Halomonas Hydrothermalis Research Articles

Highly efficient production of ectoine via an optimized combination of precursor metabolic modules in Escherichia coli BL21

Ectoine is an osmotic pressure protectant observed in various microorganisms and is widely used in cosmetics and pharmaceuticals. The market value of ectoine has increased considerably with social progress, resulting in high demand for ectoine production technology. Herein, a microbial cell factory in Escherichia coli that produces ectoine at high titers is described as developing efficient and environmentally friendly bio-based ectoine production technology. The ectoine biosynthetic pathway of Halomonas hydrothermalis was introduced into E. coli BL21 (DE3). Subsequent overexpression of precursor metabolic modules, including aspartate branching, pyruvate-oxoacetate, and glutamate biosynthesis pathways, resulted in the final strain, E. coli BCT08, which produced ectoine at a titer of 36.58 g/L during 30 h of fermentation. Sugar feeding speed optimization improved the ectoine titer to 131.8 g/L after 96 h of cultivation. This represents a remarkable achievement in ectoine production from glucose under low-salt conditions and has vast potential for industrial applications.

Natural sea salt based polyhydroxyalkanoate production by wild Halomonas hydrothermalis strain

Polyhydroxyalkanoates (PHAs) are emerging potential biopolymers replacing the hazardous petroleum based plastics with several applications and biodegradability. The major threat towards its commercialization is the substrate cost which could be replaced with crude byproducts or natural substrates for its sustainability at industrial scale. A wild marine bacterial strain Halomonas hydrothermalis (MTCC 5445) was used as a source for PHA accumulation. An effort has been made to use a sea salt formulation which is a natural substrate used both as growth and production media in our current study. To check the feasibility of this sea salt formulation, it was used in combination with glycerol and peptone as carbon and nitrogen source respectively in production media. A shake flask experiment with 20 run was set up with maximum PHA productivity of 2.61 g/L in 3% Glycerol, 3% DSM and 0.55% peptone set. The extracted PHA properties were checked through various analytical tools and compared with standard PHB indicating similarity in both structural and mechanical properties.

Effects of ocean acidification on the growth and biochemical composition of a green alga (Ulva fasciata) and its associated microbiota.

In marine ecosystems, fluctuations in surface-seawater carbon dioxide (CO2), significantly influence the whole metabolism of marine algae, especially during the early stages of macroalgal development. In this study, the response of the green alga Ulva fasciata for elevating ocean acidification was investigated using four levels of pCO2 ~ 280, 550, 750 and 1050 µatm. Maximum growth rate (6.6% day−1), protein (32.43 %DW) and pigment (2.9 mg/g) accumulation were observed at pCO2-550 with an increase of ~2-fold compared to control. On the other hand, lipid and carbohydrate contents recorded their maximum production (4.23 and 46.96 %DW, respectively) at pCO2-750 while control showed 3.70 and 42.37 %DW, respectively. SDS-PAGE showed the presence of unique bands in response to pCO2, especially at 550 µatm. Dominant associated bacteria was shifted from Halomonas hydrothermalis of control to Vibrio toranzoniae at pCO2-1050. These findings suggest that ocean acidification at 550 µatm might impose noticeable effects on growth, protein, pigments, and protein profile of U. fasciata, which could be a good source for fish farming. While, pCO2-750 was recommended for energetic purpose, due to its high lipid and carbohydrate contents.

