Late Exponential Phase Research Articles

Heavy Vacuum Gas Oil Upregulates the Rhamnosyltransferases and Quorum Sensing Cascades of Rhamnolipids Biosynthesis in Pseudomonas sp. AK6U.

We followed a comparative approach to investigate how heavy vacuum gas oil (HVGO) affects the expression of genes involved in biosurfactants biosynthesis and the composition of the rhamnolipid congeners in Pseudomonas sp. AK6U. HVGO stimulated biosurfactants production as indicated by the lower surface tension (26 mN/m) and higher yield (7.8 g/L) compared to a glucose culture (49.7 mN/m, 0.305 g/L). Quantitative real-time PCR showed that the biosurfactants production genes rhlA and rhlB were strongly upregulated in the HVGO culture during the early and late exponential growth phases. To the contrary, the rhamnose biosynthesis genes algC, rmlA and rmlC were downregulated in the HVGO culture. Genes of the quorum sensing systems which regulate biosurfactants biosynthesis exhibited a hierarchical expression profile. The lasI gene was strongly upregulated (20-fold) in the HVGO culture during the early log phase, whereas both rhlI and pqsE were upregulated during the late log phase. Rhamnolipid congener analysis using high-performance liquid chromatography-mass spectrometry revealed a much higher proportion (up to 69%) of the high-molecularweight homologue Rha–Rha–C10–C10 in the HVGO culture. The results shed light on the temporal and carbon source-mediated shifts in rhamonlipids’ composition and regulation of biosynthesis which can be potentially exploited to produce different rhamnolipid formulations tailored for specific applications.

Identification of the Genes Related to the Glycogen Metabolism in Hyperthermophilic Archaeon, Sulfolobus acidocaldarius.

Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC–gaa–glgX and amyA–glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC–gaa–glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

Co-cultivation of Streptomyces and microalgal cells as an efficient system for biodiesel production and bioflocculation formation

The phytohormone producing Streptomyces rosealbus MTTC 12,951 (S.R) and green microalga Chlorella vulgaris MSU-AGM 14 (C.V) were cultivated in co-culture system to evaluate exogenous hormonal activity. Biosynthesis of indole-3-acetic acid (IAA) and their precursors were quantitatively evaluated by employing High Performance Liquid Chromatography (HPLC). The concentration of IAA (0.72 ± 0.02 µg mL−1) was observed to be elevated in co-cultivation system due to symbiotic interaction between Streptomyces and microalgae. In exchange, microalgae produced adequate volume of tryptophan (Trp) to induce IAA biosynthesis. The Trp stress in late exponential phase encouraged lipid accumulation (175 ± 10 mg g−1). The bioflocculation property of microalgae ensures potential and economic viable harvesting process by reducing 148% input energy compared to conventional method. The overall results evidenced that C.V co-cultivation with S.R exhibits promotional behavior and serves as a promising cultivation process for microalgae in terms of cost efficiency and energy conservation.

Enhanced ascomycin production in Streptomyces hygroscopicus var. ascomyceticus by employing polyhydroxybutyrate as an intracellular carbon reservoir and optimizing carbon addition

