Removal of cadmium and arsenic from water through biomineralization.

Due to anthropogenic activities, heavy metals such as cadmium (Cd) and arsenic (As) are one of the most toxic xenobiotics contaminating water, thus affecting human health and the environment. The objective of the present investigation was to study the effect of ureolytic bacteria Bacillus paramycoides-MSR1 for the bioremediation of Cd and As from contaminated water. The B. paramycoides showed high resistance to heavy metals, Cd and As, with minimum inhibitory concentration (MIC) of 12.84 µM and 48.54 µM, respectively. The urease activity and calcium carbonate (CaCO3) precipitation were evaluated in artificial wastewater with different concentrations of Cd (0, 10, 20, 30, 40, 50, and 60 µM) and As (0, 20, 40, 60, 80, and 100 µM). The maximum urease activity in Cd-contaminated artificial wastewater was observed after 96 hours, which showed a 76.1% decline in urease activity as the metal concentration increased from 0 to 60 µM. Similarly, 14.1% decline in urease activity was observed as the concentration of As was increased from 0 to 100 µM. The calcium carbonate precipitation at the minimum inhibitory concentration of Cd and As-contaminated artificial wastewater was 189 and 183 mg/100 ml, respectively. The percentage removal of metal from artificially contaminated wastewater with varied concentrations was analyzed using atomic absorption spectroscopy (AAS). After 168 hours of incubation, 93.13% removal of Cd and 94.25% removal of As were observed. Microstructural analysis proved the presence of calcium carbonate in the form of calcite, confirming removal of cadmium and arsenic by microbially induced calcium carbonate precipitation (MICCP) to be promising technique for water decontamination.

Multi-metal tolerance of DHHC palmitoyl transferase-like protein isolated from metal contaminated soil.

The microbiota inhabiting in metal polluted environment develops strong defense mechanisms to combat pollution and sustain life. Investigating the functional genes of the eukaryotic microbiota inhabiting in these environments by using metatranscriptomics approach was the focus of this study. Size fractionated eukaryotic cDNA libraries (library A, < 0.5 kb, library B, 0.5-1.0 kb, and library C, > 1.0 kb) were constructed from RNA isolated from the metal contaminated soil. The library C was screened for Cadmium (Cd) tolerant genes by using Cd sensitive yeast mutant ycf1Δ by functional complementation assay, which yielded various clones capable of growing in Cd amended media. One of the Cd tolerant clones, PLCg39 was selected because of its ability to grow at high concentrations of Cd. Sequence analysis of PLCg39 showed homology with DHHC palmitoyl transferases, which are responsible for addition of palmitoyl groups to proteins and usually possess metal coordination domains. The cDNA PLCg39 was able to confer tolerance to Cd-sensitive (ycf1Δ), Copper-sensitive (cup1Δ) and Zn-sensitive (zrc1Δ) yeast mutants when grown at different concentrations of Cd (40-100 µM), Cu (150-1000 µM) and Zn (10-13 mM), respectively. The DHHC mutant akr1Δ transformed with PLCg39 rescued from the metal sensitivity indicating the role of DHHC palmitoyl transferase in metal tolerance. This study demonstrated that PLCg39 acts as a potential metal tolerant gene which could be used in bioremediation, biosensing and other biotechnological fields.

A robust genetic transformation protocol to obtain transgenic shoots of Solanum tuberosum L. cultivar 'Kufri Chipsona 1'.

