Genomic insights on carotenoid synthesis by extremely halophilic archaea Haloarcula rubripromontorii BS2, Haloferax lucentense BBK2 and Halogeometricum borinquense E3 isolated from the solar salterns of India.

Haloarchaeal cultures were isolated from solar salterns of Goa and Tamil Nadu and designated as BS2, BBK2 and E3. These isolates grew with a characteristic bright orange to pink pigmentation and were capable of growing in media containing upto 25% (w/vol) NaCl. Whole genome sequencing (WGS) of the three haloarchaeal strains BS2, BBK2 and E3 indicated an assembled genomic size of 4.1 Mb, 3.8 Mb and 4 Mb with G + C content of 61.8, 65.6 and 59.8% respectively. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the archaeal isolates belong to Haloarcula, Haloferax and Halogeometricum genera. Haloarcula rubripromontorii BS2  was predicted to have 4292 genes with 4242 CDS regions, 46 tRNAs, 6 rRNAs and 3 misc_RNAs. In case of Haloferax lucentense  BBK2,, 3840 genes with 3780 CDS regions were detected along with 52 tRNAs, 5 rRNAs and 3 misc_RNAs. Halogeometricum borinquense  E3 contained 4101 genes, 4043 CDS regions, 52 tRNAs, 4 rRNAs, and 2 misc_RNAs. The functional annotation and curation of the haloarchaeal genome, revealed C50 carotenoid biosynthetic genes like phytoene desaturase/carotenoid 3' -4' desaturase (crtI), lycopene elongase (ubiA/lyeJ) and carotenoid biosynthesis membrane protein (cruF) in the three isolates. Whereas crtD (C-3',4' desaturase), crtY (lycopene cyclase) and brp/blh (ß-carotene dioxygenase) genes were identified only in BS2.

Composites of Bacillus megaterium H16 derived poly-3-hydroxybutyrate as a biomaterial for skin tissue engineering.

Composite films of Bacillus megaterium H16 derived PHB with 1%Poly-L-lactic acid (PLLA), 1%Poly-ε-caprolactone (PCL), and 0.3 % graphene nanoplatelets (GNP) were produced by solvent cast method. The composite films were characterized by SEM, DSC-TGA, XRD, and ATR-FTIR. The ultrastructure of PHB and its composites depicted an irregular surface morphology with pores after the evaporation of chloroform. The GNPs were seen to be integrated inside the pores. The B. megaterium H16 derived-PHB and its composites demonstrated good biocompatibility which was evaluated in vitro on HaCaT and L929 cells by MTT assay. The cell viability was best for PHB followed by PHB/PLLA/PCL > PHB/PLLA/GNP > PHB/PLLA. PHB and its composites were highly hemocompatible as it resulted in <1 % hemolysis. The PHB/PLLA/PCL and PHB/PLLA/GNP composites can serve as ideal biomaterials for skin tissue engineering.

Transient Dynamics of Archaea and Bacteria in Sediments and Brine Across a Salinity Gradient in a Solar Saltern of Goa, India.

The microbial fluctuations along an increasing salinity gradient during two different salt production phases - initial salt harvesting (ISH) phase and peak salt harvesting (PSH) phase of Siridao solar salterns in Goa, India were examined through high-throughput sequencing of 16S rRNA genes on Illumina MiSeq platform. Elemental analysis of the brine samples showed high concentration of sodium (Na+) and chloride (Cl-) ions thereby indicating its thalassohaline nature. Comparison of relative abundance of sequences revealed that Archaea transited from sediment to brine while Bacteria transited from brine to sediment with increasing salinity. Frequency of Archaea was found to be significantly enriched even in low and moderate salinity sediments with their relative sequence abundance reaching as high as 85%. Euryarchaeota was found to be the dominant archaeal phylum containing 19 and 17 genera in sediments and brine, respectively. Phylotypes belonging to Halorubrum, Haloarcula, Halorhabdus, and Haloplanus were common in both sediments and brine. Occurence of Halobacterium and Natronomonas were exclusive to sediments while Halonotius was exclusive to brine. Among sediments, relative sequence frequency of Halorubrum, and Halorhabdus decreased while Haloarcula, Haloplanus, and Natronomonas increased with increasing salinity. Similarly, the relative abundance of Haloarcula and Halorubrum increased with increasing salinity in brine. Sediments and brine samples harbored about 20 and 17 bacterial phyla, respectively. Bacteroidetes, Proteobacteria, and Chloroflexi were the common bacterial phyla in both sediments and brine while Firmicutes were dominant albeit in sediments alone. Further, Gammaproteobacteria, Alphaproteobacteria, and Deltaproteobacteria were observed to be the abundant class within the Proteobacteria. Among the bacterial genera, phylotypes belonging to Rubricoccus and Halomonas were widely detected in both brine and sediment while Thioalkalispira, Desulfovermiculus, and Marinobacter were selectively present in sediments. This study suggests that Bacteria are more susceptible to salinity fluctuations than Archaea, with many bacterial genera being compartment and phase-specific. Our study further indicated that Archaea rather than Bacteria could withstand the wide salinity fluctuation and attain a stable community structure within a short time-frame.