BackgroundAscomycin is a multifunctional antibiotic produced by Streptomyces hygroscopicus var. ascomyceticus. As a secondary metabolite, the production of ascomycin is often limited by the shortage of precursors during the late fermentation phase. Polyhydroxybutyrate is an intracellular polymer accumulated by prokaryotic microorganisms. Developing polyhydroxybutyrate as an intracellular carbon reservoir for precursor synthesis is of great significance to improve the yield of ascomycin.ResultsThe fermentation characteristics of the parent strain S. hygroscopicus var. ascomyceticus FS35 showed that the accumulation and decomposition of polyhydroxybutyrate was respectively correlated with cell growth and ascomycin production. The co-overexpression of the exogenous polyhydroxybutyrate synthesis gene phaC and native polyhydroxybutyrate decomposition gene fkbU increased both the biomass and ascomycin yield. Comparative transcriptional analysis showed that the storage of polyhydroxybutyrate during the exponential phase accelerated biosynthesis processes by stimulating the utilization of carbon sources, while the decomposition of polyhydroxybutyrate during the stationary phase increased the biosynthesis of ascomycin precursors by enhancing the metabolic flux through primary pathways. The comparative analysis of cofactor concentrations confirmed that the biosynthesis of polyhydroxybutyrate depended on the supply of NADH. At low sugar concentrations found in the late exponential phase, the optimization of carbon source addition further strengthened the polyhydroxybutyrate metabolism by increasing the total concentration of cofactors. Finally, in the fermentation medium with 22 g/L starch and 52 g/L dextrin, the ascomycin yield of the co-overexpression strain was increased to 626.30 mg/L, which was 2.11-fold higher than that of the parent strain in the initial medium (296.29 mg/L).ConclusionsHere we report for the first time that polyhydroxybutyrate metabolism is beneficial for cell growth and ascomycin production by acting as an intracellular carbon reservoir, stored as polymers when carbon sources are abundant and depolymerized into monomers for the biosynthesis of precursors when carbon sources are insufficient. The successful application of polyhydroxybutyrate in increasing the output of ascomycin provides a new strategy for improving the yields of other secondary metabolites.

RNA release via membrane vesicles in Pseudomonas aeruginosa PAO1 is associated with the growth phase.

Pseudomonas aeruginosa PAO1 membrane vesicles (MVs) are known to play a role in cell-to-cell communication. Several studies have shown that the MV composition and physicochemical properties vary according to the bacterial growth stage, but the impact this might have on the externalization of RNA via MVs has not been addressed. Therefore, a study to characterize the RNA content from MVs retrieved at different growth phases was conducted. First, the transcriptome analyses revealed a higher abundance of around 300 RNA species in MVs when compared with the cells. The vesiculation rate along the growth curve was determined, showing that the release of MVs increased during the transition to the stationary phase, whereas it decreased in the late stationary phase. RNA-seq of MVs retrieved along the transition to the stationary phase demonstrated that the RNA cargo of vesicles did not vary. However, the amount of smaller RNAs (<200 nt) inside MVs retrieved in the late exponential phase was higher than in the stationary phase MVs. These results indicate that the externalization of RNA via MVs occurs during late exponential phase and implies the secretion of different types of MVs during growth.

Combination of colistin and tobramycin inhibits persistence of Acinetobacter baumannii by membrane hyperpolarization and down-regulation of efflux pumps

Acinetobacter baumannii, a leading cause of nosocomial infections, is a serious health threat. Limited therapeutic options due to multi-drug resistance and tolerance due to persister cells have urged the scientific community to develop new strategies to combat infections caused by this pathogen effectively. Since combination antibiotic therapy is an attractive strategy, the effect of combinations of antibiotics, belonging to four classes, was investigated on eradication of persister cells in A. baumannii. Among the antibiotics included in the study, tobramycin-based combinations were found to be the most effective. Tobramycin, in combination with colistin or ciprofloxacin, eradicated persister cells in A. baumannii in late exponential and stationary phases of growth. Mechanistically, colistin facilitated the entry of tobramycin into cells by increasing membrane permeability and inducing hyperpolarization of the inner membrane accompanied by increase in ROS production. Expression of the genes encoding universal stress protein and efflux pumps was down-regulated in response to tobramycin and colistin, suggesting increased lethality of their combination that might be responsible for eradication of persister cells. Thus, a combination of tobramycin and colistin could be explored as a promising option for preventing the relapse of A. baumannii infections due to persister cells.

