Role of carbon-dioxide sequestering bacteria for clean air environment and prospective production of biomaterials: a sustainable approach.

The increase in demand of fossil fuel uses for developmental activity and manufacturing of goods have resulted a huge emission of global warming gases (GWGs) in the atmosphere. Among all GWGs, CO2 is the major contributor that inevitably causes global warming and climate change. Mitigation strategies like biological CO2 capture through sequestration and their storage into biological organic form are used to minimize the concentration of atmospheric CO2 with the goal to control climate change. Since increasing atmospheric CO2 level supports microbial growth and productivity thus microbial-based CO2 sequestration has remarkable advantages as compared to plant-based sequestration. This review focuses on CO2 sequestration mechanism in bacteria through different carbon fixation pathways, involved enzymes, their role in calcite, and other environmentally friendly biomaterials such as biofuel, bioplastic, and biosurfactant.

Biological fixation of carbon dioxide and biodiesel production using microalgae isolated from sewage waste water.

The present research investigates potential of microalgae isolated from sewage treatment plant to utilize sodium bicarbonate as carbon source for CO2 sequestration and biodiesel production. Eight algal isolates were isolated from waste water of sewage treatment plant, Amity University Haryana, India. The most potent algal isolates were identified and characterized on the basis of growth and lipid content. The efficient isolates ASW1 and ASW2 were identified as Chlorella sp. and Arthronema sp. by 18srRNA and 16srRNA sequencing method. In both isolates, maximum growth was observed under 20-W fluorescent bulb (3500 flux light intensity) with continuous light cycle of 24 h at pH 9.0 and 25 °C on the 20th day of incubation period. CO2 utilization efficiency of both algal isolates were observed in terms of total CO2 consumption rate. Under optimized culture conditions, total lipid content and lipid yield was higher in Arthronema sp. (180 mg l-1; 32.14%) as compared to Chlorella sp. (98 mg l-1; 29.6%) in 50 mM NaHCO3. Transesterified lipids were analysed by GC-MS. The fatty acid methyl ester profile of Arthronema sp. was 34.42% saturated and 65.58% unsaturated fatty acid. Chlorella sp. produces 29.80% saturated and 70.20% unsaturated fatty acid. In both isolates, C16 and C18 fatty acids dominated, which is a promising component for biodiesel. Graphical abstract.

Cloning, expression and characterization of ß- and γ­carbonic anhydrase from Bacillus sp. SS105 for biomimetic sequestration of CO2.

Bacterium Bacillus sp. SS105, isolated from Free Air CO2 Enriched (FACE) soil was previously screened for carbonic anhydrase activity and CO2 sequestration. In this study, strain was selected to amplify carbonic anhydrase encoding genes. The CA genes from Bacillus sp. SS105 were found to be homologous with beta­carbonic anhydrase (ß-CA) and gamma­carbonic anhydrase (γ-CA). Both types of CA genes was cloned in pET30b (+) and expressed in E coliBL21 (DE3) with His-tag at the N-terminus. The recombinant proteins were purified by Ni-NTA affinity chromatography. The molecular size of ß-CA and γ-CA were approximately 27 kDa and 25 kDa respectively. The optimum pH and temperature were found to be 8.0 and 37 °C respectively. The Zn+ was enhancing the CAs enzyme activity. Anions and modulators showed inhibitory effect on CAs at specific concentration. Functional domain analysis of both CA proteins showed conserved region of respective proteins. Recombinant enzymes were used for bio-mineralization based conversion of atmospheric CO2 into valuable calcite. Calcite formation was evaluated with or without use of enzymes and confirmed by SEM and XRD analysis. SEM result confirmed the conversion of flower-shaped unstable form of vaterite to hexagonal cubic stable form of calcite in presence of enzymes.

Expression and characterization of novel laccase gene from Pandoraea sp. ISTKB and its application.

