Ectoine Production Research Articles

Valorisation of cheese whey for ectoine production by Halomonas neptunia

The production of ectoine, a valuable compound with significant potential as an osmoprotectant, antioxidant, macromolecule protector and anti‐inflammatory agent, was achieved by a lactose‐utilising bacterial strain. We explore the feasibility of using cheese whey, a waste product rich in lactose and amino acids, as a growth medium. The optimisation of various parameters was conducted to maximise ectoine yield. The highest ectoine production was achieved using 50% diluted whey, pH 6.50, 10% NaCl, 150 rpm and 30°C. This is the first study in which whey was efficiently utilised to produce ectoine by Halomonas neptunia.

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.

Multiple Functions of Compatible Solute Ectoine and Strategies for Constructing Overproducers for Biobased Production.

Ectoine and its derivative 5-hydroxyectoine are compatible solutes initially found in the hyperhalophilic bacterium Ectothiorhodospira halochloris, which inhabits the desert in Egypt. The habitat of ectoine producers implies the primary function of ectoine as a cytoprotectant against harsh conditions such as high salinity, drought, and high radiation. More extensive and in-depth studies have revealed the multiple functions of ectoine in its native producer bacterial cells and other types of cells and its biomolecular components (such as proteins and DNA) as a general protective agent. Its chemical properties as a bio-based amino acid derivative make it attractive for basic scientific research and related industries, such as the food/agricultural industry, cosmetic manufacturing, biologics, and therapeutic agent preparation. This article first discusses the functions and applications of ectoine and 5-hydroxyectoine. Subsequently, more emphasis was placed on advances in bio-based ectoine and/or 5-hydroxyectoine production. Strategies for developing more robust cell factories for highly efficient ectoine and/or 5-hydroxyectoine production are further discussed. We hope this review will provide a valuable reference for studies on the bio-based production of ectoine and 5-hydroxyectoine.

Machine learning methods for predicting the key metabolic parameters of Halomonas elongata DSM 2581T.

Ectoine is generally produced by the fermentation process of Halomonas elongata DSM 2581T, which is one of the primary industrial ectoine production techniques. To effectively monitor and control the fermentation process, the important parameters require accurate real-time measurement. However, for ectoine fermentation, three critical parameters (cell optical density, glucose, and product concentration) cannot be measured conveniently in real-time due to time variation, strong coupling, and other constraints. As a result, our work effectively created a series of hybrid models to predict the values of these three parameters incorporating both fermentation kinetics and machine learning approaches. Compared with the traditional machine learning models, our models solve the problem of insufficient data which is common in fermentation. In addition, a simple kinetic modeling is only applicable to specific physical conditions, so different physical conditions require refitting the function, which is tedious to operate. However, our models also overcome this limitation. In this work, we compared different hybrid models based on 5 feature engineering methods, 11 machine-learning approaches, and 2 kinetic models. The best models for predicting three key parameters, respectively, are as follows: CORR-Ensemble (R2: 0.983 ± 0.0, RMSE: 0.086 ± 0.0, MAE: 0.07 ± 0.0), SBE-Ensemble (R2: 0.972 ± 0.0, RMSE: 0.127 ± 0.0, MAE: 0.078 ± 0.0), and SBE-Ensemble (R2:0.98 ± 0.0, RMSE: 0.023 ± 0.001, MAE: 0.018 ± 0.001). To verify the universality and stability of constructed models, we have done an experimental verification, and its results showed that our proposed models have excellent performance. KEY POINTS: • Using the kinetic models for producing simulated data • Through different feature engineering methods for dimension reduction • Creating a series of hybrid models to predict the values of three parameters in the fermentation process of Halomonas elongata DSM 2581T.