Potential Applications of Native Cyanobacterium Isolate (Arthrospira platensis NIOF17/003) for Biodiesel Production and Utilization of Its Byproduct in Marine Rotifer (Brachionus plicatilis) Production

To achieve strong, successful and commercial aqua-biotechnological microalgae applications, screening, isolation, molecular identification, and physiological characterizations are needed. In the current study, a native cyanobacteria strain Arthrospira platensis NIOF17/003 was isolated from the surface water of El-Khadra Lake, a saline-alkaline lake located in Wadi El-Natrun, Egypt. The cyanobacterium was phylogenetically identified by 16S rRNA molecular marker and deposited in the GenBank database (accession number MW396472). The late exponential phase of A. platensis NIOF17/003 was reached at the 8th day of growth using Zarrouk medium, with a recorded dry weight (DW) of 0.845 g L−1. The isolated strain showed 52% of protein, 14% of carbohydrate, biomass productivity of 143.83 mg L−1 day−1, 8.5% of lipid, and lipid productivity of 14.37 mg L−1 day−1. In general, the values of cetane number, iodine value, cold filter plugging point (52.9, 85.5 g I2/100 g oil, and −2.2 °C, respectively) of the isolated fatty acid methyl esters are in accordance with those suggested by international standards. Besides, applying algal-free lipid (FL) as biodiesel byproduct in the production of rotifer (Brachionus plicatilis) revealed that a 0.6 g L−1 FL significantly increased the rotifer population females carrying eggs, confirming that FL can be used efficiently for B. plicatilis production. The current study concluded that the new isolate A. platensis NIOF17/003 is a promising strain for double sustainable use in biodiesel production and aquaculture feed.

Isolation of Phaeobacter sp. from Larvae of Atlantic Bonito (Sarda sarda) in a Mesocosmos Unit, and Its Use for the Rearing of European Seabass Larvae (Dicentrarchus labrax L.).

The target of this study was to use indigenous probiotic bacteria in the rearing of seabass larvae. A Phaeobacter sp. strain isolated from bonito yolk-sac larvae (Sarda sarda) and identified by amplification of 16S rDNA showed in vitro inhibition against Vibrio anguillarum. This Phaeobacter sp. strain was used in the rearing of seabass larvae (Dicentrarchus labrax L.) in a large-scale trial. The survival of seabass after 60 days of rearing and the specific growth rate at the late exponential growth phase were significantly higher in the treatment receiving probiotics (p < 0.05). Microbial community richness as determined by denaturing gradient gel electrophoresis (DGGE) showed an increase in bacterial diversity with fish development. Changes associated with the administration of probiotics were observed 11 and 18 days after hatching but were not apparent after probiotic administration stopped. In a small challenge experiment, seabass larvae from probiotic treatment showed increased survival (p < 0.05) after experimental infection with a mild pathogen (Vibrio harveyi). Overall, our results showed that the use of an indigenous probiotic strain had a beneficial impact on larval rearing in industry-like conditions.

Interactive effects of light quality and culturing temperature on algal cell size, biomass doubling time, protein content, and carbohydrate content.

Light management strategy can be used to improve algal biomass and nutrient production. However, the response of algal metabolism to different light qualities, especially their interaction with other environmental factors, is not well understood. This study focuses on the interactive effects of light quality and culturing temperature on algal protein content and carbohydrate content of C. reinhardtii. Three LED light sources (blue light, red-orange light, and white-yellow light) were applied to grow algae in batch cultures with a light intensity of 105 μmol/m2s under the temperatures of 24 °C to 32 °C. The protein and carbohydrate content were measured in both the late exponential growth phase and the late stationary growth phase. The results revealed that there was an interactive effect of light quality and culturing temperature on the protein and carbohydrate content. The combined conditions of blue light and a temperature of 24 °C or 28 °C, which induced a larger algal cell size with a prolonged cell cycle and a low division rate, resulted in the highest protein content; the protein mass fraction and concentration were 32% and 52% higher than that under white-yellow light at 32 °C. The combined conditions of red-orange light and a temperature of 24 °C, which promoted both the cell division and size growth, enhanced the carbohydrate content; the carbohydrate mass fraction and concentration were 161% and 155% higher than that under white-yellow light at 24 °C. When there was temperature stress (32 °C) or nutrient stress, the effect of light quality reduced, and the difference of protein and carbohydrate content among the three light qualities decreased. KEY POINTS: • Studied light quality-temperature interactive effect on protein, carbohydrate synthesis. • Protein content was high under low cell division rate. • Carbohydrate content was high under high cell division and cell size growth rate.