Halomonas maris sp. nov., a moderately halophilic bacterium isolated from sediment in the southwest Indian Ocean

A halophilic, Gram-staining-negative, rod-shaped, flagellated and motile bacterium, strain QX-1T, was isolated from deep-sea sediment at a depth of 3332m in the southwestern Indian Ocean. Strain QX-1T growth was observed at 4-50°C (optimum 37°C), pH 5.0-11.0 (optimum pH 7.0), 3-25% NaCl (w/v; optimum 7%), and it did not grow without NaCl. A phylogenetic analysis based on the 16S rRNA gene placed strain QX-1T in the genus Halomonas and most closely related to Halomonas sulfidaeris (97.9%), Halomonas zhaodongensis (97.8%), Halomonas songnenensis (97.6%), Halomonas hydrothermalis (97.4%), Halomonas subterranea (97.3%), Halomonas salicampi (97.1%), and Halomonas arcis (97.0%). DNA-DNA hybridization (< 26.5%) and average nucleotide identity values (< 83.5%) between strain QX-1T and the related type strains meet the accepted criteria for a new species. The principal fatty acids (> 10%) of strain QX-1T are C16:0 (25.5%), C17:0 cyclo (14.0%), C19:0 cyclo ω8c (18.7%), and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c, 18.1%). The polar lipids of strain QX-1T are mainly diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unidentified phospholipid, unidentified aminophospholipid, and five unidentified lipids. The main respiratory quinone is Q-9. The G + C content of its chromosomal DNA is 54.4mol%. Its fatty acid profile, respiratory quinones, and G + C content also support the placement of QX-1T in the genus Halomonas. These phylogenetic, phenotypic, and chemotaxonomic analyses indicate that QX-1T is a novel species, for which the name Halomonas maris is proposed. The type strain is QX-1T (= MCCC 1A17875T = KCTC 82198T = NBRC 114670T).

Promoter engineering for high ectoine production in a lower saline medium by Halomonas hydrothermalis Y2.

For the stress from fermenters, downstream processing equipment, and wastewater treatment to be alleviated, lowering salt-dependence in the ectoine synthesis process is of great significance in the moderately halotolerant Halomonas hydrothermalis Y2. In H. hydrothermalis Y2, the σ70- and σ38-controlled promoters of ectA are predicted to be involved in the osmotic regulation of ectoine synthesis. By substituting the ectA promoter with a promoter P265 that identified in the outer membrane pore protein E of H. hydrothermalis Y2, the salt dependence of ectoine synthesis was significantly decreased. In the 500-ml flask containing various NaCl contents, the engineered strain (p/Y2/△ectD/△doeA) showed a remarkably enhanced ability in ectoine synthesis, especially under lower saline stress. After a 36-h fed-batch fermentation in the 1-l fermenter, p/Y2/△ectD/△doeA synthesized 11.5g ectoine l-1 in the presence of 60g NaCl-1l, with a high 0.32g ectoine l-1 h-1 productivity, a specific productivity of 512.2mg ectoine per g cell dry weight (CDW)-1, and an excretion ratio of 67 % ectoine. As no impaired growth was observed in strain p/Y2/△ectD/△doeA while ectoine synthesis was increased, this promoter engineering strategy provides a practical protocol for lowering the salt-dependence of ectoine synthesis in this moderately halotolerant strain.

Halomonas humidisoli Sp. Nov., Isolated From Saline-Alkaline Soil.

A Gram staining-negative, halophilic, aerobic, non-motile bacteria, designated strain WN018T, were isolated from the natural saline-alkali wetland soil of Binhai new district, Tianjin, China (38°46'N, 117°13'E). Cells of strain WN018T were short rod-shaped, 0.3-0.4µm wide and 0.5-1.9µm long, and growth occurred optimally at 30-33°C, pH 7.5-8.0, and in the presence of 4-8% (w/v) NaCl. Based on 16S rRNA gene sequence analysis, the isolates could be affiliated to the genus Halomonas, exhibiting highest sequence similarity of 97.50% to Halomonas venusta DSM 4743T. The DNA G+C content of the strain was 63.8%. The distinct phylogenetic position and phenotypic traits distinguished the novel isolate from its nearest neighbors. The major respiratory quinone of strain WN018T was Q-9 (91.0%) and Q-8 (9.0%), and the dominant fatty acids were C16:0, C14:0, C10:0, C12:0 3-OH. The major polar lipids were diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), three phospholipids (PL), aminolipid (AL), and two unidentified lipids (L). The average nucleotide identity (ANI) based on whole-genome sequences of strain WN018T and Halomonas hydrothermalis DSM 15725T was 93.02%, and the dDDH value between these two strains was determined to be 49.7%. Therefore, we propose a novel species in the genus Halomonas to accommodate the novel isolate: Halomonas humidisoli sp. nov. (type strain WN018T = ACCC 19975T = KCTC 52854T).

