RESUMEN
Biodegradable plastics are urgently needed to replace petroleum-derived polymeric materials and prevent their accumulation in the environment. To this end, we isolated and characterized a halophilic and alkaliphilic bacterium from the Great Salt Lake in Utah. The isolate was identified as a Halomonas species and designated "CUBES01." Full-genome sequencing and genomic reconstruction revealed the unique genetic traits and metabolic capabilities of the strain, including the common polyhydroxyalkanoate (PHA) biosynthesis pathway. Fluorescence staining identified intracellular polyester granules that accumulated predominantly during the strain's exponential growth, a feature rarely found among natural PHA producers. CUBES01 was found to metabolize a range of renewable carbon feedstocks, including glucosamine and acetyl-glucosamine, as well as sucrose, glucose, fructose, and further glycerol, propionate, and acetate. Depending on the substrate, the strain accumulated up to ~60% of its biomass (dry wt/wt) in poly(3-hydroxybutyrate), while reaching a doubling time of 1.7 h at 30°C and an optimum osmolarity of 1 M sodium chloride and a pH of 8.8. The physiological preferences of the strain may not only enable long-term aseptic cultivation but also facilitate the release of intracellular products through osmolysis. The development of a minimal medium also allowed the estimation of maximum polyhydroxybutyrate production rates, which were projected to exceed 5 g/h. Finally, also, the genetic tractability of the strain was assessed in conjugation experiments: two orthogonal plasmid vectors were stable in the heterologous host, thereby opening the possibility of genetic engineering through the introduction of foreign genes. IMPORTANCE: The urgent need for renewable replacements for synthetic materials may be addressed through microbial biotechnology. To simplify the large-scale implementation of such bio-processes, robust cell factories that can utilize sustainable and widely available feedstocks are pivotal. To this end, non-axenic growth-associated production could reduce operational costs and enhance biomass productivity, thereby improving commercial competitiveness. Another major cost factor is downstream processing, especially in the case of intracellular products, such as bio-polyesters. Simplified cell-lysis strategies could also further improve economic viability.
Asunto(s)
Halomonas , Poliésteres , Halomonas/genética , Halomonas/metabolismo , Halomonas/crecimiento & desarrollo , Poliésteres/metabolismo , Polihidroxialcanoatos/metabolismo , Polihidroxialcanoatos/biosíntesis , Utah , Hidroxibutiratos/metabolismo , Plásticos Biodegradables/metabolismo , Lagos/microbiología , Genoma Bacteriano , PolihidroxibutiratosRESUMEN
Microbial instability is a common problem during bio-production based on microbial hosts. Halomonas bluephagenesis has been developed as a chassis for next generation industrial biotechnology (NGIB) under open and unsterile conditions. However, the hidden genomic information and peculiar metabolism have significantly hampered its deep exploitation for cell-factory engineering. Based on the freshly completed genome sequence of H. bluephagenesis TD01, which reveals 1889 biological process-associated genes grouped into 84 GO-slim terms. An enzyme constrained genome-scale metabolic model Halo-ecGEM was constructed, which showed strong ability to simulate fed-batch fermentations. A visible salt-stress responsive landscape was achieved by combining GO-slim term enrichment and CVT-based omics profiling, demonstrating that cells deploy most of the protein resources by force to support the essential activity of translation and protein metabolism when exposed to salt stress. Under the guidance of Halo-ecGEM, eight transposases were deleted, leading to a significantly enhanced stability for its growth and bioproduction of various polyhydroxyalkanoates (PHA) including 3-hydroxybutyrate (3HB) homopolymer PHB, 3HB and 3-hydroxyvalerate (3HV) copolymer PHBV, as well as 3HB and 4-hydroxyvalerate (4HB) copolymer P34HB. This study sheds new light on the metabolic characteristics and stress-response landscape of H. bluephagenesis, achieving for the first time to construct a long-term growth stable chassis for industrial applications. For the first time, it was demonstrated that genome encoded transposons are the reason for microbial instability during growth in flasks and fermentors.
