase were 2.16 and 2.88IU/mg respectively. It could grow and accumulate lipids in substrates including glycerol (42.04%), starch (41.54%), xylose (36.24%), maltose (26.31%), fructose (24.29%), lactose (21.91%) and sucrose (21.72%). The lipid profile of the organism was suitable for obtaining biodiesel with desirable fuel properties.

Microbial fuel cell assisted nitrate nitrogen removal using cow manure and soil.

Microbial fuel cells (MFCs) are emerging wastewater treatment systems with a proven potential for denitrification. In this study, we have developed a high-rate denitrifying MFC. The anode consisted of cow manure and fruit waste and the cathode consisted of cow manure and soil. The initial chemical oxygen demand (COD)/nitrate nitrogen (NO3 (-)-N) was varied from 2 to 40 at the cathode while keeping the anode ratio fixed at 100. NO3 (-)-N removal rate of 7.1 ± 0.9 kg NO3 (-)-N/m(3) net cathodic compartment (NCC)/day was achieved at cathode COD/NO3 (-)-N ratio 7.31 with the current density of 190 ± 9.1 mA/m(2) and power density of 31.92 ± 4 mW/m(2) of electrode surface area. We achieved an open-circuit voltage (OCV) of 410 ± 20 mV at initial cathodic NO3 (-)-N of 0.345 g/l. The cathode COD/NO3 (-)-N ratio had a significant influence on MFC's OCV and nitrate removal rate. Lower OCV (<150 mV) and NO3 (-)-N removal rates were observed at COD/NO3 (-)-N ratio >12 and <7. Experiments done at different cathode pH values indicated that the optimum pH for denitrification was 7. Under optimized biochemical conditions, nitrate removal rate of 6.5 kg NO3 (-)-N/m(3) net cathodic compartment (NCC)/day and power density of 210 mW/m(2) were achieved in a low resistance MFC. The present study thus demonstrates the utility of MFCs for the treatment of high nitrate wastes.

Immobilized laccase mediated dye decolorization and transformation pathway of azo dye acid red 27.

BACKGROUND: Laccases have good potential as bioremediating agents and can be used continuously in the immobilized form like many other enzymes. METHODS: In the present study, laccase from Cyathus bulleri was immobilized by entrapment in Poly Vinyl Alcohol (PVA) beads cross-linked with either nitrate or boric acid. Immobilized laccase was used for dye decolorization in both batch and continuous mode employing a packed bed column. The products of degradation of dye Acid Red 27 were identified by LC MS/MS analysis. RESULTS: The method led to very effective (90%) laccase immobilization and also imparted significant stability to the enzyme (more than 70% after 5 months of storage at 4°C). In batch decolorization, 90-95% decolorization was achieved of the simulated dye effluent for up to 10-20 cycles. Continuous decolorization in a packed bed bioreactor led to nearly 90% decolorization for up to 5 days. The immobilized laccase was also effective in decolorization and degradation of Acid Red 27 in the presence of a mediator. Four products of degradation were identified by LC-MS/MS analysis. CONCLUSIONS: The immobilized laccase in PVA-nitrate was concluded to be an effective agent in treatment of textile dye effluents.

Laccase/mediator assisted degradation of triarylmethane dyes in a continuous membrane reactor.

Laccase/mediator systems are important bioremediation agents as the rates of reactions can be enhanced in the presence of the mediators. The decolorization mechanism of two triarylmethane dyes, namely, Basic Green 4 and Acid Violet 17 is reported using Cyathus bulleri laccase. Basic Green 4 was decolorized through N-demethylation by laccase alone, while in mediator assisted reactions, dye breakdown was initiated from oxidation of carbinol form of the dye. Benzaldehyde and N,N-dimethyl aniline were the major end products. With Acid Violet 17, laccase carried out N-deethylation and in mediator assisted reactions, oxidation of the carbinol form of the dye occurred resulting in formation of formyl benzene sulfonic acid, carboxy benzene sulfonic acid and benzene sulfonic acid. Toxicity analysis revealed that Basic Green 4 was toxic and treatment with laccase/mediators resulted in 80-100% detoxification. The treatment of the textile dye solution using laccase and 2,2'-azino-di-(-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was demonstrated in an enzyme membrane reactor. At a hydraulic retention time of 6h, the process was operated for a period of 15 days with nearly 95% decolorization, 10% reduction in flux and 70% recovery of active ABTS.

Mediator-assisted decolorization and detoxification of textile dyes/dye mixture by Cyathus bulleri laccase.

Laccase from basidiomycete fungus Cyathus bulleri was evaluated for its ability to decolorize a number of reactive and acidic dyes in the presence of natural and synthetic mediators. The extent of decolorization was monitored at different mediator/dye concentrations and incubation time. Among the synthetic mediators, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was effective at low mediator/dye ratios and resulted in 80-95% decolorization at rates that varied from 226 +/- 4 nmol min(-1) mg(-1) for Reactive Orange 1 to 1,333 +/- 15 nmol min(-1) mg(-1) for Reactive Red 198. Other synthetic mediators like 1-hydroxybenzotriazole and violuric acid showed both concentration- and time-dependent increases in percent decolorization. Natural mediators like vanillin, on the other hand, were found to be less effective on all the dyes except Reactive Orange 1. Computed rates of decolorization were about twofold lower than that with ABTS. The laccase-ABTS system also led to nearly 80% decolorization for the simulated dye mixture. No clear correlation between laccase activity on the mediator and its ability to decolorize dyes was found, but pH had a significant effect: Optimum pH for decolorization coincided with the optimum pH for mediator oxidation. The treated samples were also evaluated for toxicity in model microbial systems. The laccase-mediator system appears promising for treatment of textile wastewaters.