Analysis of the microbiome of the Bolivian high-altitude Lake Pastos Grandes.

Lake Pastos Grandes in Bolivia is mainly composed of salt flats, which are sporadically and only partially submerged during the wet season. In the present study, the chemical composition of water samples of the lake and some influent rivers was determined. We found that it is likely that the lake was influenced by the dilution of metals from ancient evaporites. We performed the first metagenomic studies of this lake. Analyses of shotgun metagenomics revealed that the relative abundances of Burkholderiales and Pseudomonadales were noteworthy in the water samples, whereas the archaea belonging to the Halobacteriales and Cyanobacteria from subsection III had high abundances in the salt flat. The eukaryotes Crustacea and Diatomea exhibited the highest abundances in the water samples. We investigated further the potential effect of human activities on the nitrogen cycle mobilization in the lake and the propagation of antimicrobial resistance genes. This is the first report about the cycle in the lake. Additionally, rifamycin resistance genes and genes related to efflux pumps, which are not considered a hazard when identified in metagenomes, had the uppermost relative abundances in all sampling points. We found that Lake Pastos Grandes hitherto does not show an appreciable influence by anthropogenic actions.

The microbiome of a polluted urban lake harbors pathogens with diverse antimicrobial resistance and virulence genes.

Bacterial resistance to antibiotics is one of the greatest threats to the modern human population. Paradoxically, urban settlements are often culpable in generating such resistance by influencing the adaptation of bacterial communities via pollution of natural ecosystems. Urban lakes are well-known examples of this problem, as they often receive discharges of both domestic and industrial wastewater. In this study, we used shotgun metagenome sequencing to examine the microbial diversity of water and sediment samples of Lake Alalay, a polluted urban lake near Cochabamba, Bolivia. We found that Proteobacteria dominated the relative abundance of both water and sediment samples at levels over 25% and that a significant proportion of the microbial diversity could not be classified (about 9% in water and 22% in sediment). Further metagenomic investigation of antimicrobial resistance (AR) genes identified 277 and 150 AR genes in water and sediment samples, respectively. These included genes with functional annotations for resistance to fluoroquinolones, tetracyclines, phenicols, macrolides, beta-lactams, and rifamycin. A high number of genes involved in bacterial virulence also occurred in both water and sediment samples (169 and 283, respectively), where the virulence gene pscP normally found in the Pseudomonas aeruginosa type III secretion system had the highest relative abundance. Isolated and identified bacteria from water samples also revealed the presence of pathogenic bacteria among the microbiota of Lake Alalay. Seeing as most AR and virulence genes detected in this study are commonly described in nosocomial infections, we provide evidence suggesting that the microbial ecosystem of Lake Alalay presents a severe health risk to the surrounding population.

Comparison of engineered Escherichia coli AF1000 and BL21 strains for (R)-3-hydroxybutyrate production in fed-batch cultivation.

Accumulation of acetate is a limiting factor in recombinant production of (R)-3-hydroxybutyrate (3HB) by Escherichia coli in high-cell-density processes. To alleviate this limitation, this study investigated two approaches: (i) deletion of phosphotransacetylase (pta), pyruvate oxidase (poxB), and/or the isocitrate lyase regulator (iclR), known to decrease acetate formation, on bioreactor cultivations designed to achieve high 3HB concentrations. (ii) Screening of different E. coli strain backgrounds (B, BL21, W, BW25113, MG1655, W3110, and AF1000) for their potential as low acetate-forming, 3HB-producing platforms. Deletion of pta and pta-poxB in the AF1000 strain background was to some extent successful in decreasing acetate formation, but also dramatically increased excretion of pyruvate and did not result in increased 3HB production in high-cell-density fed-batch cultivations. Screening of the different E. coli strains confirmed BL21 as a low acetate-forming background. Despite low 3HB titers in low-cell-density screening, 3HB-producing BL21 produced five times less acetic acid per mole of 3HB, which translated into a 2.3-fold increase in the final 3HB titer and a 3-fold higher volumetric 3HB productivity over 3HB-producing AF1000 strains in nitrogen-limited fed-batch cultivations. Consequently, the BL21 strain achieved the hitherto highest described volumetric productivity of 3HB (1.52 g L-1 h-1) and the highest 3HB concentration (16.3 g L-1) achieved by recombinant E. coli. Screening solely for 3HB titers in low-cell-density batch cultivations would not have identified the potential of this strain, reaffirming the importance of screening with the final production conditions in mind.