Asunto(s)
Halomonas , Halomonas/genética , Halomonas/metabolismo , Halomonas/enzimología , Halomonas/crecimiento & desarrollo , Ingeniería Metabólica , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Redes y Vías Metabólicas/genética , Eliminación de Gen , Modelos BiológicosRESUMEN
The mutant strain Halomonas bluephagenesis (TDH4A1B5P) was found to produce PHA under low-salt, non-sterile conditions, but the yield was low. To improve the yield, different nitrogen sources were tested. It was discovered that urea was the most effective nitrogen source for promoting growth during the stable stage, while ammonium sulfate was used during the logarithmic stage. The growth time of H. bluephagenesis (TDH4A1B5P) and its PHA content were significantly prolonged by the presence of sulfate ions. After 64 hr in a 5-L bioreactor supplemented with sulfate ions, the dry cell weight (DCW) of H. bluephagenesis weighed 132 g/L and had a PHA content of 82%. To promote the growth and PHA accumulation of H. bluephagenesis (TDH4A1B5P), a feeding regimen supplemented with nitrogen sources and sulfate ions with ammonium sodium sulfate was established in this study. The DCW was 124 g/L, and the PHA content accounted for 82.3% (w/w) of the DCW, resulting in a PHA yield of 101 g/L in a 30-L bioreactor using the optimized culture strategy. In conclusion, stimulating H. bluephagenesis (TDH4A1B5P) to produce PHA is a feasible and suitable strategy for all H. bluephagenesis.
Asunto(s)
Reactores Biológicos , Medios de Cultivo , Halomonas , Nitrógeno , Polihidroxialcanoatos , Sulfatos , Halomonas/metabolismo , Halomonas/crecimiento & desarrollo , Halomonas/genética , Sulfatos/metabolismo , Polihidroxialcanoatos/metabolismo , Medios de Cultivo/química , Nitrógeno/metabolismo , Sulfato de Amonio/metabolismo , Urea/metabolismo , FermentaciónRESUMEN
Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform. KEY POINTS: A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed. Experiments and simulations predict N limitation in minimal media under aerobiosis. In silico media design increased experimental biomass yield and PHB titer.
Asunto(s)
Halomonas , Hidroxibutiratos , Nitrógeno , Poliésteres , Polihidroxibutiratos , Halomonas/metabolismo , Halomonas/genética , Halomonas/crecimiento & desarrollo , Nitrógeno/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Redes y Vías Metabólicas/genética , Biomasa , Glucosa/metabolismoRESUMEN
Poly-3-hydroxyalkanoic acids (PHAs) are bacterial storage polymers commonly used in bioplastic production. Halophilic bacteria are industrially interesting organisms, as their salinity tolerance and psychrophilic nature lowers sterility requirements and subsequent production costs. We investigated PHA synthesis in two bacterial strains, Halomonas sp. 363 and Paracoccus sp. 392, isolated from Southern Ocean sea ice and elucidated the related PHA biopolymer accumulation and composition with various approaches, such as transcriptomics, microscopy, and chromatography. We show that both bacterial strains produce PHAs at 4°C when the availability of nitrogen and/or oxygen limited growth. The genome of Halomonas sp. 363 carries three phaC synthase genes and transcribes genes along three PHA pathways (I to III), whereas Paracoccus sp. 392 carries only one phaC gene and transcribes genes along one pathway (I). Thus, Halomonas sp. 363 has a versatile repertoire of phaC genes and pathways enabling production of both short- and medium-chain-length PHA products. IMPORTANCE Plastic pollution is one of the most topical threats to the health of the oceans and seas. One recognized way to alleviate the problem is to use degradable bioplastic materials in high-risk applications. PHA is a promising bioplastic material as it is nontoxic and fully produced and degraded by bacteria. Sea ice is an interesting environment for prospecting novel PHA-producing organisms, since traits advantageous to lower production costs, such as tolerance for high salinities and low temperatures, are common. We show that two sea-ice bacteria, Halomonas sp. 363 and Paracoccus sp. 392, are able to produce various types of PHA from inexpensive carbon sources. Halomonas sp. 363 is an especially interesting PHA-producing organism, since it has three different synthesis pathways to produce both short- and medium-chain-length PHAs.