The genetic transformation of plants is an important biotechnological tool used for crop improvement for many decades. The present study was focussed to investigate various factors affecting genetic transformation of potato cultivar 'Kufri Chipsona 1'. It was observed that explants pre-cultured for 2 days on MS2 medium (MS medium containing 10 µM silver nitrate, 10 µM BA, 15 µM GA3), injured with a surgical blade and co-cultivated with Agrobacterium tumefaciens strain EHA105 [O.D600 (0.6)] for 2 days results in maximum transient ß-glucuronidase (GUS) expression. The addition of 100 µM acetosyringone in MS2 medium also increased rate of transient GUS expression in both the explants. Clumps of putative transgenic shoots were regenerated using the optimised culture conditions from leaf and internodal explants. The stable integration of T-DNA was established using histochemical staining for GUS and amplification of DNA fragment specific to nptII and uidA genes. Within the clumps, around 67.85% of shoots showed uniform GUS expression in all the tissues and about 32.15% shoots show intermittent GUS expression establishing chimeric nature. Uniform GUS staining of the tissue was used as initial marker of non-chimeric transgenic shoots. Quantitative expression of nptII transgene was found to be directly proportional to uniformity of GUS staining in transgenic shoots. The present investigation indicated that manipulation of culture conditions and the medium composition may help to get transgenic shoots with uniform expression of transgene in all the tissues of potato cultivar 'Kufri Chipsona 1'.

Computational screening of potential drug targets for pathogens causing bacterial pneumonia.

Streptococcus pneumoniae is widely recognized as the main cause of bacterial pneumonia among all age groups. Other important gram-positive, gram-negative and atypical bacteria causing pneumonia majorly infect children and infants. Despite abundant occurrence of bacterial pneumonia, there is no specific antibiotic therapy available. On the other hand non-specific therapies are less effective and may influence bacterial resistance. Therefore, search for novel drug targets for pathogen is highly necessary. The current study suggested novel potential drug targets through the subtractive and comparative genomics approach. Putative drug targets were identified from highly virulent strain of Streptococcus pneumoniae using target identification (TiD) software and compared with other 12 pneumonia causing pathogens. The putative targets were prioritized through druggability analysis, virulence analysis, metabolic pathway enrichment followed by functional annotations and interactome network. Prioritization of 74 drug targets revealed that 42 of them were enzymes which included 29 new targets and seven chokepoint enzymes. Twenty (out of 74) potential targets are proposed as hub genes through interactome analysis and explored their significance in survival of the pathogen. Comparative analysis of 20 hub genes represents that 15 are enzymes and five are non-enzymes. Functional annotation of two chokepoint hub enzymes namely, peptidoglycan bridge formation alanyltransferase MurN (fibB) and PTS mannitol transporter subunit IIA (mltF) were significantly enriched in peptidoglycan biosynthesis and phosphotransferase system (PTS) respectively. Therefore these enzymes would be of prior interest for rational design of targeted therapy against bacterial pneumonia.

Cadmium induced glutathione bioaccumulation mediated by γ-glutamylcysteine synthetase in ectomycorrhizal fungus Hebeloma cylindrosporum.

Ectomycorrhizal fungi hold a potential role in bioremediation of heavy metal polluted areas because of its metal accumulation and detoxification property. We investigated the cadmium (Cd) induced bioaccumulation of glutathione (GSH) mediated by γ-glutamylcysteine synthetase (γ-GCS) in the ectomycorrhizal fungus Hebeloma cylindrosporum. In H. cylindrosporum, a demand driven synthesis of GSH has been observed in response to Cd. The expression and enzyme activity of H. cylindrosporum γ-GCS (Hcγ-GCS) increased as a function of external Cd stress resulting in increased GSH production. The function of Hcγ-GCS in providing heavy metal tolerance to H. cylindrosporum was justified by complementing the gene in gsh1Δ mutant of Saccharomyces cerevisiae. The metal sensitive mutant gsh1Δ successfully restored its metal tolerance ability when transformed with Hcγ-GCS gene. Sequence analysis of Hcγ-GCS showed homology with most of the reported γ-GCS proteins from basidiomycetes family. The active site of the Hcγ-GCS protein is composed of amino acids that were found to be conserved not only in fungi, but also in plants and mammals. From these results, it was concluded that Hcγ-GCS plays an important role in bioaccumulation of GSH, which is a core component in the mycorrhizal defense system under Cd stress for Cd homeostasis and detoxification.

Corn steep liquor as a nutritional source for biocementation and its impact on concrete structural properties.