Metabolic engineering of Halomonas campaniensis strain XH26 to remove competing pathways to enhance ectoine production

Ectoine has gained considerable attention as a high-value chemical with significant application potential and market demand. This study aimed to increase ectoine yields by blocking the metabolic shunt pathway of l-aspartate-4-semialdehyde, the precursor substrate in ectoine synthesis. The homoserine dehydrogenase encoded by hom in H. campaniensis strain XH26 is responsible for the metabolic shunt of l-aspartate-4-semialdehyde to glycine. CRISPR/Cas9 technology was used to seamlessly knockout hom, blocking the metabolic shunt pathway to increase ectoine yields. The ectoine yield of XH26/Δhom was 351.13 mg (g CDW)−1 after 48 h of incubation in 500 mL shake flasks using optimal medium with 1.5 mol L−1 NaCl, which was significantly higher than the 239.18 mg (g CDW)−1 of the wild-type strain. Additionally, the absence of the ectoine metabolic shunt pathway affects betaine synthesis, and thus the betaine yields of XH26/Δhom was 19.98 mg (g CDW)−1, considerably lower than the 69.58 mg (g CDW)−1 of the wild-type strain. Batch fermentation parameters were optimized, and the wild-type strain and XH26/Δhom were fermented in 3 L fermenters, resulting in a high ectoine yield of 587.09 mg (g CDW)−1 for the defective strain, which was significantly greater than the ectoine yield of 385.03 mg (g CDW)−1 of the wild-type strain. This study showed that blocking the metabolic shunt of synthetic substrates effectively increases ectoine production, and a reduction in the competitively compatible solute betaine appears to promote increased ectoine synthesis.

Metabolic engineering for improving ectoine production in Escherichia coli

Metabolic engineering for improving ectoine production in Escherichia coli

DNA scaffold assisted ectoine production in Escherichia coli

DNA scaffold assisted ectoine production in Escherichia coli

Comparative genomic analysis of Halomonas campaniensis wild-type and ultraviolet radiation-mutated strains reveal genomic differences associated with increased ectoine production

Ectoine is a natural amino acid derivative and one of the most widely used compatible solutes produced by Halomonas species that affects both cellular growth and osmotic equilibrium. The positive effects of UV mutagenesis on both biomass and ectoine content production in ectoine-producing strains have yet to be reported. In this study, the wild-type H. campaniensis strain XH26 (CCTCCM2019776) was subjected to UV mutagenesis to increase ectoine production. Eight rounds of mutagenesis were used to generate mutated XH26 strains with different UV-irradiation exposure times. Ectoine extract concentrations were then evaluated among all strains using high-performance liquid chromatography analysis, alongside whole genome sequencing with the PacBio RS II platform and comparison of the wild-type strain XH26 and the mutant strain G8-52 genomes. The mutant strain G8-52 (CCTCCM2019777) exhibited the highest cell growth rate and ectoine yields among mutated strains in comparison with strain XH26. Further, ectoine levels in the aforementioned strain significantly increased to 1.51 ± 0.01 g L−1 (0.65 g g−1 of cell dry weight), representing a twofold increase compared to wild-type cells (0.51 ± 0.01 g L−1) when grown in culture medium for ectoine accumulation. Concomitantly, electron microscopy revealed that mutated strain G8-52 cells were obviously shorter than wild-type strain XH26 cells. Moreover, strain G8-52 produced a relatively stable ectoine yield (1.50 g L−1) after 40 days of continuous subculture. Comparative genomics analysis suggested that strain XH26 harbored 24 mutations, including 10 nucleotide insertions, 10 nucleotide deletions, and unique single nucleotide polymorphisms. Notably, the genes orf00723 and orf02403 (lipA) of the wild-type strain mutated to davT and gabD in strain G8-52 that encoded for 4-aminobutyrate-2-oxoglutarate transaminase and NAD-dependent succinate-semialdehyde dehydrogenase, respectively. Consequently, these genes may be involved in increased ectoine yields. These results suggest that continuous multiple rounds of UV mutation represent a successful strategy for increasing ectoine production, and that the mutant strain G8-52 is suitable for large-scale fermentation applications.