In the present study, a non-blue laccase gene from previously reported lignin degrading bacterium, Pandoraea sp. ISTKB, was isolated, cloned and expressed in E. coli. Bioinformatics analysis of sequence discovered twin-arginine translocation signal sequence, copper binding motifs and presence of more random coil compare to helices and sheets in structure. The enzyme was found to be active on wide pH range and the pH optima was observed at pH 4 and 8 on substrate 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 2,6-Dimethoxyphenol respectively. This is a thermophilic enzyme with maximum activity around 50-70 °C. The enzyme was further characterized by spectroscopy, reaction kinetics and effect of metal ions and inhibitors were studied. Compared to laccase alone; the treatment of dyes with laccase plus mediator resulted in enhanced decolorization of crystal violet, methylene blue, azure B, carmine and Congo red but the effect of mediator was not observed on trypan blue. Laccase treatment triggered polymerization on vanillic acid (VA) and kraft lignin (KL). Laccase plus mediator treatment reversed the polymerization and resulted in transformation or degradation of VA and KL. This thermophilic and alkalophilic non-blue laccase from Pandoraea sp. ISTKB is promising with prospective biotechnological application.

Production, process optimization and molecular characterization of polyhydroxyalkanoate (PHA) by CO2 sequestering B. cereus SS105.

Carbon dioxide sequestering bacterial strains were previously isolated from free air CO2 enriched (FACE) soil. In the present study, these strains were screened for PHA accumulation and Bacillus cereus SS105 was found to be the most prominent PHA accumulating strain on sodium bicarbonate and molasses as carbon source. This strain was further characterized by Spectrofluorometric method and Confocal microscopy after staining with Nile red. PHA granules in inclusion bodies were visualized by Transmission Electron Microscopy. The PHA and its monomer composition were characterized by GC-MS followed by FTIR and NMR. The genetic basis of PHA production was confirmed by the amplification, cloning and analysis of PHA biosynthesis genes phaR, phaB and phaC from B. cereus with the degenerate primers. The PHA production was further optimized by Response Surface Methodology and the percent increase observed after optimization was 55.16% (w/v).

Biofuel production and phycoremediation by Chlorella sp. ISTLA1 isolated from landfill site.

The present study aims to investigate the biofuel production ability and potential of heavy metal remediation of Chlorella sp. ISTLA1 isolated from a landfill site. The strain was cultured in Bold's Basal medium at different concentration of NaHCO3 and pH. Response surface methodology was employed for the optimization of nutrient sources for higher lipid production. Under the optimized conditions, the yield of lipid and biomass was 365.42 and 833.14 mg L-1 respectively. GC-MS analysis of lipid indicated the presence of C8 to C31 organic compounds consisting mainly of palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1). Additionally, remediation of heavy metals like Zn, Cu, Mn and Fe from waste water was observed by AAS and EDX. The removal efficiency was 82.6% for Zn, 56.5% for Cu, 79.8% for Mn and 40% for Fe. The study revealed simultaneous biodiesel production and waste water treatment by Chlorella sp. ISTLA1.

Recycling of carbon dioxide by free air CO2 enriched (FACE) Bacillus sp. SS105 for enhanced production and optimization of biosurfactant.

Carbon dioxide utilizing bacterium Bacillus sp. SS105 was isolated from FACE (free air CO2 enriched) sample. The strain was grown in shake flask containing minimal salt medium with 50mM NaHCO3 as autotrophic carbon source and molasses as a low cost byproduct for mixotrophic growth. Carbon dioxide sequestration property of Bacillus sp. SS105 was determined by enzyme assay of carbonic anhydrase and ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO). Along with CO2 sequestration this strain produced biosurfactant and its characterization by FTIR and 1H NMR indicated lipopeptide nature. Optimization of process parameter along with nutrient sources for higher biosurfactant production was done by Response Surface Methodology (RSM). Under optimized conditions, the yield of biosurfactant and biomass was 2.65 and 2.78gL-1 respectively. The study revealed simultaneous CO2 sequestration and biosurfactant production by Bacillus sp. SS105.