Microbial-induced carbonate precipitation (MICP) has a potential to improve the durability properties and remediate cracks in concrete. In the present study, the main emphasis is placed upon replacing the expensive laboratory nutrient broth (NB) with corn steep liquor (CSL), an industrial by-product, as an alternate nutrient medium during biocementation. The influence of organic nutrients (carbon and nitrogen content) of CSL and NB on the chemical and structural properties of concrete structures is studied. It has been observed that cement-setting properties were unaffected by CSL organic content, while NB medium influenced it. Carbon and nitrogen content in concrete structures was significantly lower in CSL-treated specimens than in NB-treated specimens. Decreased permeability and increased compressive strength were reported when NB is replaced with CSL in bacteria-treated specimens. The present study results suggest that CSL can be used as a replacement growth medium for MICP technology at commercial scale.

Microbial healing of cracks in concrete: a review.

Concrete is the most widely used construction material of the world and maintaining concrete structures from premature deterioration is proving to be a great challenge. Early age formation of micro-cracking in concrete structure severely affects the serviceability leading to high cost of maintenance. Apart from conventional methods of repairing cracks with sealants or treating the concrete with adhesive chemicals to prevent the cracks from widening, a microbial crack-healing approach has shown promising results. The unique feature of the microbial system is that it enables self-healing of concrete. The effectiveness of microbially induced calcium carbonate precipitation (MICCP) in improving durability of cementitious building materials, restoration of stone monuments and soil bioclogging is discussed. Main emphasis has been laid on the potential of bacteria-based crack repair in concrete structure and the applications of different bacterial treatments to self-healing cracks. Furthermore, recommendations to employ the MICCP technology at commercial scale and reduction in the cost of application are provided in this review.

Efficient, one step and cultivar independent shoot organogenesis of potato.

An efficient, one step and genotype independent protocol of shoot organogenesis was developed from leaf and internodal explants taken from microshoots of different cultivars of potato (Solanum tuberosum L.). Initially, microshoots were cultured on basal Murashige and Skoog medium additionally supplemented with 10 µM AgNO3 (MS1 medium) to achieve healthy shoot growth required to get the quality explants. Shoot organogenesis was induced from both types of explants (leaf and internodal) on MS1 medium variously supplemented with 6-benzyladenine (BA) and gibberellic acid (GA3). Maximum explants were induced shoot organogenesis on MS1 medium supplemented with 10 µM BA and 15.0 µM GA3 from both the cultivars namely 'Kufri Chipsona 1' and 'Kufri Pukhraj'. Among the types of explants used, better response was observed from internodal segments as compared to leafs. This optimized medium combination was found to be equally effective for all the eight cultivars tested namely 'Kufri Pukhraj', 'Kufri Chipsona 1', 'Kufri Chipsona 2', 'Kufri Jyoti', 'Kufri Surya', 'Kufri Chandramukhi', 'Kufri Khyati' and 'Desiree'. The clonal uniformity of the regenerated shoots was confirmed using random amplified polymorphic DNA and inter-simple sequence repeats markers.

Metal induction of a Pisolithus albus metallothionein and its potential involvement in heavy metal tolerance during mycorrhizal symbiosis.

Metallothioneins (MTs) are small, cysteine-rich peptides involved in intracellular sequestration of heavy metals in eukaryotes. We examined the role in metal homeostasis and detoxification of an MT from the ectomycorrhizal fungus Pisolithus albus (PaMT1). PaMT1 encodes a 35 amino acid-long polypeptide, with 7 cysteine residues; most of them part of a C-x-C motif found in other known basidiomycete MTs. The expression levels of PaMT1 increased as a function of increased external Cu and Cd concentrations and were higher with Cu than with Cd. Heterologous complementation assays in metal-sensitive yeast mutants indicated that PaMT1 encodes a polypeptide capable of conferring higher tolerance to both Cu and Cd. Eucalyptus tereticornis plantlets colonized with P. albus grown in the presence of Cu and Cd showed better growth compared with those with non-mycorrhizal plants. Higher PaMT1 expression levels were recorded in mycorrhizal plants grown in the presence of Cu and Cd compared with those in control mycorrhizal plants not exposed to heavy metals. These data provide the first evidence to our knowledge that fungal MTs could protect ectomycorrhizal fungi from heavy metal stress and in turn help the plants to establish in metal-contaminated sites.