Production and Recovery of Ectoine: A Review of Current State and Future Prospects

Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a revolutionizing substance with vast applications in the cosmetic and food industries. Ectoine is often sourced from halobacteria. The increasing market demand for ectoine has urged the development of cost-effective and sustainable large-scale production of ectoine from microbial sources. This review describes the existing and potential microbial sources of ectoine and its derivatives, as well as microbial production and fermentation approaches for ectoine recovery. In addition, conventional methods and emerging technologies for enhanced production and recovery of ectoine from microbial fermentation with a focus on the aqueous biphasic system (ABS) are discussed. The ABS is a practically feasible approach for the integration of fermentation, cell disruption, bioconversion, and clarification of various biomolecules in a single-step operation. Nonetheless, the implementation of the ABS on an industrial-scale basis for the enhanced production and recovery of ectoine is yet to be exploited. Therefore, the feasibility of the ABS to integrate the production and direct recovery of ectoine from microbial sources is also highlighted in this review.

Optimization of ectoine production from Nesterenkonia xinjiangensis and one-step ectoine purification

In the current study, the optimization of ectoine production byNesterenkonia xinjiangensisand purification of ectoine from the bacterial cell extract were performed for the first time. Various carbon sources (glucose, sucrose, maltose, lactose, mannitol, and xylose) and nitrogen sources (ammonium nitrate, ammonium phosphate, ammonium chloride, ammonium oxalate, ammonium sulphate, and ammonium acetate), were used to optimize ectoine production. Subsequently, the effects of salt, pH and, concentrations of carbon and nitrogen source on ectoine production were optimized by response surface methodology (RSM). Ultimately, high pure (over 99%) and yield (98%) of ectoine from bacterial cells extracted was obtained by a single-step process using cation exchange chromatography. This study provides information that higher ectoine production can be achieved from this bacterial isolate by optimizing the factors influencing ectoine production and thus can be used as a new and alternative ectoine producer.

Draft Genomes of Halophilic Chromohalobacter and Halomonas Strains Isolated from Brines of The Carpathian Foreland, Poland.

Chromohalobacter and Halomonas are genera of bacterial microorganisms belonging to the group of halophiles. They are characterized by high diversity and the ability to produce bioproducts of biotechnological importance, such as ectoine, biosurfactants and carotenoids. Here, we report three draft genomes of Chromohalobacter and two draft genomes of Halomonas isolated from brines. The length of the genomes ranged from 3.6 Mbp to 3.8 Mbp, and GC content was in the 60.11%-66.46% range. None of the analysed genomes has been assigned to any previously known species of the genus Chromohalobacter or Halomonas. Phylogenetic analysis revealed that Chromohalobacter 296-RDG and Chromohalobacter 48-RD10 belonged to the same species, and Chromohalobacter 11-W is more distantly related to the other two analysed strains than to Chromohalobacter canadensis. Halomonas strains 11-S5 and 25-S5 were clustered together and located close to Halomonas ventosae. Functional analysis revealed BGCs related to ectoine production in all genomes analysed. This study increases our overall understanding of halophilic bacteria and is also consistent with the notion that members of this group have significant potential as useful natural product producers.

Identification of novel halophilic/halotolerant bacterial species producing compatible solutes.

Ectoine and hydroxyectoine are compatible solutes with enormous potential for use in the medical and cosmetic industries. Considering the excellent osmoprotective properties of these compatible solutes, we investigate the presence of four compatible solutes (ectoine, hydroxyectoine, proline, and glutamic acid) quantitatively by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in forty-five halophilic/halotolerant bacterial isolates. We determined ectoine production by Marinibacillus sp., Nesterenkonia xinjiangensis, Halobacillus sp., Bacillus patagoniensis, Virgibacillus picturae, Halomonas neptunia, Bacillus patagoniensis, Gracilibacillus sp., Thalassobacillus devorans, Microbacterium sp., Nesterenkonia sp., and Bacillus agaradhaerens, and this production was NaCl dependent. Additionally, the production of hydroxyectoine was observed in six bacterial isolates (Nesterenkonia xinjiangensis, Halobacillus sp., Halomonas neptunia, Thalassobacillus devorans, Nesterenkonia sp., and Bacillus agaradhaerens) which was NaCl and temperature dependent. The study identified new bacterial isolates producing ectoine or hydroxyectoine. While the ectoine production in many different Bacillus members and a few Nesterenkonia have been documented before, ectoine production by Bacillus patagoniensis and Nesterenkonia xinjiangensis has not been shown so far. Further, ectoine production by a member of the genus Thalassobacillus (Thalassobacillus devorans) was demonstrated experimentally for the first time. The findings reported in the study may serve as a basis for the large-scale production of ectoine and hydroxyectoine in the future.