Cloning and expression of gamma carbonic anhydrase from Serratia sp. ISTD04 for sequestration of carbon dioxide and formation of calcite.

Bacterial strains isolated from marble mines rock and enriched in the chemostat culture with different concentrations of sodium bicarbonate. The enriched consortium had six bacterial isolates. One of bacterium isolate showed carbonic anhydrase (CA) activity by catalyzing the reversible hydration reaction of carbon dioxide to bicarbonate. The bacterium was identified as Serratia sp. by 16S rRNA sequence analysis. The carbonic anhydrase gene from Serratia sp. was found to be homologous with gamma carbonic anhydrase. The carbonic anhydrase gene was cloned in PET21b(+) and expressed it in recombinant Escherichia coli BL21 (DE3) with His-tag at the C-terminus. The recombinant protein was purified efficiently by using one-step nickel affinity chromatography. Expected size of carbonic anhydrase was approximately 29 kDa in SDS-PAGE gel. Recombinant carbonic anhydrase enzyme was used for biomineralization-based conversion of atmospheric CO2 into valuable calcite minerals. The calcification was confirmed by using XRD, FTIR, EDX and SEM analysis.

Characterization of bacteria isolated from palaeoproterozoic metasediments for sequestration of carbon dioxide and formation of calcium carbonate.

Bacterial community of palaeoproterozoic metasediments was enriched in the chemostat in the presence of different concentrations of NaHCO3. Six bacterial isolates were isolated from the chemostat on nutrient agar plates on the basis of distinct morphology. Denaturing gradient gel electrophoresis (DGGE) proved the presence of six operational taxonomic units (OTUs) at 50 and 100 mM NaHCO3. The OTU was reduced to three and one at enrichment concentration of 150 and 200 mM NaHCO3 respectively. These six isolates were tested for sequestration of carbon dioxide by (14)C metabolic labeling of NaH(14)CO3. Among the six isolates, one of the bacterium showed better potency to fix radiolabeled NaH(14)CO3. The isolate (ISTD04) was identified as Serratia sp. by 16S ribosomal RNA (16S rRNA) sequence analysis and was found to be same as the DGGE OTU sequence at 200-mM NaHCO3 concentration. The bacterium was tested for product formation in form of calcite crystals in presence of 5 % CO2. Scanning electron microscopy (SEM) of product formed by the bacterium revealed defined faceted rhombohedral structure which resembled calcite and vaterite phases of the crystal. Formation of calcium carbonate crystals was further confirmed by Fourier transform infrared (FTIR) spectroscopy as carbonate group showing strong vibration at 1,456 cm(-1). Major calcite phase diffraction peaks were determined by X-ray diffraction (XRD) analysis, and energy-dispersive X-ray (EDX) analysis showed the presence of CaO (72 %) and carbon (18 %). Bacterium use bicarbonate as carbon source for their growth as well as by-product formation in form of calcite shows carbon circulation and storage.

Proteomic analysis of carbon concentrating chemolithotrophic bacteria Serratia sp. for sequestration of carbon dioxide.

A chemolithotrophic bacterium enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was identified as Serratia sp. by 16S rRNA sequencing. Carbon dioxide sequestering capacity of bacterium was detected by carbonic anhydrase enzyme and ribulose-1, 5- bisphosphate carboxylase/oxygenase (RuBisCO). The purified carbonic anhydrase showed molecular weight of 29 kDa. Molecular weight of RuBisCO was 550 kDa as determined by fast protein liquid chromatography (FPLC), however, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) showed presence of two subunits whose molecular weights were 56 and 14 kDa. The Western blot analysis of the crude protein and purified sample cross reacted with RuBisCO large-subunit polypeptides antibodies showed strong band pattern at molecular weight around 56 kDa regions. Whole cell soluble proteins of Serratia sp. grown under autotrophic and heterotrophic conditions were resolved by two-dimensional gel electrophoresis and MALDI-TOF/MS for differential expression of proteins. In proteomic analysis of 63 protein spots, 48 spots were significantly up-regulated in the autotrophically grown cells; seven enzymes showed its utilization in autotrophic carbon fixation pathways and other metabolic activities of bacterium including lipid metabolisms indicated sequestration potency of carbon dioxide and production of biomaterials.

