Ectoine production from a novel bacterial strain and high-purity purification with a cost-effective and single-step method.

This study marks the exploration into the production of ectoine, a valuable compound with significant potential as an antioxidant, osmoprotectant, anti-inflammatory agent, and stabilizer of cell membranes, proteins, and DNA integrity. Our focus centred on investigating the presence of ectoine and optimizing its production by the novel ectoine producer bacterial strain, Piscibacillus halophilus. For the optimization of ectoine production the effects of carbon and nitrogen sources, salt, pH, agitation and incubation period were optimized by one-factor-at-a-time. We started with an initial ectoine content of 46.92 mg/L, and through a series of optimization processes, we achieved a remarkable increase, resulting in an ectoine content of 1498.2 mg/L. The bacterial species P. halophilus achieved its highest ectoine production after 48 h of incubation, with conditions set at 10 % (w/v) salinity, pH of 7.50, and an agitation speed of 160 rpm. These precise conditions were found to be the most favourable for maximizing ectoine production by this strain. Besides, we have achieved successful purification of ectoine from the crude extract through a streamlined single-step process. This purification method has delivered an exceptional level of purity, surpassing 99.15 %, and an impressive yield of over 99 %. Importantly, we accomplished this using readily available and cost-effective strong acids (HCl) and strong bases (NaOH) to arrange pH gradients. The use of acid and base in the purification process of ectoine reflects an innovative and sustainable methodology.

Engineering low-salt growth Halomonas Bluephagenesis for cost-effective bioproduction combined with adaptive evolution.

Halophilic Halomonas bluephagenesis has been engineered to produce various added-value bio-compounds with reduced costs. However, the salt-stress regulatory mechanism remained unclear. H. bluephagenesis was randomly mutated to obtain low-salt growing mutants via atmospheric and room temperature plasma (ARTP). The resulted H. bluephagenesis TDH4A1B5 was constructed with the chromosomal integration of polyhydroxyalkanoates (PHA) synthesis operon phaCAB and deletion of phaP1 gene encoding PHA synthesis associated protein phasin, forming H. bluephagenesis TDH4A1B5P, which led to increased production of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-4-hydrobutyrate) (P34HB) by over 1.4-fold. H. bluephagenesis TDH4A1B5P also enhanced production of ectoine and threonine by 50% and 77%, respectively. A total 101 genes related to salinity tolerance was identified and verified via comparative genomic analysis among four ARTP mutated H. bluephagenesis strains. Recombinant H. bluephagenesis TDH4A1B5P was further engineered for PHA production utilizing sodium acetate or gluconate as sole carbon source. Over 33% cost reduction of PHA production could be achieved using recombinant H. bluephagenesis TDH4A1B5P. This study successfully developed a low-salt tolerant chassis H. bluephagenesis TDH4A1B5P and revealed salt-stress related genes of halophilic host strains.

Assessment of Shoot Priming Efficiency to Counteract Complex Metal Stress in Halotolerant Lobularia maritima.

The study investigated whether short-term priming supports plant defense against complex metal stress and multiple stress (metals and salinity) in halophyte Lobularia maritima (L.) Desv. Plants were pre-treated with ectoine (Ect), nitric oxide donor-sodium nitroprusside (SNP), or hydrogen sulfide donor-GYY4137 for 7 days, and were transferred onto medium containing a mixture of metal ions: Zn, Pb, and Cd. To test the effect of priming agents in multiple stress conditions, shoots were also subjected to low salinity (20 mM NaCl), applied alone, or combined with metals. Hydropriming was a control priming treatment. Stress impact was evaluated on a basis of growth parameters, whereas defense responses were on a basis of the detoxification activity of glutathione S-transferase (GST), radical scavenging activity, and accumulation of thiols and phenolic compounds. Exposure to metals reduced shoot biomass and height but had no impact on the formation of new shoots. Priming with nitric oxide annihilated the toxic effects of metals. It was related to a sharp increase in GST activity, glutathione accumulation, and boosted radical scavenging activity. In NO-treated shoots level of total phenolic compounds (TPC) and flavonoids remained unaffected, in contrast to other metal-treated shoots. Under combined metal stress and salinity, NO and H2S were capable of restoring or improving growth parameters, as they stimulated radical scavenging activity. Ect and H2S did not exert any effect on metal-treated shoots in comparison to hydropriming. The results revealed the stimulatory role of nitric oxide and low doses of NaCl in combating the toxic effects of complex metal stress in L. maritima. Both NO and NaCl interfered with thiol metabolism and antioxidant activity, whereas NaCl also contributed to the accumulation of phenolic compounds.