Bioactivity profiling of the extremolyte ectoine as a promising protectant and its heterologous production.

Ectoine is a compatible solutes that is diffusely dispersed in bacteria and archaea. It plays a significant role as protectant against various external pressures, such as high temperature, high osmolarity, dryness and radiation, in cells. Ectoine can be utilized in cosmetics due to its properties of moisturizing and antiultraviolet. It can also be used in the pharmaceutical industry for treating various diseases. Therefore, strong protection of ectoine creates a high commercial value. Its current market value is approximately US$1000kg-1. However, traditional ectoine production in high-salinity media causes high costs of equipment loss and wastewater treatment. There is a growing attention to reduce the salinity of the fermentation broth without sacrificing the production of ectoine. Thus, heterologous production of ectoine in nonhalophilic microorganisms may represent the new generation of the industrial production of ectoine. In this review, we summarized and discussed the biological activities of ectoine on cell and human health protection and its heterologous production.

Physiological metabolic topology analysis of Halomonas elongata DSM 2581T in response to sodium chloride stress.

The halophilic bacterium Halomonas elongata DSM 2581T generally adapts well to high level of salinity by biosynthesizing ectoine, which functions as an important compatible solute protecting the cell against external salinity environment. Halophilic bacteria have specific metabolic activities under high-salt conditions and are gradually applied in various industries. The present study focuses on investigating the physiological and metabolic mechanism of H. elongata DSM 2581T driven by the external salinity environment. The physiological metabolic dynamics under salt stress were investigated to evaluate the effect of NaCl stress on the metabolism of H. elongata. The obtained results demonstrated that ectoine biosynthesis transited from a nongrowth-related process to a growth-related process when the NaCl concentration varied from 1% to 13% (w/v). The maximum biomass (Xm = 41.37 g/L), and highest ectoine production (Pm = 12.91 g/L) were achieved under 8% NaCl. Moreover, the maximum biomass (Xm ) and the maximum specific growth rates (μm ) showed a first rising and then declining trend with the increased NaCl stress. Furthermore, the transcriptome analysis of H. elongata under different NaCl concentrations demonstrated that both 8% and 13% NaCl conditions resulted in increased expressions of genes involved in the pentose phosphate pathway, Entner-Doudoroffpathway, flagellar assembly pathway, and ectoine metabolism, but negatively affected the tricarboxylic acidcycle and fatty acid metabolism. At last, the proposed possible adaptation mechanism under the optimum NaCl concentration in H. elongata was described.

Metabolic engineering of Halomonas elongata: Ectoine secretion is increased by demand and supply driven approaches.