Neoteric Solvent Systems as Sustainable Media for Dissolution and Film Preparation of Poly-[(R)-3-hydroxybutyrate

Polyhydroxyalkanoates (PHAs) have a wide range of applicability due to their biodegradable and biocompatible characteristics. This makes them suitable for commercialization as an alternative to petroleum-based synthetic plastics. Solvents such as chloroform are reported to solubilize polyhydroxyalkanoates. With chloroform being hazardous for both health and the environment, this work attempts to replace this chlorinated solvent with green solvents like ionic liquids. In this study, poly-[(R)-3-hydroxybutyrate] (PHB) was extracted from Halomonas hydrothermalis, and its solubility, stability, and film-forming ability were investigated in the presence of ammonium-based ionic liquids (ILs). Among the studied ILs, maximum solubility (3% w/v at 70 °C) along with chemical and structural stability of the polymer was observed in diethanol ammonium acetate (DA), while the lowest dissolution (0.4% w/v) was exhibited in 2-hydroxyethylammonium formate (HF). The molecular docking studies have established a critical role of the anions of the ILs in the dissolution process. Further, PHB dissolved in DA formed a film in the presence of gelatin (G). The recovered ILs were found to be soil compatible, and due to their high nitrogen content, they can be used as a fortifier for nitrogen-deficient seaweed-based liquid plant biostimulants making them suitable soil nutrients for agriculture applications.

Complete Genome Sequence of Halomonas hydrothermalis Strain Slthf2, a Halophilic Bacterium Isolated from a Deep-Sea Hydrothermal-Vent Environment.

Halomonas hydrothermalis strain Slthf2 is a Gram-negative bacterium isolated from low-temperature hydrothermal fluids in South Pacific Ocean vent fields located at 2,580-m depth. Here, we report the complete genome sequence of this strain, which has a genome size of 4.12 Mb, with a GC content of 53.2%.

Biocharacteristics and draft genome sequence of Halomonas hydrothermalis C22, a pyruvate-producing halophilic bacterium isolated from a commercial Spirulina culture in Vietnam.

Halophilic bacteria are receiving increasing attention for industrial chemical production processes due to their unique properties. Herein, an alkaliphilic and halophilic bacterium was isolated from a commercial Spirulina culture at Nghe An province in Vietnam and found to secrete pyruvate. Pyruvate is widely used as a starting material in the industrial biosynthesis of pharmaceuticals, and is employed for production of crop protection agents, polymers, cosmetics, and food additives. Phenotypic and chemotaxonomic characterization, and the 16S rRNA gene sequence homology with Halomonas hydrothermalis strain DSM 15,725 (99.2%) predicted that the strain belongs to the Halomonas genus, thus we named this strain as H. hydrothermalis strain C22. We investigated the biocharacteristics and capacity of strain C22 and determined the draft genome sequence comprising 3,934,166bp with a G + C content of 60.2% encoding 3,668 proteins, 58 tRNAs, 9 rRNAs, and 1 tmRNA. Maximal pyruvate secretion reached 51.1g/l after 84h of cultivation. The results will facilitate future studies on the genetic and metabolic diversity of halophilic bacteria and expand our understanding of important bioprocesses in this microorganism.