The role of the acyl-CoA thioesterase "YciA" in the production of (R)-3-hydroxybutyrate by recombinant Escherichia coli.

Biotechnologically produced (R)-3-hydroxybutyrate is an interesting pre-cursor for antibiotics, vitamins, and other molecules benefitting from enantioselective production. An often-employed pathway for (R)-3-hydroxybutyrate production in recombinant E. coli consists of three-steps: (1) condensation of two acetyl-CoA molecules to acetoacetyl-CoA, (2) reduction of acetoacetyl-CoA to (R)-3-hydroxybutyrate-CoA, and (3) hydrolysis of (R)-3-hydroxybutyrate-CoA to (R)-3-hydroxybutyrate by thioesterase. Whereas for the first two steps, many proven heterologous candidate genes exist, the role of either endogenous or heterologous thioesterases is less defined. This study investigates the contribution of four native thioesterases (TesA, TesB, YciA, and FadM) to (R)-3-hydroxybutyrate production by engineered E. coli AF1000 containing a thiolase and reductase from Halomonas boliviensis. Deletion of yciA decreased the (R)-3-hydroxybutyrate yield by 43%, whereas deletion of tesB and fadM resulted in only minor decreases. Overexpression of yciA resulted in doubling of (R)-3-hydroxybutyrate titer, productivity, and yield in batch cultures. Together with overexpression of glucose-6-phosphate dehydrogenase, this resulted in a 2.7-fold increase in the final (R)-3-hydroxybutyrate concentration in batch cultivations and in a final (R)-3-hydroxybutyrate titer of 14.3 g L-1 in fed-batch cultures. The positive impact of yciA overexpression in this study, which is opposite to previous results where thioesterase was preceded by enzymes originating from different hosts or where (S)-3-hydroxybutyryl-CoA was the substrate, shows the importance of evaluating thioesterases within a specific pathway and in strains and cultivation conditions able to achieve significant product titers. While directly relevant for (R)-3-hydroxybutyrate production, these findings also contribute to pathway improvement or decreased by-product formation for other acyl-CoA-derived products.

Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply.

BACKGROUND: In a recently discovered microorganism, Halomonas boliviensis, polyhydroxybutyrate production was extensive and in contrast to other PHB producers, contained a set of alleles for the enzymes of this pathway. Also the monomer, (R)-3-hydroxybutyrate (3HB), possesses features that are interesting for commercial production, in particular the synthesis of fine chemicals with chiral specificity. Production with a halophilic organism is however not without serious drawbacks, wherefore it was desirable to introduce the 3HB pathway into Escherichia coli. RESULTS: The production of 3HB is a two-step process where the acetoacetyl-CoA reductase was shown to accept both NADH and NADPH, but where the V max for the latter was eight times higher. It was hypothesized that NADPH could be limiting production due to less abundance than NADH, and two strategies were employed to increase the availability; (1) glutamate was chosen as nitrogen source to minimize the NADPH consumption associated with ammonium salts and (2) glucose-6-phosphate dehydrogenase was overexpressed to improve NADPH production from the pentose phosphate pathway. Supplementation of glutamate during batch cultivation gave the highest specific productivity (q3HB = 0.12 g g(-1) h(-1)), while nitrogen depletion/zwf overexpression gave the highest yield (Y3HB/CDW = 0.53 g g(-1)) and a 3HB concentration of 1 g L(-1), which was 50% higher than the reference. A nitrogen-limited fedbatch process gave a concentration of 12.7 g L(-1) and a productivity of 0.42 g L(-1) h(-1), which is comparable to maximum values found in recombinant E. coli. CONCLUSIONS: Increased NADPH supply is a valuable tool to increase recombinant 3HB production in E. coli, and the inherent hydrolysis of CoA leads to a natural export of the product to the medium. Acetic acid production is still the dominating by-product and this needs attention in the future to increase the volumetric productivity further.

Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation.

The chiral compound (R)-3-hydroxybutyrate (3HB) is naturally produced by many wild type organisms as the monomer for polyhydroxybutyrate (PHB). Both compounds are commercially valuable and co-polymeric polyhydroxyalkanoates have been used e.g., in medical applications for skin grafting and as components in pharmaceuticals. In this paper we investigate cultivation strategies for production of 3HB in the previously described E. coli strain AF1000 pJBGT3RX. This strain produces extracellular 3HB by expression of two genes from the PHB pathway of Halomonas boliviensis. H. boliviensis is a newly isolated halophile that forms PHB as a storage compound during carbon excess and simultaneous limitation of another nutrient like nitrogen and phosphorous. We hypothesize that a similar approach can be used to control the flux from acetyl-CoA to 3HB also in E. coli; decreasing the flux to biomass and favoring the pathway to the product. We employed ammonium- or phosphate-limited fed-batch processes for comparison of the productivity at different nutrient limitation or starvation conditions. The feed rate was shown to affect the rate of glucose consumption, respiration, 3HB, and acetic acid production, although the proportions between them were more difficult to affect. The highest 3HB volumetric productivity, 1.5 g L(-1) h(-1), was seen for phosphate-limitation.

Production of poly(3-hydroxybutyrate) by Halomonas boliviensis in an air-lift reactor.

BACKGROUND: Microbial polyesters, also known as polyhydroxyalkanoates (PHAs), closely resemble physical and mechanical features of petroleum derived plastics. Recombinant Escherichia coli strains are being used in industrial production of PHAs in Stirred Tank Bioreactors (STRs). However, use of Air-Lift Reactors (ALRs) has been known to offer numerous technical operating options over STRs, and as such has been successfully implemented in many bioprocesses. Halomonas boliviensis is a halophilic bacterium that is known to assimilate various carbohydrates and convert them into a particular type of PHA known as poly(3-hydroxybutyrate) (PHB). Owing to this capability, it has been used to synthesize the polyester using hydrolysates of starch or wheat bran in stirred tank bioreactors. RESULTS: This research article firstly describes the production of PHB in shake flasks by H. boliviensis using different combinations of carbohydrates and partially hydrolyzed starch as carbon sources. The highest PHB yields, between 56 and 61 % (wt.), were achieved when either starch hydrolysate or a mixture of glucose and xylose were used as carbon sources. The starch hydrolysate obtained in this study was then used as carbon source in an ALR. The largest amount of PHB, 41 % (wt.), was attained after 24 hrs of cultivation during which maltose in the hydrolysate was assimilated more rapidly than glucose during active cell growth; however, the rate of assimilation of both the carbohydrates was found to be similar during synthesis of PHB. An incomplete pentose phosphate pathway, which lacks 6-phosphogluconate dehydrogenase, was deduced from the genome sequence of this bacterium and may result in the characteristic assimilation of glucose and maltose by the cells. CONCLUSIONS: This study showed that the production of PHB by H. boliviensis using cheap substrates such as starch hydrolysate in a simple production system involving an ALR is feasible. Both maltose and glucose in the hydrolysate induce cell growth and PHB synthesis; most likely the cells balance adequately CoA and NAD(P)H during the assimilation of these carbohydrates. The combination of cheap substrates, simple production systems and the use of non-strict sterile conditions by the halophile H. boliviensis are desirable traits for large scale production of PHB, and should lead to a competitive bioprocess.

Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli.