Biocalcification by halophilic bacteria for remediation of concrete structures in marine environment.

Microbial carbonate precipitation has emerged as a promising technology for remediation and restoration of concrete structures. Deterioration of reinforced concrete structures in marine environments is a major concern due to chloride-induced corrosion. In the current study, halophilic bacteria Exiguobacterium mexicanum was isolated from sea water and tested for biomineralization potential under different salt stress conditions. The growth, urease and carbonic anhydrase production significantly increased under salt stress conditions. Maximum calcium carbonate precipitation was recorded at 5 % NaCl concentration. Application of E. mexicanum on concrete specimens significantly increased the compressive strength (23.5 %) and reduced water absorption about five times under 5 % salt stress conditions compared to control specimens. SEM and XRD analysis of bacterial-treated concrete specimens confirmed the precipitation of calcite. The present study results support the potential of this technology for improving the strength and durability properties of building structures in marine environments.

Pseudomonas sp. CL7 from Sludge Removed 2,3,4,6-Tetrachlorophenol in Vivo and in Vitro Condition.

The present research focused on 2,3,4,6-Tetrachlorophenol (2,3,4,6-TeCP) mineralizing bacterium from the sludge of pulp and paper industry and identified as Pseudomonas sp. CL7 by 16s rRNA gene sequences analysis. This isolate degraded 2,3,4,6-TeCP as indicated by stoichiometric release of chloride and biomass formation. High pressure liquid chromatography (HPLC) analysis showed that Pseudomonas sp. (CL7) was able to mineralize a higher concentration of 2,3,4,6-TeCP (600 mg/l or 2.5 mM) than any previously reported 2,3,4,6-TeCP degrading bacteria. As the concentration of 2,3,4,6-TeCP increased from 50 (0.21 mM) to 600 mg/l (2.5 mM), the reduction in the cell growth was observed and the 2,3,4,6-TeCP degradation was more than 85% in all the concentrations in the present study. CL7 was able to remove 100% of 2,3,4,6-TeCP from the sludge (in Vitro condition) when supplemented with 100 mg/l (0.42 mM) of 2,3,4,6-TeCP and grown for two weeks. This study showed that CL7 can be used for bioremediation of 2,3,4,6-TeCP.

Seasonal variations in harvest index and bacoside A contents amongst accessions of Bacopa monnieri (L.) Wettst. collected from wild populations.

Bacoside A, a major active principle of Bacopa monnieri known for its cognitive effects is a mixture of saponins like bacoside A3, bacopaside II, isomer of bacopasaponin C and bacopasaponin C. Seasonal changes in biomass and bacoside A levels in fourteen accessions of B. monnieri were evaluated after maintaining these at a common site at Thapar University campus, Patiala (30°19'36.12″N and 76°24'1.08″E) for 1 year. Harvestable biomass and total bacoside A contents varied significantly between the accessions and also in a particular accession during different seasons of the year. The maximum dry weight of plant (biomass 1.64 g) and bacoside A levels (6.82 mg/plant) were recorded in accession BM1. Harvestable biomass was highest during summer in accessions BM1 and BM7 (FW 4.2 g/plant), whereas bacoside A levels were also highest during summer and in accession BM1 (6.82 mg/plant). The lowest bacoside A level (0.06 mg/plant) was recorded in accession BM14 during winter. Principal component analysis showed that samples of summer were positively correlated with both the components suggesting an appropriate time for the harvest.

Differential expression of metallothioneins in response to heavy metals and their involvement in metal tolerance in the symbiotic basidiomycete Laccaria bicolor.