High ectoine production by an engineered Halomonas hydrothermalis Y2 in a reduced salinity medium

BackgroundAs an attracted compatible solute, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) showed great potentials in various field. However, lower productivity and high saline medium seriously hinder its wide applications.ResultsThe entire ectoine metabolism, including pathways for ectoine synthesis and catabolism, was identified in the genome of an ectoine-excreting strain Halomonas hydrothermalis Y2. By in-frame deletion of genes encoding ectoine hydroxylase (EctD) and (or) ectoine hydrolase (DoeA) that responsible for ectoine catabolism, the pathways for ectoine utilization were disrupted and resulted in an obviously enhanced productivity. Using an optimized medium containing 100 g L−1 NaCl in a 500-mL flask, the double mutant of Y2/ΔectD/ΔdoeA synthesized 3.13 g L−1 ectoine after 30 h cultivation. This is much higher than that of the wild type strain (1.91 g L−1), and also exceeds the production of Y2/ΔectD (2.21 g L−1). The remarkably enhanced accumulation of ectoine by Y2/ΔectD/ΔdoeA implied a critical function of Doe pathway in the ectoine catabolism. Furthermore, to reduce the salinity of fermentation medium and overcome the wastewater treatment difficulty, mutants that lacking key Na+/H+ antiporter, Mrp and (or) NhaD2, were constructed based on strain Y2/ΔectD/ΔdoeA. As a result, the Mrp-deficient strain could synthesize equal amount of ectoine (around 7 g L−1 or 500 mg (g DCW) −1) in the medium containing lower concentration of NaCl. During a fed-batch fermentation process with 60 g L−1 NaCl stress, a maximum 10.5 g L−1 ectoine was accumulated by the Mrp-deficient strain, with a specific production of 765 mg (g DCW)−1 and a yield of 0.21 g g−1 monosodium glutamate.ConclusionThe remarkably enhanced production of ectoine by Y2/ΔectD/ΔdoeA implied the critical function of Doe pathway in the ectoine catabolism. Moreover, the reduced salinity requirement of Mrp-deficient strain implied a feasible protocol for many compatible solute biosynthesis, i.e., by silencing some Na+/H+ antiporters in their halophilic producers and thus lowering the medium salinity.

Production of polyhydroxyalkanoates on waste frying oil employing selected Halomonas strains

The aim of this work was to study the potential of selected Halomonas species for conversion of waste frying oil into polyhydroxyalkanoates (PHA). In total nine Halomonas strains were experimentally screened for their capability of PHA production. Among them, Halomonas neptunia and Halomonas hydrothermalis were identified as potent PHA producers. Initial concentration of NaCl was identified as parameter influencing PHA yields as well as molecular weight of the polymer. In addition, H. hydrothermalis was capable of biosynthesis of a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate P(3HB-co-3HV). When valerate was utilized as a precursor, the 3HV fraction in the copolymer reached high values of 50.15 mol.%. PHA production on lipid substrates by Halomonas has not been reported so far. Bearing in mind all the positive aspects of employing extremophiles in industrial biotechnology, H. hydrothermalis seems to be a very interesting halophilic strain for production of PHA using lipid substrates.

Genome sequence of Halomonas hydrothermalis Y2, an efficient ectoine-producer isolated from pulp mill wastewater

Halophilic microorganisms have great potentials towards biotechnological applications. Halomonas hydrothermalis Y2 is a halotolerant and alkaliphilic strain that isolated from the Na+-rich pulp mill wastewater. The strain is dominant in the bacterial community of pulp mill wastewater and exhibits metabolic diversity in utilizing various substrates. Here we present the genome sequence of this strain, which comprises a circular chromosome 3,933,432 bp in size and a GC content of 60.2%. Diverse genes that encoding proteins for compatible solutes synthesis and transport were identified from the genome. With a complete pathway for ectoine synthesis, the strain could produce ectoine from monosodium glutamate and further partially secreted into the medium. In addition, around 20% ectoine was increased by deleting the ectoine hydroxylase (EctD). The genome sequence we report here will provide genetic information regarding adaptive mechanisms of strain Y2 to its harsh habitat, as well as facilitate exploration of metabolic strategies for diverse compatible solutes, e.g., ectoine production.