Characterization of dynamic regulation in Chinese hamster ovary (CHO) cell cultures in the late exponential phase

Chinese hamster ovary (CHO) cells are the most commonly used host cells to produce biologics in the biopharmaceutical industry. In a batch or fed-batch process, cells grow at a relatively constant rate at the exponential growth phase, a period that is commonly assumed as a “metabolically quasi steady-state”. However, the cell culture environment, such as medium composition, does not maintain the same during this time; instead, the medium composition changes with nutrient decrease, cell density increase, and byproduct accumulation. Therefore, cellular microenvironment may respond to such process changes. This study examined the transcriptome across the late exponential growth phase of three CHO cell lines to illuminate the dynamic regulation at glycolysis, tricarboxylic acid (TCA) cycle, glycosylation, and antioxidant pathways. We found that the expression of a series of genes has a concurrent shift around 91 h, coincident with lactate shift from its accumulation to consumption. The gene expressions altogether suggest a homeostasis behavior before the 91 h, presumably a response to the high abundance of nutrients at the exponential growth phase.

Single-Nucleotide RNA Maps for the Two Major Nosocomial Pathogens Enterococcus faecalis and Enterococcus faecium.

Enterococcus faecalis and faecium are two major representative clinical strains of the Enterococcus genus and are sadly notorious to be part of the top agents responsible for nosocomial infections. Despite their critical implication in worldwide public healthcare, essential and available resources such as deep transcriptome annotations remain poor, which also limits our understanding of post-transcriptional control small regulatory RNA (sRNA) functions in these bacteria. Here, using the dRNA-seq technique in combination with ANNOgesic analysis, we successfully mapped and annotated transcription start sites (TSS) of both E. faecalis V583 and E. faecium AUS0004 at single nucleotide resolution. Analyzing bacteria in late exponential phase, we capture ~40% (E. faecalis) and 43% (E. faecium) of the annotated protein-coding genes, determine 5′ and 3′ UTR (untranslated region) length, and detect instances of leaderless mRNAs. The transcriptome maps revealed sRNA candidates in both bacteria, some found in previous studies and new ones. Expression of candidate sRNAs is being confirmed under biologically relevant environmental conditions. This comprehensive global TSS mapping atlas provides a valuable resource for RNA biology and gene expression analysis in the Enterococci. It can be accessed online at www.helmholtz-hiri.de/en/datasets/enterococcus through an instance of the genomic viewer JBrowse.

Effect of gibberellin, methyl jasmonate and myoinositol on biomass and eicosapentaenoic acid productivities in the eustigmatophyte Monodopsis subterranea CCALA 830

Omega-3 fatty acids sourced from microalgae are in demand due to their therapeutic potential and sustainability of the source. The present study was focused towards enhancing eicosapentaenoic acid (EPA) productivity in the marine Chrysophyte Monodopsis subterranea through media supplementation using the plant growth regulators gibberellic acid, methyl jasmonate and myoinositol. The effect of three concentrations (1 μM, 10 μM and 100 μM) of supplements on biomass yield, fatty acid composition, total fatty acid (TFA) and EPA productivity were analyzed at three phases of microalgal growth. Supplementation with 100 μM GA increased biomass yield and TFA productivity by 3.3-fold and 3.9-fold respectively at the mid-exponential phase and EPA productivity increased up to 3.2-fold at the late-exponential phase. The addition of 1 μM MeJA stimulated biomass accumulation to 2.7-fold at the late-stationary phase, but this effect was reduced to 1.1–1.4-fold at higher concentrations. The addition of 10 μM MI also showed a positive effect on TFA productivity and thus increased EPA productivity by 1.8-fold at the late-stationary phase. Supplementation with 100 μM GA, 100 μM MeJA and 10 μM MI at the late-stationary phase increased all fatty acids except 18:0 and 18:2. EPA was negatively correlated with 18:2 at all three growth phases. Principal components analysis indicated a strong relationship between key fatty acids and EPA at all three growth stages and associated with 100 μM GA.