Cysteine-rich peptides such as metallothioneins (MTs) are involved in metal homeostasis and detoxification in many eukaryotes. We report the characterization and expression of two MT genes, LbMT1 and LbMT2 from the ectomycorrhizal fungus Laccaria bicolor under metal stress conditions. LbMT1 and LbMT2 differ with respect to the length of the encoded peptides (58 versus 37 aa, respectively) and also by their expression patterns in response to metals. The expression levels of both LbMT1 and LbMT2 increased as a function of increased external Cu concentration, the expression levels for LbMT2 were always significantly higher compared with those of LbMT1. Only LbMT1, but not LbMT2, responded to Cd supply in the range of 25-100 µM while Zn did not affect the transcription of either LbMT1 or LbMT2. Both genes also responded to oxidative stress, but to a lesser extent compared to their responses to either Cu or Cd stress. Heterologous complementation assays in metal-sensitive yeast mutants indicated that both LbMT1 and LbMT2 encode peptides capable of conferring higher tolerance to both Cu and Cd. The present study identified LbMTs as potential determinants of the response of this mycorrhizal fungus to Cu and Cd stress.

Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

BACKGROUND: Construction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs. RESULTS: We report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases. CONCLUSIONS: A simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

Bacterial diversity of extremely alkaline bauxite residue site of alumina industrial plant using culturable bacteria and residue 16S rRNA gene clones.

Bauxite residue (red mud), generated during the extraction of alumina from bauxite ore is characterized by high pH, high concentrations of soluble ions with low or virtually no organic matter. These extreme conditions along with numerous nutrient deficiencies, limit the microbial growth and vegetation establishment. In the present study, diversity of both cultivable and non-cultivable bacteria present in the red mud was investigated by 16S rDNA sequence analyses. The cultivable bacteria were identified as Agromyces indicus, Bacillus litoralis, B. anthracis, Chungangia koreensis, Kokuria flava, K. polaris, Microbacterium hominis, Planococcus plakortidis, Pseudomonas alcaliphila and Salinococcus roseus based on their 16S rDNA sequence analysis. These isolates were alkali tolerant, positive for one or more of the enzyme activities tested, able to produce organic acids and oxidize wide range of carbon substrates. For non-cultivable diversity of bacteria, DNA was extracted from the bauxite residue samples and 16S rDNA clone library was constructed. The 16S rDNA clones of this study showed affiliation to three major phyla predominant being betaproteobacteria (41.1%) followed by gammaproteobacteria (37.5%) and bacteroidetes (21.4%). We are reporting for the first time about the bacterial diversity of this unique and extreme environment.

Influence of sclerotia formation on ligninolytic enzyme production in Morchella crassipes.

Morels are wild edible ascomycetous mushrooms that are highly prized because of their medicinal and nutritional qualities. Ligninolytic enzymes are considered as one of the most important enzymes in fungi due to their involvement in fruiting body formation during artificial cultivation on different substrates. In the life cycle of morels, sclerotia are the intermediate stage to form a fruiting body from mycelia. We have studied the production of ligninolytic enzymes by Morchella crassipes MR8 growing on different substrates and during sclerotia formation. This fungus is able to produce ligninolytic enzymes such as laccase (Lac), lignin peroxidase (LiP), and manganese-dependent peroxidase (MnP) when grown on different substrates. Maximum Lac activity was observed when grown in wheat grains whereas maximum activities of MnP and LiP were observed when grown in rice straw. Laccase enzyme was produced in high titers during sclerotia formation and maturation when grown in combinations of soil and substrates. A large number of sclerotia was observed in soil and wheat grains, along with high titers of laccase. Cellulase activity was observed to be constant during sclerotia formation and maturation. The present study results suggest that laccase enzyme might play an important role in sclerotia formation in morels.

Alkalistable xylanase production by alkalitolerant Paenibacillus montaniterrae RMV1 isolated from red mud.

The alkalitolerant and xylanolytic bacterial strain (RMV1) isolated from red mud pond was identified as Paenibacillus montaniterrae based on both biochemical and 16S rDNA sequence analysis. The RMV1 bacterial isolate produced alkalistable and thermostable endoxylanase active over a broad range of pH (4.0-11.0) and temperature (20-100 °C), with optima at 50 °C and pH 9.0 with a T1/2 of 6.7 hours at 50 °C. This is the first report on the isolation of P. montaniterrae from bauxite residue with quite a high xylanase producing ability.