Extraction of extracellular lipids from chemoautotrophic bacteria Serratia sp. ISTD04 for production of biodiesel.

A CO2 sequestering bacterial strain, Serratia sp. ISTD04, that produces a significant amount of extracellular lipids was isolated from marble mine rocks. (14)C labeling analysis revealed that the rate of assimilation of CO2 by the strain is 0.756×10(-9)µmolCO2fixedcell(-1)h(-1). It was found to produce 466mg/l of extracellular lipid which was characterized using (1)H NMR. After transesterification of lipids, the total saturated and unsaturated FAME was found to be 51% and 49% respectively. The major FAME contained in the biodiesel were palmitic acid methyl ester (C16:0), oleic acid methyl ester (C18:1) and 10-nonadecenoic acid methyl ester (C19:1). Biodiesel produced by Serratia sp. ISTD04 is balanced in terms of FAME composition of good quality. It also contained higher proportion of oleic acid (35%) which makes it suitable for utilization in existing engines. Thus, the strain can be harnessed commercially to sequester CO2 into biodiesel.

Production and characterization of biodiesel from carbon dioxide concentrating chemolithotrophic bacteria, Serratia sp. ISTD04.

A chemolithotrophic bacterium, Serratia sp. ISTD04, enriched in the chemostat in presence of sodium bicarbonate as sole carbon source was evaluated for potential of carbon dioxide (CO2) sequestration and biofuel production. CO2 sequestration efficiency of the bacterium was determined by enzymatic activity of carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Further, Western blot analysis confirmed presence of RuBisCO. The bacterium produced 0.487 and 0.647mgmg(-1) per unit cell dry weight of hydrocarbons and lipids respectively. The hydrocarbons were within the range of C13-C24 making it equivalent to light oil. GC-MS analysis of lipids produced by the bacterium indicated presence of C15-C20 organic compounds that made it potential source of biodiesel after transesterification. GC-MS, FTIR and NMR spectroscopic characterization of the fatty acid methyl esters revealed the presence of 55% and 45% of unsaturated and saturated organic compounds respectively, thus making it a balanced biodiesel composition.

A physiologically regulated multidomain cystatin of wheat shows stage-dependent immunity against Karnal Bunt (Tilletia indica).

To identify novel components of basal resistance against the Tellitia indica of wheat, breeding for disease resistance was carried out on resistant and susceptible genotype of Karnal Bunt. The different members of wheat cystatin gene families were cloned, and their role in triggering differential resistance through co-expression was analyzed in our lab. The multidomain wheat cystatin (WCM) is a proteinase inhibitor characterized by cloning the gene from susceptible (WH542) and resistant genotype (HD 29). A WCM cDNA was isolated from both genotypes and sequenced. The WCM had a highly conserved N-terminal cystatin domain and a long C-terminal extension containing a second region, which exhibited similarity to the cystatin domain. The expression level was significantly (P > 0.001) higher in resistant compared to susceptible genotype at all the physiological stages of wheat spikes. In order to characterize the biochemical properties of WCM, the coding sequence was expressed in Escherichia coli using pET expression vector. The recombinant WCM was purified from soluble fraction of the cell extract by using affinity chromatography. WCM, with 23 KDa molecular mass, showed cysteine proteinase inhibitory activity against papain (Ki 3.039 × 10(-7) M) as determined by using BAPNA as substrate. Furthermore, it was able to arrest the fungal mycelial growth of T. indica. Hyphae growth was inhibited, and morphological changes such as swelling and fragmentation of the fungus were observed. Overall, these observations suggest an endogenous high expression of cystatin, possibly associated with the resistance of wheat against Karnal bunt.

Dibenzofuran induces oxidative stress, disruption of trans-mitochondrial membrane potential (ΔΨm) and G1 arrest in human hepatoma cell line.