Interactive effect of light quality and temperature on Chlamydomonas reinhardtii growth kinetics and lipid synthesis

The efficient operation of an algal photobioreactor (PBR) requires appropriate lighting conditions. Despite recent advantages in PBR design and operation, optimal lighting conditions are still not fully understood. This work focuses on the effect of light quality and culturing temperature on the algal growth kinetics and lipid content. The green alga Chlamydomonas reinhardtii was grown in batch culture in a constant-temperature incubator-shaker under LED light. Three light wavelength regimes were studied: blue light (peaking at 433 nm–447 nm, 458 nm–470 nm), white-yellow light (peaking at 456–458 nm in blue, 545 nm in green and 570 nm in yellow) and red-orange light (peaking at 580 nm–594 nm and 604 nm in yellow-orange, 630 nm and 656 nm in red, and 735 nm in IR). Results show that red-orange light obtained 38% higher biomass productivity than blue light at 24 °C, and blue light obtained 13% higher biomass productivity than red-orange light at 32 °C. Red-orange light or temperatures of 30–32 °C favored lipid accumulation with a lipid mass fraction and lipid concentration of 44% and 80% higher, respectively, compared to blue light at 24 °C. In the late exponential phase, when nutrients in the medium were still sufficient, light quality was the predominant factor controlling the lipid synthesis and accumulation. While in the late stationary phase under nutrient starvation, a strong interactive effect was detected between the light quality and temperature (P = 0.02) for lipid synthesis and accumulation. Under both temperature and nutrient stress, there was a reduced effect of the light quality on the lipid synthesis.

Active Transposition of Insertion Sequences by Oxidative Stress in Deinococcus geothermalis.

Radiation-resistant bacterium Deinococcus geothermalis has a total of 73 insertion sequences (ISs) in genomes, and some of them are actively transposed to other loci with replicative mode due to oxidative stress of hydrogen peroxide treatment. Here, we detected two transposition events in wild-type (WT) strain and LysR family member gene disrupted strain (Δdgeo_2840). Similar to our previous report (Lee et al., 2019), phytoene desaturase (dgeo_0524), a key enzyme of carotenoid biosynthesis, was disrupted by the integration of IS element, thereby detected a single phenotypically non-pigmented colony in each WT and Δdgeo_2840 strain. Two separate types of IS element have been integrated into non-pigmented clones: ISDge11 for WT and ISDge6 for Δdgeo_2840 strain. Surprisingly, Δdgeo_2840 mutant strain revealed higher resistance to oxidative stress than WT strain at late exponential growth phase. From the qRT-PCR analysis, OxyR (dgeo_1888) was highly up-regulated to 30-fold by oxidative stress through hydrogen peroxide treatment in both WT and Δdgeo_2840 mutant strains. However, the oxidative stress response enzyme, catalase or superoxide dismutase, was not significantly induced by overexpressed OxyR. Thus, a putative LysR family regulator Dgeo_2840 controlled the expression of ISDge6 type transposase and the induction of OxyR under oxidative condition. There is LysR family DNA-binding protein dependent active transposition of specific type IS and the up-regulated OxyR has not positively controlled ROS scavenger enzymes in D. geothermalis.