Halomonas spp., as chassis for low-cost production of chemicals.

Halomonas spp. are the well-studied platform organisms or chassis for next-generation industrial biotechnology (NGIB) due to their contamination-resistant nature combined with their fast growth property. Several Halomonas spp. have been studied regarding their genomic information and molecular engineering approaches. Halomonas spp., especially Halomonas bluephagenesis, have been engineered to produce various biopolyesters such as polyhydroxyalkanoates (PHA), proteins including surfactants and enzymes, small molecular compounds including amino acids and derivates, as well as organic acids. This paper reviews all the progress reported in the last 10 years regarding this robust microbial cell factory. KEY POINTS: • Halomonas spp. are robust chassis for low-cost production of chemicals • Genomic information of some Halomonas spp. has been revealed • Molecular tools and approaches for Halomonas spp. have been developed • Halomonas spp. are becoming more and more important for biotechnology.

Primary purification of intracellular Halomonas salina ectoine using ionic liquids-based aqueous biphasic system.

Ectoine is a zwitterionic amino acid derivative that can be naturally sourced from halophilic microorganisms. The increasing demands of ectoine in various industries have urged the researches on the cost-effective approaches on production of ectoine. Ionic liquids-based aqueous biphasic system (ILABS) was applied to recover Halomonas salina ectoine from cells hydrolysate. The 1-butyl-3-methylimidazolium tetrafluoroborate (Bmim)BF4 was used in the ILABS and the recovery efficiency of ILABS to recover ectoine from H. salina cells lysate was evaluated by determining the effects of phase composition; pHs; crude loading and additional neutral salt (NaCl). The hydrophilic ectoine was targeted to partition to the hydrophilic salt-rich phase. A total yield (YB) of 96.32% ± 1.08 of ectoine was obtained with ILABS of phase composition of 20% (w/w) (Bmim)BF4 and 30% (w/w) sulfate salts; system pH of 5.5 when the 20% (w/w) of crude feedstock was applied to the ILABS. There was no significant enhancement on the ectoine recovery efficiency using the ILABS when NaCl was added, therefore the ILABS composition without the additional neutral salt was recommended for the primary purification of ectoine. Partition coefficient (KE) of 30.80 ± 0.42, purity (PE) of 95.82% and enrichment factor (Ef) of 1.92 were recorded with the optimum (Bmim)BF4/sulfate ILABS. These findings have provided an insight on the feasibility of recovery of intracellular biomolecules using the green solvent-based aqueous system in one single-step operation.

Bio-conversion of methane into high profit margin compounds: an innovative, environmentally friendly and cost-effective platform for methane abatement.

Despite the environmental relevance of CH4 and forthcoming stricter regulations, the development of cost-efficient and environmentally friendly technologies for CH4 abatement is still limited. To date, one of the most promising solutions for the mitigation of this important GHG consists of the bioconversion of CH4 into bioproducts with a high profit margin. In this context, methanotrophs have been already proven as cell-factories of some of the most expensive products synthesized by microorganisms. In the case of ectoine (1000 $ kg-1), already described methanotrophic genera such as Methylomicrobium can accumulate up to 20% (ectoine wt-1) using methane as the only carbon source. Moreover, α-methanotrophs, such as Methylosynus and Methylocystis, are able to store bioplastic concentrations up to 50-60% of their total cell content. More than that, methanotrophs are one of the greatest potential producers of methanol and exopolysaccharides. Although this methanotrophic factory could be enhanced throughout metabolic engineering, the valorization of CH4 into valuable metabolites has been already consistently demonstrated under continuous and discontinuous mode, producing more than one compound in the same bioprocess, and using both, single strains and specific consortia. This review states the state-of-the-art of this innovative biotechnological platform by assessing its potential and current limitations.