The application of naturally-derived biomolecules in everyday products, replacing conventional synthetic manufacturing, is an ever-increasing market. An example of this is the compatible solute ectoine, which is contained in a plethora of treatment formulations for medicinal products and cosmetics. As of today, ectoine is produced in a scale of tons each year by the natural producer Halomonas elongata. In this work, we explore two complementary approaches to obtain genetically improved producer strains for ectoine production. We explore the effect of increased precursor supply (oxaloacetate) on ectoine production, as well as an implementation of increased ectoine demand through the overexpression of a transporter. Both approaches were implemented on an already genetically modified ectoine-excreting strain H. elongata KB2.13 (ΔteaABC ΔdoeA) and both led to new strains with higher ectoine excretion. The supply driven approach led to a 45% increase in ectoine titers in two different strains. This increase was attributed to the removal of phosphoenolpyruvate carboxykinase (PEPCK), which allowed the conversion of 17.9% of the glucose substrate to ectoine. For the demand driven approach, we investigated the potential of the TeaBC transmembrane proteins from the ectoine-specific Tripartite ATP-Independent Periplasmic (TRAP) transporter as export channels to improve ectoine excretion. In the absence of the substrate-binding protein TeaA, an overexpression of both subunits TeaBC facilitated a three-fold increased excretion rate of ectoine. Individually, the large subunit TeaC showed an approximately five times higher extracellular ectoine concentration per dry weight compared to TeaBC shortly after its expression was induced. However, the detrimental effect on growth and ectoine titer at the end of the process hints toward a negative impact of TeaC overexpression on membrane integrity and possibly leads to cell lysis. By using either strategy, the ectoine synthesis and excretion in H. elongata could be boosted drastically. The inherent complementary nature of these approaches point at a coordinated implementation of both as a promising strategy for future projects in Metabolic Engineering. Moreover, a wide variation of intracelllular ectoine levels was observed between the strains, which points at a major disruption of mechanisms responsible for ectoine regulation in strain KB2.13.

High-yield ectoine production in engineered Corynebacterium glutamicum by fine metabolic regulation via plug-in repressor library

Ectoine is a high-value protective and stabilizing agent with different applications in biopharmaceuticals, biotechnology, and fine chemicals. Here, efficient production of ectoine in Corynebacterium glutamicum was achieved by combination of metabolic engineering and plug-in repressor library strategy. First, the ectBAC cluster from Pseudomonas stutzeri was introduced into strain K02, and the titer of the obtained strain was 2.12 g/L. Metabolic engineering was then performed for further optimization, including removal of competing pathways (pck and ldh knockout), deletion of glycolysis repressor (sugR knockout), and enhancement of precursor supply (overexpression of Ecasd and CglysCS301Y). Next, two repressor libraries were designed for targeted flux control to improve ectoine production. Finally, strain CB5L6 produced 45.52 g/L ectoine and had the highest yield in C. glutamicum. For the first time, plug-in repressor library was employed to engineer C. glutamicum for metabolites production, which will provide a guideline for the construction of microbial cell factories.

Ectoine production from biogas: A sensitivity analysis. Effect of local commodity prices, economy of scale, market trends and biotechnological limitations

The utilization of methanotrophic haloalkaliphilic bacteria for the production of ectoine in waste treatment plants has demonstrated a great potential to upgrade the traditional use of biogas as energy vector and the current industrial routes for the production of this high added-value chemical (600–1000 €·kg−1). However, the influence of socio-economic aspects such as the location of the plant, the economy of scale and the market fluctuations on the profitability of the process remains unknown. A techno-economic and sensitivity analysis of the bioconversion of CH4 into ectoine in a bubble column bioreactor and the extraction and purification of ectoine via ionic exchange chromatography was herein conducted using Madrid as base-case scenario. The geographical assessment performed in 13 representative cities revealed high differences in the ectoine production costs, ranging from 158 to 231 €·kg−1. The economy of scale analysis evidenced a high dependence of the ectoine production costs towards the production scale, amounting to 782 and 164 €·kg−1 when manufacturing 0.1 and 89.6 t ectoine·y−1, respectively. The techno-economic study also showed a high robustness of CH4-ectoine profitability towards future market fluctuations, with all the scenarios analyzed guaranteeing internal rates of return >15% and payback periods <10 y. Finally, the sensitivity analysis identified the improvement of CH4 elimination capacity in bioreactors, the development of highly efficient microbial strains and the selection of the highest quality ionic exchange resins as key factors impacting the profitability of future biogas-to-ectoine biorefineries.