Insights into oleaginous phenotype of the yeast Papiliotrema laurentii

Oleaginous yeasts have stood out due to their ability to accumulate oil, which can be used for fatty acid-derived biofuel production. Papiliotrema laurentii UFV-1 is capable of starting the lipid accumulation in the late exponential growth phase and achieves maximum lipid content at 48 h of growth; it is, therefore, interesting to study how its oleaginous phenotype is regulated. Herein, we provide for the first time insights into the regulation of this phenotype in P. laurentii UFV-1. We sequenced and assembled its genome, performed comparative genomic analyses and investigated its phylogenetic relationships with other yeasts. Gene expression and metabolomic analyses were carried out on the P. laurentii UFV-1 cultivated under a nitrogen-limiting condition. Our results indicated that the lipogenesis of P. laurentii might have taken place during evolution after the divergence of genera in the phylum Basidiomycota. Metabolomic data indicated the redirection of the carbon flux towards fatty acid synthesis in response to the nitrogen limitation. Furthermore, purine seems to be catabolized to recycle nitrogen and leucine catabolization may provide acetyl-CoA for fatty acid synthesis. Analyses of the expression of genes encoding certain enzymes involved with the oleaginous phenotype indicated that the NADP+-dependent malic enzyme seems to play an important role in the supply of NADPH for fatty acid synthesis. There was a surprising decrease in the expression of the ACC1 gene, which encodes acetyl-CoA carboxylase, during lipid accumulation. Taken together, our results provided a basis for understanding lipid accumulation in P. laurentii under nitrogen limiting conditions.

Effects of phenylalanine and methyl jasmonate on verbascoside production in Buddleja cordata Kunth cell suspension cultures

Buddleja cordata Kunth (Scrophulariaceae) is an important medicinal herb widespread in Mexico that produces diverse phenolic compounds; among others, the pharmacologically important phenylethanoid glycoside (PeG) verbascoside (VB, also named acteoside). This work describes the effects of the metabolic precursor, L-phenylalanine (L-Phe), and the elicitor, methyl jasmonate (MeJa), on biomass accumulation and VB production in a B. cordata cell-line under suspension culture conditions. Cultures were exogenously fed with different concentrations of the precursor and elicitor at the early and late stages of the exponential growth phase. Precursor or elicitor addition was most effective when added in the late-exponential phase. Cultures fed with 600 µM L-Phe reached the highest production of VB after a 10-day exposure time (1.96 g L−1, which was 130% higher than in control cultures). Maximum VB production after elicitation (4.8 g L−1, with an overall increase of 213% above control level) occurred with 50 µM MeJa after an exposure time of 48 h. VB production yields reported in this work exceed those of previously reported biotechnological efforts using cell suspension cultures and other in vitro systems to increase phenylethanoid glycosides production, especially, VB.

Development of a species-specific transformation system using the novel endogenous promoter calreticulin from oleaginous microalgae Ettlia sp.

Microalgae not only serve as raw materials for biofuel but also have uses in the food, pharmaceutical, and cosmetic industries. However, regulated gene expression in microalgae has only been achieved in a few strains due to the lack of genome information and unstable transformation. This study developed a species-specific transformation system for an oleaginous microalga, Ettlia sp. YC001, using electroporation. The electroporation was optimized using three parameters (waveform, field strength, and number of pulses), and the final selection was a 5 kV cm−1 field strength using an exponential decay wave with one pulse. A new strong endogenous promoter CRT (Pcrt) was identified using transcriptome and quantitative PCR analysis of highly expressed genes during the late exponential growth phase. The activities of this promoter were characterized using a codon optimized cyan fluorescent protein (CFP) as a reporter. The expression of CFP was similar under Pcrt and under the constitutive promoter psaD (PpsaD). The developed transformation system using electroporation with the endogenous promoter is simple to prepare, is easy to operate with high repetition, and utilizes a species-specific vector for high expression. This system could be used not only in molecular studies on microalgae but also in various industrial applications of microalgae.

Effects of NaCl and ethanol stresses on γ-aminobutyric acid synthesis in Kocuria kristinae