1-Ethyl-3-methylimidazolium Diethylphosphate Based Extraction of Bioplastic “Polyhydroxyalkanoates” from Bacteria: Green and Sustainable Approach

An eco-friendly approach toward downstream processing of bacterial biomass for extraction of an intracellular potential bioplastic material polyhydroxyalkanoates replacing chlorinated organic solvents is reported using ionic liquid (IL) 1-ethyl-3-methylimidazolium diethylphosphate ([C2mim][(C2)2OPO3]) as an extractant. [C2mim][(C2)2OPO3] dissolved wet bacterial biomass of Halomonas hydrothermalis (MTCC accession no. 5445; NCBI Genbank accession no. GU938192), with ease on account of its high hydrogen bond basicity (β = 1.07). The recovered polymer with a yield of 60% ± 2% was characterized using 1H NMR, FT-IR, and TGA techniques and confirmed to be polyhydroxybutyrate (PHB). The properties of PHB were found to be in close proximity to the standard PHB. The [C2mim][(C2)2OPO3] was recovered with 60% yield and retention of chemical structure after two consecutive dissolution cycles, which minimized the cost of developed process. The recovered PHB was used to prepare a bioplastic film, which showed good therm...

Astrobiology as a framework for investigating antibiotic susceptibility: a study of Halomonas hydrothermalis.

Physical and chemical boundaries for microbial multiplication on Earth are strongly influenced by interactions between environmental extremes. However, little is known about how interactions between multiple stress parameters affect the sensitivity of microorganisms to antibiotics. Here, we assessed how 12 distinct permutations of salinity, availability of an essential nutrient (iron) and atmospheric composition (aerobic or microaerobic) affect the susceptibility of a polyextremotolerant bacterium, Halomonas hydrothermalis, to ampicillin, kanamycin and ofloxacin. While salinity had a significant impact on sensitivity to all three antibiotics (as shown by turbidimetric analyses), the nature of this impact was modified by iron availability and the ambient gas composition, with differing effects observed for each compound. These two parameters were found to be of particular importance when considered in combination and, in the case of ampicillin, had a stronger combined influence on antibiotic tolerance than salinity. Our data show how investigating microbial responses to multiple extremes, which are more representative of natural habitats than single extremes, can improve our understanding of the effects of antimicrobial compounds and suggest how studies of habitability, motivated by the desire to map the limits of life, can be used to systematically assess the effectiveness of antibiotics.

Halomonas lutescens sp. nov., a halophilic bacterium isolated from a lake sediment.

A novel, Gram-stain-negative, facultatively anaerobic, halophilic bacterium, designated strain Q1UT, was isolated from a sediment sample collected from Qinghai Lake, PR China. The cells of the strain were short rod-shaped (0.2-0.3×0.6-2.5 µm) and non-motile. Strain Q1UT formed yellowish colonies and grew at temperatures of 2-37 °C (optimum 30-33 °C), at pH 6.0-9.0 (optimum pH 7.0) and in the presence of 0-20 % (w/v) NaCl (optimum 7.5 %). The major cellular fatty acids were C18 : 1ω7c (58.6 %), C16 : 1ω7c and/or C16 : 1ω6c (14.8 %) and C16 : 0 (10.1 %). The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, unknown phospholipid and unknown lipids. The genomic DNA G+C content was 61.5 mol%, and the predominant respiratory ubiquinone Q-9. Based on phylogenetic analysis of the 16S rRNA gene sequences and concatenated 16S rRNA, gyrB and rpoD gene sequences, the isolate was found to belong to the genus Halomonas in the class Gammaproteobacteria. The most closely related species were Halomonas venusta DSM 4743T (98.3 % 16S rRNA sequence similarity), Halomonas songnenensis DSM 25870T (98.2 %) and Halomonas hydrothermalis DSM 15725T (98.2 %). DNA-DNA relatedness values between strain Q1UT and the type strains of eight other species of the genus Halomonas ranged from 21.3 % to 10.1 %. On the basis of phenotypic, phylogenetic and chemotaxonomic analyses, and DNA-DNA hybridization relatedness values, strain Q1UT is considered to represent a novel species of the genus Halomonas; the name Halomonas lutescens sp. nov. is proposed. The type strain is Q1UT (=CGMCC 1.15122T=KCTC 42517T).