Asunto(s)
Halomonas/metabolismo , Cubierta de Hielo/microbiología , Paracoccus/metabolismo , Polihidroxialcanoatos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Frío , Genoma Bacteriano , Halomonas/genética , Halomonas/crecimiento & desarrollo , Halomonas/aislamiento & purificación , Paracoccus/genética , Paracoccus/crecimiento & desarrollo , Paracoccus/aislamiento & purificación , Filogenia , Polihidroxialcanoatos/química , Agua de Mar/microbiología , TemperaturaRESUMEN
OBJECTIVES: 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. RESULTS: 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/â³ectD/â³doeA) 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/â³ectD/â³doeA synthesized 11.5 g ectoine l-1 in the presence of 60 g NaCl-1 l, with a high 0.32 g ectoine l-1 h-1 productivity, a specific productivity of 512.2 mg ectoine per g cell dry weight (CDW)-1, and an excretion ratio of 67 % ectoine. CONCLUSIONS: As no impaired growth was observed in strain p/Y2/â³ectD/â³doeA 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.
Asunto(s)
Aminoácidos Diaminos/biosíntesis , Proteínas Bacterianas/genética , Técnicas de Cultivo Celular por Lotes/métodos , Halomonas/crecimiento & desarrollo , Proteínas de la Membrana Bacteriana Externa/genética , Reactores Biológicos/microbiología , Medios de Cultivo/química , Fermentación , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Ingeniería Genética , Halomonas/metabolismo , Regiones Promotoras Genéticas , Cloruro de Sodio/químicaRESUMEN
Ectoine, a compatible solute synthesized by many halophiles for hypersalinity resistance, has been successfully produced by metabolically engineered Halomonas bluephagenesis, which is a bioplastic poly(3-hydroxybutyrate) producer allowing open unsterile and continuous conditions. Here we report a de novo synthesis pathway for ectoine constructed into the chromosome of H. bluephagenesis utilizing two inducible systems, which serve to fine-tune the transcription levels of three clusters related to ectoine synthesis, including ectABC, lysC and asd based on a GFP-mediated transcriptional tuning approach. Combined with bypasses deletion, the resulting recombinant H. bluephagenesis TD-ADEL-58 is able to produce 28 g L-1 ectoine during a 28 h fed-batch growth process. Co-production of ectoine and PHB is achieved to 8 g L-1 ectoine and 32 g L-1 dry cell mass containing 75% PHB after a 44 h growth. H. bluephagenesis demonstrates to be a suitable co-production chassis for polyhydroxyalkanoates and non-polymer chemicals such as ectoine.
Asunto(s)
Aminoácidos Diaminos/biosíntesis , Halomonas/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Biomasa , Vías Biosintéticas/genética , Cromatografía Liquida/métodos , Halomonas/genética , Halomonas/crecimiento & desarrollo , Ingeniería Metabólica/métodos , Polihidroxialcanoatos/química , Polihidroxialcanoatos/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
A systematic study on the lack of dissimilatory nitrate reductase (NAR) properties in Halomonas strains had been reported so far. The effects of different factors on Halomonas sp. B01 NAR activity were investigated. The salt tolerance of NAR was characterized. The denitrification process under high salt conditions was reported. Halomonas sp. B01 expressed membrane-bound NAR under induced culture by nitrate. The optimum pH of the enzyme reaction system was 8, and the optimum temperature was 30 °C. The mRNA expression abundance of narH in NAR encoding gene was highest in the 60 g/L NaCl inducing matrix. The NaCl concentration of optimum growth and induction of NAR were both 60 g/L. The ectoine added to the NAR vitro enzyme reaction system could maintain NAR activity under high NaCl concentration. In the range of 0-60 g/L NaCl, the NAR activity was stable at 17.7 (± 0.3) U/mg. The denitrification was performed by Halomonas sp. B01 at 60 g/L NaCl, and the denitrification rate reached 97.1% at 24 h. This study reveals for the first time the NAR properties of Halomonas strains, which provides a theoretical and technical basis for the nitrogen removal of high-salt nitrogenous wastewater using this strain.