Viability of calcifying bacterial formulations in fly ash for applications in building materials.

Evidence of bacterial involvement in precipitation of calcium carbonates has brought a revolution in the field of applied microbiology, geotechnical sciences, environmental and civil engineering with its marked success in restoration of various building materials. For applications of these calcite binder-producing bacterial cultures, different expensive carrier materials have been used but their high costs have come in the way of their successful commercialization. In the present study, we have explored the potential of cheap industrial by-product fly ash as a carrier material for bacterial cells and investigated the viability of calcifying bacterial isolates: Bacillus megaterium, Bacillus cereus, and Lysinibacillus fusiformis in fly ash carrier at varying temperatures and moisture conditions along with biomineralization efficacy of these formulations. We used laser scanning confocal microscopy to analyze the viability of bacteria by florescent dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) along with the plate count method. Results revealed that fly ash successfully served as an effective carrier material and bacterial formulations stored at 4 °C provided longer shelf life than those stored at higher temperatures. Up to 10(6) cfu/g was found to sustain in all formulations at 4 °C compared to 10(4)-10(5) cfu/g in case of higher temperatures up to 1 year. For 4 °C, higher moistures (50 %) were found to provide better survivability while for higher temperatures, lower moistures (30 %) favored higher viability. The biomineralization capability of fresh and formulated bacterial cells was compared on the basis of precipitation of carbonates and it was found that carbonate precipitation efficacy of formulated bacterial cells was comparable to fresh bacterial cells.

Removal of 2,4,5-trichlorophenol by bacterial isolates from the secondary sludge of pulp and paper mill.

2,4,5-trichlorophenol (2,4,5-TCP) mineralizing bacteria were isolated from the secondary sludge of pulp and paper industry. These isolates used 2,4,5-TCP as a source of carbon and energy and were capable of degrading this compound, as indicated by stoichimetric release of chloride and biomass formation. Based on 16S rRNA sequence analysis, these bacteria were identified as Kocuria sp. (CL2), Bacillus pumillus (CL5), Pseudomonas stutzeri (CL7). HPLC analysis revealed that these isolates were able to degrade 2,4,5-TCP at higher concentrations (600 mg/l or 3.0 mM). A consortia of these isolates completely removed 2,4,5-TCP from the sludge obtained from pulp and paper mill within 2 weeks when supplemented at a rate of 100 mg l(-1) . Bacterial consortium also significantly reduced absorbable organic halogen (AOX) and extractable organic halogen (EOX) by 61% and 63%, respectively from the sludge. These isolates have high potential to remove 2,4,5-TCP and may be used for remediation of pulp paper mill waste containing 2,4,5-TCP.

Bacillus megaterium mediated mineralization of calcium carbonate as biogenic surface treatment of green building materials.

Microbially induced calcium carbonate precipitation is a biomineralization process that has various applications in remediation and restoration of range of building materials. In the present study, calcifying bacteria, Bacillus megaterium SS3 isolated from calcareous soil was applied as biosealant to enhance the durability of low energy, green building materials (soil-cement blocks). This bacterial isolate produced high amounts of urease, carbonic anhydrase, extra polymeric substances and biofilm. The calcium carbonate polymorphs produced by B. megaterium SS3 were analyzed by scanning electron microscopy, confocal laser scanning microscopy, X-ray diffraction and Fourier transmission infra red spectroscopy. These results suggested that calcite is the most predominant carbonate formed by this bacteria followed by vaterite. Application of B. megaterium SS3 as biogenic surface treatment led to 40 % decrease in water absorption, 31 % decrease in porosity and 18 % increase in compressive strength of low energy building materials. From the present investigation, it is clear that surface treatment of building materials by B. megaterium SS3 is very effective and eco friendly way of biodeposition of coherent carbonates that enhances the durability of building materials.