Utilization of Sugarcane Bagasse by Halogeometricum borinquense Strain E3 for Biosynthesis of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Sugarcane bagasse (SCB), one of the major lignocellulosic agro-industrial waste products, was used as a substrate for biosynthesis of polyhydroxyalkanoates (PHA) by halophilic archaea. Among the various wild-type halophilic archaeal strains screened, Halogeometricum borinquense strain E3 showed better growth and PHA accumulation as compared to Haloferaxvolcanii strain BBK2, Haloarcula japonica strain BS2, and Halococcus salifodinae strain BK6. Growth kinetics and bioprocess parameters revealed the maximum PHA accumulated by strain E3 to be 50.4 ± 0.1 and 45.7 ± 0.19 (%) with specific productivity (qp) of 3.0 and 2.7 (mg/g/h) using NaCl synthetic medium supplemented with 25% and 50% SCB hydrolysate, respectively. PHAs synthesized by strain E3 were recovered in chloroform using a Soxhlet apparatus. Characterization of the polymer using crotonic acid assay, X-ray diffraction (XRD), differential scanning calorimeter (DSC), Fourier transform infrared (FT-IR), and proton nuclear magnetic resonance (¹H-NMR) spectroscopy analysis revealed the polymer obtained from SCB hydrolysate to be a co-polymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] comprising of 13.29 mol % 3HV units.

Anti-bacterial TeNPs biosynthesized by haloarcheaon Halococcus salifodinae BK3.

Microbial synthesis of highly structured metal sulfide and metallic nanoparticles is a benign approach of nanomaterial synthesis. Various microbes have been exploited for nanoparticle synthesis, but nanofabrication using haloarchaea is still in nascent stages. Here, we report the intracellular synthesis of hexagonal needle-shaped tellurium nanoparticles with an aspect ratio of 1:4.4, by the haloarcheon Halococcus salifodinae BK3. The isolate was able to tolerate up to 5.5 mM K2TeO3. The yield of tellurium nanoparticles was highest when the culture was exposed to 3 mM K2TeO3, even though the isolate exhibited slightly decreased growth rate as compared to the culture growing in the absence of K2TeO3. The enzyme tellurite reductase was responsible for tellurite resistance and nanoparticle synthesis in H. salifodinae BK3. These tellurium nanoparticles exhibited anti-bacterial activities against both Gram-positive and Gram-negative bacteria, with higher antibacterial activity towards Gram-negative bacteria. This is the first report on the synthesis of tellurium nanoparticles by Halophilic archaea.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Halogeometricum borinquense strain E3.

Polyhydroxyalkanoates (PHA's) can be a key solution for pollution problems caused by plastics derived from petrochemical sources. Extremely halophilic archaeon Halogeometricum borinquense strain E3 showed maximum PHA accumulation of 73.51 ±1.7% (wt/wt) of cell dry weight (CDW) with 2% glucose. The crotonic acid assay, XRD, FT-IR and (1)H NMR analysis revealed that the polymer was a co-polymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)] comprising of 21.47% HV units. This is the first report on P(HB-co-HV) production by an extremely halophilic archaeon Hgm. borinquense strain E3.

In vivo synthesis of selenium nanoparticles by Halococcus salifodinae BK18 and their anti-proliferative properties against HeLa cell line.

Nanoparticles synthesis by bacteria and yeasts has been widely reported, however, synthesis using halophilic archaea is still in a nascent stage. This study aimed at the intracellular synthesis of selenium nanoparticles (SeNPs) by the haloarchaeon Halococcus salifodinae BK18 when grown in the presence of sodium selenite. Crystallographic characterization of SeNPs by X-ray diffraction, Selected area electron diffraction, and transmission electron microscopy exhibited rod shaped nanoparticles with hexagonal crystal lattice, a crystallite domain size of 28 nm and an aspect ratio (length:diameter) of 13:1. Energy disruptive analysis of X-ray analysis confirmed the presence of selenium in the nano-preparation. The nitrate reductase enzyme assay and the inhibitor studies indicated the involvement of NADH-dependent nitrate reductase in SeNPs synthesis and metal tolerance. The SeNPs exhibited good anti-proliferative properties against HeLa cell lines while being non-cytotoxic to normal cell line model HaCat, suggesting the use of these SeNPs as cancer chemotherapeutic agent. This is the first study on selenium nanoparticles synthesis by haloarchaea.

Cadmium resistance in extremely halophilic archaeon Haloferax strain BBK2.