Asunto(s)
Proteínas Bacterianas/metabolismo , Halomonas/enzimología , Nitrato-Reductasa/metabolismo , Tolerancia a la Sal , Aminoácidos Diaminos/metabolismo , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Desnitrificación , Regulación Bacteriana de la Expresión Génica , Halomonas/genética , Halomonas/crecimiento & desarrollo , Halomonas/metabolismo , Concentración de Iones de Hidrógeno , Nitrato-Reductasa/genética , Nitratos/metabolismo , Cloruro de Sodio/metabolismo , TemperaturaRESUMEN
Halomonas TD01, which can grow under non-sterile and continuous processes at high pH and high salt concentrations, is a robust platform for PHA production from glucose. For extending other low-cost sustainable substrates and increasing the potential application in other value-added products, a better understanding of substrates utilization and chemicals tolerance is necessary. In this study, the substrate profiling of TD01 was analyzed via Biolog. Phenotype microarray results demonstrated that TD01 has a wide-ranging substrate spectrum and can utilize 140 of the 190 test compounds. Some cheap, abundant carbon sources, such as sodium acetate, glycerol, ethanol and lactate can well support the growth of TD01 in shake-flask, and are therefore suggested to be its alternative low-cost substrates for chemicals production in future. Furthermore, the tolerance of TD01 to various chemicals was tested. The results showed that the tolerability of TD01 to high concentrations of organic acid salts is prominent. When adding 75â¯g/L sodium acetate, 100â¯g/L succinic acid and 100â¯g/L itaconic acid in the medium, the growth rate reduced 56.14%, 52.63% and 47.37%, respectively. All these results highlight TD01 as a promising, next generation industrial workhorse in chemicals biomanufacturing from cheap organic acid salts.
Asunto(s)
Halomonas/metabolismo , Ingeniería Metabólica/métodos , Carbohidratos/farmacología , Ácidos Carboxílicos/farmacología , Halomonas/efectos de los fármacos , Halomonas/crecimiento & desarrollo , Polihidroxialcanoatos/metabolismoRESUMEN
The optimum condition at which the halophilic salt-tolerant bacterium Halomonas variabilis (MTCC 3712) produces the maximum amount of extracellular polymeric substances (EPS) was investigated experimentally using response surface methodology based on the central composite design (CCD). Hyper-saline medium containing 1.5% w/v NaCl enriched nutrient medium with 1.5% glucose as a carbon source was used to produce about 4.74 g/L of EPS in 16 h compared to various other EPS production of this kind. The metabolic heat profile confirms net EPS production by HV was a growth-associated aerobic process. There is a good agreement between metabolic heat and Oxygen Uptake Rate (OUR). The maximum observed heat release was 2.1 W. The total protein content of the sample is 53% of the total EPS (Soluble EPS, Loosely bound EPS, and tightly bound EPS). The emulsifying and flocculating activities of the EPS were measured to explore the possibility of using the biopolymer for effluent treatment.
Asunto(s)
Matriz Extracelular de Sustancias Poliméricas/metabolismo , Halomonas/crecimiento & desarrollo , Reactores Biológicos , Glucosa/metabolismo , Halomonas/metabolismo , Calor , Oxígeno/metabolismo , Tolerancia a la Sal , Cloruro de Sodio/metabolismoRESUMEN
In most halophiles, K+ generally acts as a major osmotic solute for osmotic adjustment and pH homeostasis. However, strains also need to extrude excessive intracellular K+ to avoid its toxicity. In the halotolerant and alkaliphilic Halomonas sp. Y2, an Na+-induced K+ extrusion process was observed when the cells were confronted with high extracellular K+ pressure and supplementation by millimolar Na+ ions. Among three mechanosensitive channels (KefA) and two K+/H+ antiporters founded in the genome of the strain, ke1 displayed around 3-5-fold upregulation to ion stress at pH 8.0, while much higher upregulation of Ha-mrp was observed at pH 10.0. Compared to the growth of wild-type Halomonas sp. Y2, deletion of these genes from the strain resulted in different growth phenotypes in response to the osmotic pressure of potassium. In combination with the transcriptional response of these genes, we proposed that the KefA channel of Ke1 is the main contributor to the K+-extrusion process under weak alkalinity, while the Mrp system plays critical roles in alleviating K+ contents at high pH. The combination of these strategies allows Halomonas sp. Y2 to grow over a range of extracellular pH and ion concentrations, and thus protect cells under high osmotic stress conditions.
Asunto(s)
Halomonas/fisiología , Potasio/metabolismo , Sodio/metabolismo , Proteínas Bacterianas/genética , Medios de Cultivo/química , Perfilación de la Expresión Génica , Halomonas/efectos de los fármacos , Halomonas/genética , Halomonas/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Presión Osmótica , Potasio/farmacología , Canales de Potasio/genética , Antiportadores de Potasio-Hidrógeno/genética , Eliminación de Secuencia , Sodio/análisis , Intercambiadores de Sodio-Hidrógeno/genéticaRESUMEN
For the first time, we propose the use of an extremophilic bacterium to remove nitrate salt efflorescence from the surfaces of stone samples. A haloalkaliphilic bacterium was selected "ad hoc" for its ability to reduce nitrates; i.e. Halomonas campaniensis sp. nov., strain 5AGT (DSM 15293T, ATCC BAA-966T). Quantitative monitoring of nitrate content, on untreated and treated surfaces of stone samples artificially enriched with nitrate, as a function of incubation/treatment time, was carried out by molecular spectroscopy. The results obtained reveal the good performance of Halomonas campaniensis bacterium in decreasing nitrate concentration on stone surfaces both in a controlled laboratory environment for temperature and relative humidity and in a real outdoor environmental conditions.