Dioxins are a class of extremely toxic environmentally persistent pollutant, comprised of halogenated dibenzo-p-dioxins, dibenzofurans and biphenyls. Despite significant human exposure via multiple routes, very little is known about toxicity induced by dibenzofuran (DF). Current study shed lights on the potential toxicity mechanism of DF using human hepatoma cell line (HepG2). It was observed that the exposure to DF potentiate oxidative stress, apoptosis and necrosis at 10µM within 8h in HepG2 cells. Interestingly, when we pre-incubated the cells with α-NF (1nM) for 12h, an aromatic hydrocarbon receptor antagonist, the IC(50) of DF increased by 14 folds indicating the cytoprotective ability of α-NF from DF induced toxicity. Furthermore, three additional metabolites were observed while studying the metabolic profile of DF in HepG2 cells with and without pre-incubation with α-NF using chromatography-mass spectroscopy (GC-MS). Of these, two metabolites were characterized as dihydroxylated derivative of DF and third metabolite was characterized as quinone derivative of DF. By flow cytometry and confocal laser microscopy analysis we followed the ROS formation after DF (10µM) exposure for 3h. Significantly low ROS was generated in cells which were pre-incubated with α-NF than cells which were not pre-incubated with α-NF underlining the importance of metabolism in DF toxicity. The same pattern of protection was consistent while measuring mitochondrial membrane potential (MMP), i.e., less MMP dip was observed in 'with α-NF pre-incubated and DF (10µM) exposed cells' than 'without α-NF pre-incubated but DF exposed cells'. In cell cycle studies, it was confirmed that cell population of HepG2 at G1 stage progressively increased in number (∼74%) within 24h. Thus, DF and its metabolites induce significantly higher cytotoxicity after metabolism in HepG2 cells than its parent compound (DF) by ROS formation, MMP dip and impaired cell cycle.

Biosorption and biotransformation of chromium by Serratia sp. isolated from tannery effluent.

A bacterium isolated from soil and sediment ofa leather tanning mill's effluent was identified as Serratia sp. by the analysis of 16S rDNA. Scanning electron microscopy-energy dispersive X-ray analysis (SEM-EDX) and transmission electron microscopy (TEM) were used to assess morphological changes and confirm chromium biosorption in Serratia sp. both in a shake-flask culture containing chromium and in a tannery wastewater. The SEMEDX and the elemental analysis of the chromate-containing samples confirmed the binding of chromium with the bacterial biomass. The TEM exhibited chromium accumulation throughout the bacterial cell, with some granular deposits in the cell periphery and in the cytoplasm. X-ray diffraction analysis (XRD) was used to quantify the chromium and to determine the chemical nature of the metal-microbe interaction. The XRD data showed the crystalline character of the precipitates, which consisted of mainly calcium chromium oxide, chromium fluoride phosphate and related organo-Cr(III) complex crystals. The XRD data also revealed a strong involvement of cellular carboxyl and phosphate groups in chromium binding by the bacterial biomass. The results of the study indicated that a combined mechanism of ion-exchange, complexation, croprecipitation and immobilization was involved in the biosorption of chromium by bacterial cells in contaminated environments.

Isolation and characterization of Bacillus cereus IST105 from electroplating effluent for detoxification of hexavalent chromium.