Salinity Influences the Response of Halomonas hydrothermalis to Artificial Fossilization by Evaporative Silicification

ABSTRACTEvaporative silicification can drive microbial fossilization within diverse natural habitats. Research into this process is pivotal to understanding the terrestrial fossil record and the preservation of biomarkers within extreme environments. We employed laboratory experiments to silicify the polyextremotolerant bacterium Halomonas hydrothermalis cultured at low, intermediate and high salinities (1, 3.5 or 11.8% w/v of NaCl) under iron-rich or iron-deprived conditions. Silicification was achieved by adding sodium silicate solution (30 or 150 ppm of Si) onto cultures, followed by evaporation. Scanning electron microscopy demonstrated the presence of mineralized bacteria with intact morphology across all culture conditions. However, multivariate analysis of the attenuated total reflectance Fourier-transform infrared (ATR-FT-IR) spectra of silicified cultures showed significant differences between the examined salinities, most notably between cultures silicified after incubation at high salinity and those at lower salinities. Although the spectra of mineralized low- and intermediate-salinity cultures appeared distinct from their nonsilicified counterparts, these differences were less pronounced at high salinity. By showing that differences in salinity can influence microbial responses to mineralization at the molecular level, these data indicate that the potential for evaporative silicification to contribute to microbial fossilization may differ between freshwater and hypersaline environments.

Effects of salinity on performance, extracellular polymeric substances and microbial community of an aerobic granular sequencing batch reactor

The effects of salinity on the performance, extracellular polymeric substances (EPS) and microbial community of granular sludge were evaluated in an aerobic granular sequencing batch reactor (GSBR). The chemical oxygen demand (COD) and NH4+–N removal efficiencies decreased from 92% and 93% to 25% and 21% at 0–8% salinity, respectively. The specific ammonium oxidation rate (SAOR), specific nitrite oxidation rate (SNOR), and specific nitrate reduction rate (SNRR) decreased from 4.6, 5.6 and 43.0mgNg−1MLSSh−1 to 0.5, 0.4 and 9mgNg−1MLSSh−1 at 0–8% salinity, respectively. The specific oxygen utilization rate (SOUR) of granular sludge increased from 30 to 42mgO2g−1MLSSh−1 at 0–3% salinity and subsequently decreased to 11mgO2g−1MLSSh−1 at 8% salinity. The protein (PN) and polysaccharide (PS) contents in loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) increased at 0–8% salinity. The relative hydrophobicity (RH) of granular sludge illustrated good positive linear correlations with the PN/PS ratios in TB-EPS and LB-EPS. The chemical structural changes of LB-EPS and TB-EPS at different salinities were analyzed by three-dimensional excitation–emission matrix (3D-EEM) fluorescence spectroscopy. A good linear correlation was found between sludge volume index (SVI) and LB-EPS (or TB-EPS). The denaturing gradient gel electrophoresis (DGGE) profile revealed that Lactococcus sp. YM05004, Desulfopila sp. PR5_F09 and Demequina oxidasica were present at all the salinities, suggesting these bacteria were capable of tolerating 8% salinity. Some bacteria could utilize nitrate as an electron acceptor and was present at certain salinities, such as Arcobacter defluvii, Halomonas hydrothermalis and Paracoccus denitrificans.

Reduction of the temperature sensitivity of Halomonas hydrothermalis by iron starvation combined with microaerobic conditions.