Asunto(s)
Biodegradación Ambiental , Materiales de Construcción , Halomonas/crecimiento & desarrollo , Nitratos/aislamiento & purificación , Nitratos/metabolismo , Dióxido de Silicio/química , Biotecnología , Contaminantes Ambientales/aislamiento & purificación , Contaminantes Ambientales/metabolismo , Halomonas/metabolismoRESUMEN
Halomonas has been developed as a platform for the next generation industrial biotechnology allowing open and nonsterile growth without microbial contamination under a high-salt concentration and alkali pH. To reduce downstream cost associated with continuous centrifugation and salt containing wastewater treatment, Halomonas campaniensis strain LS21 was engineered to become self-flocculating by knocking out an etf operon encoding two subunits of an electron transferring flavoprotein in the predicted electron transfer chain. Self-flocculation could be attributed to the decrease of the surface charge and increase of the cellular hydrophobicity resulted from deleted etf. A wastewaterless fermentation strategy based on the self-flocculating H. campaniensis was developed for growth and the production of poly-3-hydroxybutyrate (PHB) as an example. Most microbial cells flocculated and precipitated to the bottom of the bioreactor within 1 min after stopping the aeration and agitation. The supernatant can be used again without sterilization or inoculation for the growth of the next batch after collecting the precipitated cell mass. The wastewaterless process was conducted for four runs without generating wastewater. PHB accumulation by the self-flocculent strain was enhanced via promoter and ribosome binding site optimizations, the productivities of cell dry weight and PHB were increased from 0.45 and 0.18 g·L -1 ·hr -1 for the batch process compared to 0.82 and 0.33 g·L -1 ·hr -1 for the wastewaterless continuous process, respectively. This has clearly demonstrated the advantages of the wastewaterless process in that it not only reduces wastewater but also increases cell growth and product formation efficiency in a given period of time.
Asunto(s)
Fermentación , Halomonas/metabolismo , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Aguas Residuales/microbiología , Reactores Biológicos/microbiología , Ingeniería Celular/métodos , Floculación , Halomonas/genética , Halomonas/crecimiento & desarrollo , Microbiología Industrial/métodos , Aguas Residuales/análisis , Purificación del Agua/métodosRESUMEN
Traditional microbial chassis, including Escherichia coli, Bacillus subtilis, Ralstonia eutropha, and Pseudomonas putida, are grown under neutral pH and mild osmotic pressure for production of chemicals and materials. They tend to be contaminated easily by many microorganisms. To address this issue, next-generation industrial biotechnology employing halophilic Halomonas spp. has been developed for production of bioplastics polyhydroxyalkanoates (PHAs) and other chemicals. Halomonas spp. that can be grown contamination free under open and unsterile condition at alkali pH and high NaCl have been engineered to produce several PHA polymers in elongated or enlarged cells. New pathways can also be constructed both in plasmids and on chromosomes for Halomonas spp. Synthetic biology approaches and parts have been developed for Halomonas spp., allowing better control of their growth and product formation as well as morphology adjustment. Halomonas spp. and their synthetic biology will play an increasingly important role for industrial production of large volume chemicals.