Kocuria kristinae, one of the dominant strains in Kedong sufu, can generate γ-aminobutyric acid (GABA). NaCl and ethanol are added to sufu for preservation. This study aims to reveal the effects of NaCl and ethanol stresses on GABA synthesis in K. kristinae. The results suggested that high GABA production was obtained by adding 6% NaCl at the mid-exponential growth phase and 3% ethanol at the late exponential growth phase. Two GABA synthesis pathways were observed, including a new GABA synthesis pathway. Under NaCl stress, genes were upregulated by 1.46- to 3.67-fold in two synthesis pathways of GABA. Under ethanol stress, genes were upregulated by 1.20- to 6.39-fold in two synthesis pathways of GABA, except for speG and MAO. However, their corresponding metabolites were downregulated by 1.02- to 7.69-fold. The changes in genes and metabolites in the carbohydrate and amino acid metabolism pathways and in membrane transport suggested that different metabolic strategies were used to cope with NaCl and ethanol stresses in K. kristinae. The molecular mechanism in improving the production of GABA by NaCl and ethanol stresses was shown in K. kristinae. This study provides a theoretical basis for the development of GABA-enriched sufu by NaCl and ethanol stresses.

Isolation and Characterization of a Novel Bacterium from the Marine Environment for Trichloroacetic Acid Bioremediation

Halogenated compounds are an important class of environmental pollutants that are widely used in industrial chemicals such as solvents, herbicides, and pesticides. Many studies have been carried out to explore the biodegradation of these chemicals. Trichloroacetic acid (TCA) is one of the main halogenated compounds that are carcinogenic to humans and animals. The bacterium was isolated from the northern coastline of Johor Strait. In this study, the ability of strain MH2 to biodegrade TCA was evaluated by a growth experiment and dehalogenase enzyme assay. The growth profile of the isolated strain was examined. The doubling time for L. boronitolerans MH2 was found to be 32 h. The release of chloride ion in the degradation process was measured at 0.33 × 10−3 ± 0.03 mol∙L−1 after 96 h when the growth curve had reached its maximum within the late bacterial exponential phase. The results showed that the strain had a promising ability to degrade TCA by producing dehalogenase enzyme when cell-free extracts were prepared from growth on TCA as the sole carbon source with enzyme-specific activity, 1.1 ± 0.05 µmolCl−min−1∙mg−1 protein. Furthermore, the morphological, and biochemical aspects of the isolated bacterium were studied to identify and characterize the strain. The morphological observation of the isolated bacterium was seen to be a rod-shaped, Gram-positive, motile, heterotrophic, and spore-forming bacterium. The amplification of the 16S rRNA and gene analysis results indicated that the isolated bacterium had 98% similarity to Lysinibacillus boronitolerans. The morphological and biochemical tests supported the 16S rRNA gene amplification. To the best of the authors’ knowledge, this is the first reported case of this genus of bacteria to degrade this type of halogenated compound.

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation, Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus

Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum, temperature extremes and a lack of water and nutrients. A wide variety of terrestrial microorganisms, including those counted amongst the most ancient inhabitants of Earth, can cope with high levels of salinity, extreme temperatures, desiccation and high levels of radiation. Key among these are the haloarchaea, considered particularly relevant for astrobiological studies due to their ability to thrive in hypersaline environments. In this study, a novel haloarchaea isolated from Urmia Salt Lake, Iran, Halovarius luteus strain DA50T, was exposed to varying levels of simulated extraterrestrial conditions and compared to that of the bacteria Bacillus atrophaeus. Bacillus atrophaeus was selected for comparison due to its well-described resistance to extreme conditions and its ability to produce strong spore structures. Thin films were produced to investigate viability without the protective influence of cell multi-layers. Late exponential phase cultures of Hvr. luteus and B. atrophaeus were placed in brine and phosphate buffered saline media, respectively. The solutions were allowed to evaporate and cells were encapsulated and exposed to radiation, desiccation and vacuum conditions, and their post-exposure viability was studied by the Most Probable Number method. The protein profile using High Performance Liquid Chromatography and Matrix Assisted Laser Desorption/Ionization bench top reflector time-of-flight are explored after vacuum and UV-radiation exposure. Results showed that the change in viability of the spore-forming bacteria B. atrophaeus was only minor whereas Hvr. luteus demonstrated a range of viability under different conditions. At the peak radiation flux of 105 J/m2 under nitrogen flow and after two weeks of desiccation, Hvr. luteus demonstrated the greatest decrease in viability. This study further expands our understanding of the boundary conditions of astrobiologically relevant organisms in the harsh space environment.