Ectoine and Hydroxyectoine from Halophilic Bacteria Isolated from Traditional Solar Saltern at Pejarakan Village, Buleleng Regency, Bali

The increased use of ectoine and hydroxyectoine in cosmetics and pharmaceuticals has led to the increasing demand for these bioactive compounds. This study aims to investigate the production of ectoine and hydroxyectoine by halophilic bacteria isolated from traditional solar saltern at Pejarakan Village, Buleleng Regency, Province of Bali, Indonesia. The halophilic bacteria were grown from the brine and the sediment samples using Luria Bertani media. The production of ectoine and hydroxyectoine was determined by inoculating the bacteria in MM63 media containing 12% w/v NaCl. Ectoine and hydroxyectoine were then extracted from the bacterial cells using a mixture of methanol/chloroform/water (10/5/4). Finally, the ability of the bacteria to excrete ectoine and hydroxyectoine was investigated using the osmotic downshock technique. The results showed that a total of 88 halophilic bacteria have been isolated which showed varied salt tolerances from 0 to 27.5% w/v. Most of the bacteria (86 isolates) were able to grow in MM63 media, suggesting the potential of the bacteria producing ectoine and hydroxyectoine. Further investigation showed that 33 selected halophilic bacteria were able to produce ectoine and hydroxyectoine with levels of 9.1 to 301.8 mg/L and 3.9 to 122.1 mg/L, respectively. All the bacteria were also able to excrete ectoine and hydroxyectoine after the osmotic downshock process with levels of 9.8 to 277.5 mg/L and 14.3 to 122.7 mg/L, respectively. The best ectoine and hydroxyectoine-producing bacteria showed the closest homology relationship with Salinivibrio costicola and Salinivibrio kushneri. Our study has found several promising candidates for ectoine and hydroxyectoine producers.

Isolation of an ectoine-producing Sinobaca sp. and identification of genes that are involved in ectoine biosynthesis.

A moderate halophilic bacterium that could accumulate ectoine and hydroxyectoine was isolated from soil near a salt mine and was identified as a Sinobaca sp. (designed strain H24) according to 16S rRNA gene sequence analysis. The bacterium grew well in the presence of 1-2M NaCl, while growth in a medium that contained 2M NaCl led to higher accumulation of ectoines. The yields of ectoine and hydroxyectoine by Sinobaca sp. H24 reached 11.27mg/l and 1.34mg/l, respectively, when cultured in the following medium: NaCl (2M), peptone (5g/l), yeast extract (1g/l), NH4Cl (0.02M), KH2PO4 (1M), K2HPO4 (0.1M), and glycerol (1%w/v). Genes that are involved in ectoine biosynthesis of Sinobaca sp. H24 were also identified, and their sequences were determined by a metagenomics approach. The results demonstrated that Sinobaca sp. H24 possesses ectoine metabolism genes for both ectoine biosynthesis (ectA, ectB, ectC, and ectD) and ectoine degradation (doeA). Genes that are related to ectoine biosynthesis, such as lysC and asd, were also characterized. The identification and characterization results for ectoine/hydroxyectoine biosynthesis genes are in agreement with the physiology of Sinobaca sp. H24 as a potential candidate for ectoine production for industrial applications. This report established for the first time the accumulation of ectoine/hydroxyectoine in Sinobaca sp. and characterized the genes that are involved in ectoine/hydroxyectoine biosynthesis in Sinobaca sp. H24.

Influence of operational conditions on the performance of biogas bioconversion into ectoines in pilot bubble column bioreactors

The application of biogas as a low-priced substrate for the production of ectoines constitutes an opportunity to decrease their production costs and to enhance the viability of anaerobic digestion. The influence of operational conditions on CH4-biogas biodegradation and on ectoines production yields was assessed in continuous pilot bubble column bioreactors. The rise in biomass concentration from 1 to 3 g L−1 resulted in a decrease in the specific ectoine content from 42 ± 8 to 30 ± 4 mgectoine gVSS−1. The concentration of Cu2+ and Mg2+ did not impact process performance, while the use of ammonium as N source resulted in low CH4 biodegradation and ectoine yields (13 ± 7 mgectoine gVSS−1). The increase in CH4 content from 4.5 to 9 %v·v−1 enhanced CH4 removal efficiency. Process operation at NaCl concentrations of 3 %w·w−1 instead of 6 %w·w−1 decreased the ectoine yield to 17 mgectoine gVSS−1. Finally, Methylomicrobiumburyatense was identified as the dominant species.