Asunto(s)
Halomonas/genética , Microbiología Industrial/métodos , Ingeniería Metabólica/métodos , Polihidroxialcanoatos/genética , Vías Biosintéticas , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , Fermentación , Halomonas/citología , Halomonas/crecimiento & desarrollo , Halomonas/metabolismo , Polihidroxialcanoatos/metabolismo , Biología Sintética/métodosRESUMEN
Levan polysaccharide is an industrially important natural polymer with unique properties and diverse high-value applications. However, current bottlenecks associated with its large-scale production need to be overcome by innovative approaches leading to economically viable processes. Besides many mesophilic levan producers, halophilic Halomonas smyrnensis cultures hold distinctive industrial potential and, for the first time with this study, the advantage of halophilicity is used and conditions for non-sterile levan production were optimized. Levan productivity of Halomonas cultures in medium containing industrial sucrose from sugar beet and food industry by-product syrup, a total of ten sea, lake and rock salt samples from four natural salterns, as well as three different industrial-grade boron compounds were compared and the most suitable low-cost substitutes for sucrose, salt and boron were specified. Then, the effects of pH control, non-sterile conditions and different bioreactor modes (batch and fed-batch) were investigated. The development of a cost-effective production process was achieved with the highest yield (18.06 g/L) reported so far on this microbial system, as well as the highest theoretical bioconversion efficiency ever reported for levan-producing suspension cultures. Structural integrity and biocompatibility of the final product were also verified in vitro.
Asunto(s)
Fructanos/química , Fructanos/aislamiento & purificación , Halomonas/química , Reactores Biológicos , Halomonas/crecimiento & desarrolloRESUMEN
In this study, polyhydroxyalkanoates (PHA) accumulation by Halomonas venusta KT832796, a moderate halophilic bacteria isolated from marine source was studied. Both nutritional requirements and process parameters for submerged cultivation of the organism in bioreactor have been standardized. From the shake flask studies, glucose and ammonium citrate as carbon and nitrogen source produced maximum PHA at a ratio 20 with 3.52 g/L of dry cell weight and 70.56% of PHA content. However, ammonium sulfate as the nitrogen source was found to be more suitable for fed-batch cultivation. Several feeding strategies including pH-based fed-batch and variants of pulse feeding were studied to improve the PHA levels. pH-based feeding, although improved PHA level to 26 g/L, most of the carbon flux was diverted towards biomass formation; hence, the percent PHA was only 39.15% of the dry cell weight. Maximum PHA of 33.4 g/L, which corresponded to 88.12% of the dry cell, was obtained from high concentration single pulse method. There was a net 8.65-fold increase in PHA using this feeding strategy when compared to batch studies. According to our knowledge, this is the highest amount of PHA reported for a Halomonas venusta strain.
Asunto(s)
Reactores Biológicos , Halomonas/crecimiento & desarrollo , Polihidroxialcanoatos/biosíntesis , Polihidroxialcanoatos/química , Polihidroxialcanoatos/aislamiento & purificaciónRESUMEN
Biological removal of chromate [Cr(VI)] in the presence or absence of nitrate by granular sludge biofilms was investigated in batch experiments and in a sequencing batch reactor (SBR). Denitrifying granular sludge cultivated from activated sludge was able to directly reduce Cr(VI) in the presence of an electron donor. Bioreduction was dependent on the initial Cr(VI) and the granular sludge concentrations. Bioreduction of Cr(VI) was followed by Cr(III) precipitation or entrapment in the granular sludge which was corroborated with decrease in total soluble Cr and increase in inorganic content of biomass. Batch experiments revealed that Cr(VI) addition has no major influence on high-strength nitrate (3000 mg L-1) denitrification, but nitrite denitrification was slowed-down. However, SBR experiment demonstrated successful denitrification as well as Cr(VI) removal due to enrichment of Cr(VI)-tolerant denitrifying bacteria. In fact, stable SBR performance in terms of complete and sustained removal of 0.05, 0.1, 0.2, 0.3, 0.5 and 0.75 mM Cr(VI) and denitrification of 3000 mg L-1 was observed during 2 months of operation. Active biomass and electron donor-dependent Cr(VI) removal, detection of Cr(III) in the biomass and recovery of ~ 92% of the Cr from the granular sludge biofilms confirms bioreduction followed by precipitation or entrapment of Cr(III) as the principal chromate removal mechanism. Metagenomic bacterial community analysis showed enrichment of Halomonas sp. in denitrifying granular sludge performing either denitrification or simultaneous reduction of nitrate and chromate.
