Judith M. Bragança

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

BackgroundGoa 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.ResultsSalinity 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.ConclusionSalterns 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.

Characterization of polyhydroxyalkanoates accumulated by a moderately halophilic salt pan isolate Bacillus megaterium strain H16.

Characterization of polyhydroxyalkanoates (PHA) accumulated by halophilic bacteria isolated from solar salterns.

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.

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.

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

Polyhydroxyalkanoates (PHAs) 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.

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.

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.

Pretreatment of cottage cheese to enhance biogas production.

This study evaluated the possibility of pretreating selected solid fraction of an anaerobic digester treating food waste to lower the hydraulic retention time and increase the methane production. The study investigated the effect of different pretreatments (thermal, chemical, thermochemical and enzymatic) for enhanced methane production from cottage cheese. The most effective pretreatments were thermal and enzymatic. Highest solubilisation of COD was observed in thermal pretreatment, followed by thermochemical. In single enzyme systems, lipase at low concentration gave significantly higher methane yield than for the experiments without enzyme additions. The highest lipase dosages decreased methane yield from cottage cheese. However, in case of protease enzyme an increase in concentration of the enzyme showed higher methane yield. In the case of mixed enzyme systems, pretreatment at 1 : 2 ratio of lipase : protease showed higher methane production in comparison with 1 : 1 and 2 : 1 ratios. Methane production potentials for different pretreatments were as follows: thermal 357 mL/g VS, chemical 293 mL/g VS, and thermochemical 441 mL/g VS. The average methane yield from single enzyme systems was 335 mL/g VS for lipase and 328 mL/g VS for protease. Methane potentials for mixed enzyme ratios were 330, 360, and 339 mL/g VS for 1 : 1, 1 : 2, and 2 : 1 lipase : protease, respectively.

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.

Green Synthesis of Silver Nanoparticles by Haloarchaeon Halococcus salifodinae BK6

Nanobiotechnology is a multidisciplinary branch of nanotechnology which includes fabrication of nanosized materials using biological approaches. Highly structured metallic and metal sulfide nanoparticles have been reported to be synthesized by numerous bacteria, fungi, yeasts and viruses. However, biosynthesis of nanoparticles by Haloarchaea (salt-loving archaea) of the third domain of life, Archaea, is in its nascent stages. In this study, we report the intracellular synthesis of stable, mostly spherical silver nanoparticles (SNPs) by the haloarchaeal isolate Halococcus salifodinae BK6. The isolate adapted to silver nitrate was found to exhibit growth kinetics similar to that of cells unexposed to silver nitrate. The nitrate reductase enzyme assay and the enzyme inhibitor studies showed the involvement of NADH dependent nitrate reductase in silver tolerance, reduction, and synthesis of SNPs. UV visible spectroscopy, XRD, TEM and EDAX were used for characterization of SNPs. The XRD exhibited characteristic Bragg peaks of face centered cubic silver with crystallite domain size of 26 nm and 12 nm for SNPs synthesized in NTYE and halophilic nitrate broth, respectively. TEM analysis exhibited an average particle size of 50.3 nm and 12 nm for SNPs synthesized in NTYE and halophilic nitrate broth (HNB), respectively. The as synthesized SNPs exhibited antimicrobial activity against both Gram positive and Gram negative organisms.