The limits to biological processes on Earth are determined by physicochemical parameters, such as extremes of temperature and low water availability. Research into microbial extremophiles has enhanced our understanding of the biophysical boundaries which define the biosphere. However, there remains a paucity of information on the degree to which rates of microbial multiplication within extreme environments are determined by the availability of specific chemical elements. Here, we show that iron availability and the composition of the gaseous phase (aerobic versus microaerobic) determine the susceptibility of a marine bacterium, Halomonas hydrothermalis, to suboptimal and elevated temperature and salinity by impacting rates of cell division (but not viability). In particular, iron starvation combined with microaerobic conditions (5% [vol/vol] O2, 10% [vol/vol] CO2, reduced pH) reduced sensitivity to temperature across the 13°C range tested. These data demonstrate that nutrient limitation interacts with physicochemical parameters to determine biological permissiveness for extreme environments. The interplay between resource availability and stress tolerance, therefore, may shape the distribution and ecology of microorganisms within Earth's biosphere.

Biochemical changes induced by salt stress in halotolerant bacterial isolates are media dependent as well as species specific

ABSTRACTHalophilic bacteria respond to salt stress by regulating the cytosolic pools of organic solutes to achieve osmotic equilibrium. In order to understand the metabolic regulation of these organic solutes, for the first time, we have investigated the effect of salt on growth and biochemical changes in four major moderately halophilic bacterial strains isolated from a saltern region of the Kumta coast, India. The strains under study were Halomonas hydrothermalis VITP9, Bacillus aquimaris VITP4, Planococcus maritimus VITP21, and Virgibacillus dokdonensis VITP14, which exhibited similar salt tolerance (0% to 10% w/v NaCl) with optimal growth at 5% w/v NaCl. Biochemical analysis showed that the total intracellular organic solutes increased significantly with increasing NaCl concentration in the growth medium, and the compositions of the solutes were dependent on the type of strain and also on the nutrient richness of the growth medium. Glutamic acid levels increased in all the strains under salt stress, indicating the significance of glutamic acid as the anionic counterpart of K+/Na+ ions and precursor for other synthesized nitrogenous osmolytes. Though initial studies were performed with thin-layer chromatography, mass spectrometry was used to identify the major solutes accumulated by the strains under salt stress, such as proline (VITP4), ectoine (VITP14 and VITP9), and sugars (VITP21) under minimal medium and glycine betaine (by all the strains under study) under complex growth medium conditions. Such comparative study on the stress-dependent metabolic differences of different microbes, under identical experimental condition, helps to identify possible bacterial sources for the production of industrially important solutes.

Microbial synthesis of polyhydroxyalkanoate using seaweed-derived crude levulinic acid as co-nutrient

Production of polyhydroxyalkanoates (PHAs) from Jatropha biodiesel residues, namely crude glycerol and oil cake hydrolysate, has been reported previously. Halomonas hydrothermalis (MTCC accession no. 5445; NCBI Genbank accession no. GU938192), a wild marine strain, was used in the bio-synthesis. The present study was initiated to vary the properties of the polymer. Seaweed-derived crude levulinic acid (SDCLA), containing formic acid, residual sugars and dissolved minerals additionally, was proposed as co-feed along with the biodiesel residues. Experiments were conducted at 100mL scale in batch process. Whereas the PHA yield was only 0.40±0.01g when only biodiesel residues were employed, it rose to 1.07±0.02g in presence of 0.35% (w/v) of SDCLA. The corresponding carbon utilisation efficiencies were 29.3% and 57.5%, respectively. 3-Hydroxy valerate incorporation in the PHA was pronounced in presence of SDCLA, with associated changes in polymer properties. The microbial synthesis fared poorly when SDCLA was substituted with pure levulinic acid. Thus, Halomonas hydrothermalis had a poor response to levulinic acid, as such, and other constituents present in SDCLA appear to have played a vital role in bacterial cell division and accumulation of PHA. Biodegradability tests in moist soil were also conducted as part of the study. Marine microalgal cultivation for biodiesel and seaweed cultivation for fuels may help generate biodiesel residues and crude levulinic acid in proximity, which would open up the possibility of large scale PHA manufacture in efficient and practical manner in the future through the methodology of the present study.