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Reactores Biológicos/microbiología , Cromatos/análisis , Nitratos/análisis , Aguas del Alcantarillado/microbiología , Contaminantes Químicos del Agua/análisis , Purificación del Agua/métodos , Biomasa , Desnitrificación , Halomonas/genética , Halomonas/crecimiento & desarrollo , Metagenoma , Consorcios Microbianos/genética , Oxidación-ReducciónRESUMEN
Studies on the halotolerance of bacteria are attractive to the fermentation industry. However, a lack of sufficient genomic information has precluded an investigation of the halotolerance of Halomonas beimenensis. Here, we describe the molecular mechanisms of saline adaptation in H. beimenensis based on high-throughput omics and Tn5 transposon mutagenesis. The H. beimenensis genome is 4.05 Mbp and contains 3,807 genes, which were sequenced using short and long reads obtained via deep sequencing. Sixteen Tn5 mutants with a loss of halotolerance were identified. Orthologs of the mutated genes, such as nqrA, trkA, atpC, nadA, and gdhB, have significant biological functions in sodium efflux, potassium uptake, hydrogen ion transport for energy conversion, and compatible solute synthesis, which are known to control halotolerance. Other genes, such as spoT, prkA, mtnN, rsbV, lon, smpB, rfbC, rfbP, tatB, acrR1, and lacA, function in cellular signaling, quorum sensing, transcription/translation, and cell motility also shown critical functions for promoting a halotolerance. In addition, KCl application increased halotolerance and potassium-dependent cell motility in a high-salinity environment. Our results demonstrated that a combination of omics and mutagenesis could be used to facilitate the mechanistic exploitation of saline adaptation in H. beimenensis, which can be applied for biotechnological purposes.
Asunto(s)
Adaptación Fisiológica , Elementos Transponibles de ADN/genética , Genómica/métodos , Halomonas/genética , Halomonas/fisiología , Mutagénesis/genética , Salinidad , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Mapeo Cromosómico , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genoma Bacteriano , Halomonas/citología , Halomonas/crecimiento & desarrollo , Mutación/genética , Fenotipo , Filogenia , Potasio/farmacología , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
In living systems, environmental stress due to biotic and abiotic factors triggers the production of myriad metabolites as a potential mechanism for combating stress. Among these metabolites are the small polycationic aliphatic amine molecules - polyamines, which are ubiquitous in all living organisms. In this work, we demonstrate a correlation between cellular concentration of three major polyamines (putrescine, spermidine and spermine) with lead exposure on bacteria for a period of 6-24 h. We report that indigenously isolated Halomonas sp. strain BVR 1 exhibits lead induced fluctuations in their cellular polyamine concentration. This response to lead occurs within 6 h post metal treatment. During the same time interval there was a surge in the growth of bacteria along with an enhancement in the putrescine levels. We conclude that in Halomonas sp. strain BVR 1, an early response is seen with respect to modulation of polyamines as a result of lead treatment and hypothesize that endogenous polyamines contribute towards scavenging lead in these bacteria.
Asunto(s)
Halomonas/metabolismo , Plomo/metabolismo , Metales Pesados/metabolismo , Poliaminas/metabolismo , Estrés Fisiológico , Microbiología Ambiental , Halomonas/efectos de los fármacos , Halomonas/crecimiento & desarrollo , Plomo/toxicidad , Metales Pesados/toxicidad , Viabilidad Microbiana/efectos de los fármacos , Poliaminas/análisis , Análisis EspectralRESUMEN
We previously reported that Synechococcus elongatus PCC 7942, engineered with the sucrose transporter CscB, can export up to 85% of its photosynthetically-fixed carbon as sucrose and shows considerable promise as an alternative carbohydrate source. One approach to effectively utilize this cyanobacterium is to generate synthetic, light-driven consortia in which sucrose-metabolizing heterotrophs catalyze the conversion of the low-value carbohydrate into higher-value compounds in co-culture. Here, we report an improved synthetic photoautotroph/chemoheterotroph consortial design in which sucrose secreted by S. elongatus CscB directly supports the bacterium Halomonas boliviensis, a natural producer of the bioplastic precursor, PHB. We show that alginate encapsulation of S. elongatus CscB enhances sucrose-export rates ~2-fold within 66h, to ~290mg sucrose L-1d-1 OD750-1 and enhances the co-culture stability. Consortial H. boliviensis accumulate up to 31% of their dry-weight as PHB, reaching productivities up to 28.3mg PHB L-1d-1. This light-driven, alginate-partitioned co-culture platform achieves PHB productivities that match or exceed those of traditionally engineered cyanobacterial monocultures. Importantly, S. elongatus CscB/H. boliviensis co-cultures were continuously productive for over 5 months and resisted invasive microbial species without the application of antibiotics or other chemical selection agents.