Halophilic archaea are prevalent in highly saline habitats. Haloferax strain BBK2 is an orange pigmented, exopolysaccharide (EPS) producing extremely halophilic archaeon, isolated from solar salterns of Ribandar, Goa, India. It grew in varying pH (5-10) and NaCl concentration (10-30%). The isolate grew well in complex (NTYE) and minimal media (NGSM) in presence of heavy metal cadmium (Cd) up to 4.0 mM (805.28 mg L(-1)) concentration. The optimum growth in the presence and absence of Cd was seen at a pH range of 7-9 and salinity of 15-25%. The growth kinetics of the isolate in NTYE showed a specific growth rate (µmax) of 0.352 with generation time of 1.968 days. In presence of 1mM Cd, the µmax was 0.325 day(-1) and generation time was 2.132 days. In NGSM, the µmax decreased from 0.517 day(-1) (in control) to 0.265 day(-1) in 1mM Cd while, the doubling time increased from 1.34 days in control to 2.615 days in presence of 1 mM Cd. SDS PAGE of the whole cell protein extracts showed overexpressed proteins of 74.14 and 40 kDa. The scanning electron microscopy, energy dispersive X-ray spectroscopy (SEM-EDX) analysis of the intact cells and cells disrupted by dialysis revealed that Cd was bound onto the cells, which was further confirmed by AAS, FTIR and XRD analysis.

Community solar salt production in Goa, India.

Traditional salt farming in Goa, India has been practised for the past 1,500 years by a few communities. Goa's riverine estuaries, easy access to sea water and favourable climatic conditions makes salt production attractive during summer. Salt produced through this natural evaporation process also played an important role in the economy of Goa even during the Portuguese rule as salt was the chief export commodity. In the past there were 36 villages involved in salt production, which is now reduced to 9. Low income, lack of skilled labour, competition from industrially produced salt, losses incurred on the yearly damage of embankments are the major reasons responsible for the reduction in the number of salt pans.Salt pans (Mithagar or Mithache agor) form a part of the reclaimed waterlogged khazan lands, which are also utilised for aquaculture, pisciculture and agriculture. Salt pans in Goa experience three phases namely, the ceased phase during monsoon period of June to October, preparatory phase from December to January, and salt harvesting phase, from February to June. After the monsoons, the salt pans are prepared manually for salt production. During high tide, an influx of sea water occurs, which enters the reservoir pans through sluice gates. The sea water after 1-2 days on attaining a salinity of approximately 5ºBé, is released into the evaporator pans and kept till it attains a salinity of 23 - 25ºBé. The brine is then released to crystallizer pans, where the salt crystallises out 25 - 27ºBé and is then harvested.Salt pans form a unique ecosystem where succession of different organisms with varying environmental conditions occurs. Organisms ranging from bacteria, archaea to fungi, algae, etc., are known to colonise salt pans and may influence the quality of salt produced.The aim of this review is to describe salt farming in Goa's history, importance of salt production as a community activity, traditional method of salt production and the biota associated with salt pans.

Culturable halophilic archaea at the initial and crystallization stages of salt production in a natural solar saltern of Goa, India.

BACKGROUND: Goa is a coastal state in India and salt making is being practiced for many years. This investigation aimed in determining the culturable haloarchaeal diversity during two different phases of salt production in a natural solar saltern of Ribandar, Goa. Water and sediment samples were collected from the saltern during pre-salt harvesting phase and salt harvesting phase. Salinity and pH of the sampling site was determined. Isolates were obtained by plating of the samples on complex and synthetic haloarchaeal media. Morphology of the isolates was determined using Gram staining and electron microscopy. Response of cells to distilled water was studied spectrophotometrically at 600nm. Molecular identification of the isolates was performed by sequencing the 16S rRNA. RESULTS: Salinity of salt pans varied from 3-4% (non-salt production phase) to 30% (salt production phase) and pH varied from 7.0-8.0. Seven haloarchaeal strains were isolated from water and sediment samples during non-salt production phase and seventeen haloarchaeal strains were isolated during the salt production phase. All the strains stained uniformly Gram negative. The orange-red acetone extract of the pigments showed similar spectrophotometric profile with absorption maxima at 393, 474, 501 and 535 nm. All isolates obtained from the salt dilute phase were grouped within the genus Halococcus. This was validated using both total lipid profiling and 16S rRNA data sequencing. The isolates obtained from pre-salt harvesting phase were resistant to lysis. 16S rRNA data showed that organisms belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained during the salt concentrated phase. The isolates obtained from salt harvesting phase showed varied lysis on suspension in distilled water and /or 3.5% NaCl. CONCLUSION: Salterns in Goa are transiently operated during post monsoon season from January to May. During the pre-salt harvesting phase, all the isolates obtained belonged to Halococcus sp. During the salt harvesting phase, isolates belonging to Halorubrum, Haloarcula, Haloferax and Halococcus genera were obtained. This study clearly indicates that Halococcus sp. dominates during the low salinity conditions.

Interspecific Interactions Among Members of Family Halobacteriaceae from Natural Solar Salterns.

Solar salterns are crystallizer ponds with highly diverse extremophilic microbial flora living individually or in consortium. Twenty-four culturable haloarchaeal isolates were obtained from solar salterns of Goa, which were grouped under Halococcus, Haloferax, Haloarcula and Halorubrum. Cell-free supernatants of different isolates were checked against each other by pour plate technique combined with agar well diffusion method. This resulted in a zone of growth inhibition or stimulation around wells, indicating that some isolates had antagonistic and/or a beneficial effect on the other genera. Thus, members of family Halobacteriaceae were found to secrete extracellular metabolites, which can act as growth enhancers or repressors.