PURPOSE: Electroplating industries are the main sources of heavy metals, chromium, nickel, lead, zinc, cadmium and copper. The highest concentrations of chromium (VI) in the effluent cause a direct hazards to human and animals. Therefore, there is a need of an effective and affordable biotechnological solution for removal of chromium from electroplating effluent. METHODS: Bacterial strains were isolated from electroplating effluent to find out higher tolerant isolate against chromate. The isolate was identified by 16S rDNA sequence analysis. Absorbed chromium level of bacterium was determined by inductively coupled plasma-atomic emission spectrometer (ICP-AES), atomic absorption spectrophotometer (AAS), scanning electron microscope (SEM), transmission electron microscope (TEM) and energy dispersive X-ray analysis (EDX). Removal of metals by bacterium from the electroplating effluent eventually led to the detoxification of effluent confirmed by MTT assay. Conformational changes of functional groups of bacterial cell surface were studied through Fourier transform infrared spectroscopy. RESULTS: The chromate tolerant isolate was identified as Bacillus cereus. Bacterium has potency to remove more than 75% of chromium as measured by ICP-AES and AAS. The study indicated the accumulation of chromium (VI) on bacterial cell surface which was confirmed by the SEM-EDX and TEM analysis. The biosorption of metals from the electroplating effluent eventually led to the detoxification of effluent. The increased survivability of Huh7 cells cultured with treated effluent also confirmed the detoxification as examined by MTT assay. CONCLUSION: Isolated strain B. cereus was able to remove and detoxify chromium (VI). It would be an efficient tool of the biotechnological approach in mitigating the heavy metal pollutants.

Evaluation of biosorption potency of Acinetobacter sp. for removal of hexavalent chromium from tannery effluent.

Two bacterial consortia were developed by continuous enrichment of microbial population of tannery and pulp and paper mill effluent contained Serratia mercascens, Pseudomonas fluorescence, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter sp. identified by 16S rDNA method. The consortia evaluated for removal of chromate [(Cr(VI)] in shake flask culture indicated pulp and paper mill consortium had more potential for removal of chromate. Acinetobacter sp. isolated from pulp and paper mill consortium removed higher amount of chromate [Cr(VI)] under aerobic conditions. Parameters optimized in different carbon, nitrogen sources, and pH, indicated maximum removal of chromate in sodium acetate (0.2%), sodium nitrate (0.1%) and pH 7 by Acinetobacter sp. Bacteria was applied in 2-l bioreactor significantly removed chromate after 3 days. The results of the study indicated removal of more than 75% chromium by Acinetobacter sp. determined by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometer after 7 days. Study of microbial [Cr(VI)] removal and identification of reduction intermediates has been hindered by the lack of analytical techniques. Therefore, removal of chromium was further substantiated by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) which indicated bioaccumulation of chromium in the bacterial cells.

Removal of chromium and pentachlorophenol from tannery effluents.

Three bacterial strains, including one Acinetobacter sp. PCP3, grown in the presence of minimal salt medium and pentachlorophenol (PCP) as sole carbon source in the chemostat showed higher utilization of PCP and adsorption of chromium. In sequential bioreactor, tannery effluents treated initially by bacterial consortium followed by fungus removed 90% and 67% chromium and PCP respectively, whereas in another set of bioreactor in which effluents was treated initially by fungi followed by bacteria could remove 64.7% and 58% chromium and PCP, respectively.

Biosorption potency of Aspergillus niger for removal of chromium (VI).

Aspergillus niger isolated from soil and effluent of leather tanning mills had higher activity to remove chromium. The potency of Aspergillus niger was evaluated in shake flask culture by absorption of chromium at pH 6 and temperature 30 degrees C. The results of the study indicated removal of more than 75% chromium by Aspergillus niger determined by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometry after 7 days. Study of microbial Cr(VI) reduction and identification of reduction intermediates has been hindered by the lack of analytical techniques that can identify the oxidation state with subcellular spatial resolution. Therefore, removal of chromium was further substantiated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX), which indicated an accumulation of chromium in the fungal mycelium.

Isolation and process parameter optimization of Aspergillus sp. for removal of chromium from tannery effluent.

Five morphologically different fungi were isolated from leather tanning effluent in which Aspergillus sp. and Hirsutella sp. had higher potential to remove chromium. The potential of Aspergillus sp. for removal of chromium was evaluated in shake flask culture in different pH, temperature, inoculums size, carbon and nitrogen source. The maximum chromium was removed at pH 6, temperature 30 degrees C, sodium acetate (0.2%) and yeast extract (0.1%). Aspergillus sp. was applied in 2l bioreactor for removal of chromium, and it was observed that 70% chromium was removed after 3 days.