BACKGROUND: Lignocellulosic waste is a desirable biomass for use in second generation biorefineries. Up to 40% of its sugar content consist of pentoses, which organisms either take up sequentially after glucose depletion, or not at all. A previously described Escherichia coli strain, PPA652ara, capable of simultaneous consumption of glucose, xylose and arabinose was in the present work utilized for production of (R)-3-hydroxybutyric acid (3HB) from a mixture of glucose, xylose and arabinose. RESULTS: The Halomonas boliviensis genes for 3HB production were for the first time cloned into E. coli PPA652ara, leading to product secretion directly into the medium. Process design was based on comparisons of batch, fed-batch and continuous cultivation, where both excess and limitation of the carbon mixture was studied. Carbon limitation resulted in low specific productivity of 3HB (<2 mg g(-1) h(-1)) compared to carbon excess (25 mg g(-1) h(-1)), but the yield of 3HB/cell dry weight (Y3HB/CDW) was very low (0.06 g g(-1)) during excess. Nitrogen-exhausted conditions could be used to sustain a high specific productivity (31 mg g(-1) h(-1)) and to increase the yield of 3HB/cell dry weight to 1.38 g g(-1). Nitrogen-limited fed-batch process design led to further increased specific productivity (38 mg g(-1) h(-1)) but also to additional cell growth (Y3HB/CDW=0.16 g g(-1)). Strain PPA652ara did under all processing conditions simultaneously consume glucose, xylose and arabinose, which was not the case for a reference wild type E. coli, which also gave a higher carbon flux to acetic acid. CONCLUSIONS: It was demonstrated that by using E. coli PPA652ara, it was possible to design a production process for 3HB from a mixture of glucose, xylose and arabinose where all sugars were consumed. An industrial 3HB production process is proposed to be divided into a growth and a production phase, and nitrogen depletion/limitation is a potential strategy to maximize the yield of 3HB/CDW in the latter. The specific productivity of 3HB reported here from glucose, xylose and arabinose by E. coli is further comparable to the current state of the art for production from glucose sources.

Genomic studies on nitrogen metabolism in Halomonas boliviensis: metabolic pathway, biochemistry and evolution.

Halomonas boliviensis LC1(T)=DSM 15516(T) is a halophilic bacterium that copiously produces osmolytes and polyesters. The growth of H. boliviensis is restricted when glutamate or glutamine is not included in its culture medium. The concentration of glutamate in the medium can regulate the production of either osmolytes or polyesters. However, genomic studies on the nitrogen assimilation have not been performed on H. boliviensis and other members of the family Halomonadaceae. Glutamate metabolism in H. boliviensis was discerned based on genome sequence analysis. The genome sequences of other Halomonadaceae members revealed similar enzymes to those found in H. boliviensis. H. boliviensis and H. elongata DSM 2581(T) acquired distinct glutamate dehydrogenase genes through horizontal gene transfer from a different bacterium. Two alleles of glutamine synthetase could be found in H. boliviensis, one of which was obtained from a thermophilic archaeon via horizontal gene transfer. Two subunits of glutamate synthase were also present in H. boliviensis. The small ß-subunit had a molecular weight of 52 kDa and was phylogenetically closely affiliated to proteins of other halomonads and Gammaproteobacteria. The large (161 kDa) α-subunit of the halomonads gathered in a separate phylogenetic group, hence glutamate synthase α-subunits of halomonads may be included a novel group of enzymes. Furthermore, putative enzymes obtained from the genome of H. boliviensis should permit complete glutamate metabolism. A similar metabolism should be followed by other halomonads. However, some phenotypic differences between halomonads, such as the ability to assimilate ammonia, resulted as a consequence of horizontal gene transfer. Each enzyme that forms part of the glutamate metabolism in prokaryotes evolved following a different pattern. Yet, most enzymes of halomonads diverged in phylogenetic clusters composed of Proteobacteria, as might be expected.

Marinococcus tarijensis sp. nov., a moderately halophilic bacterium isolated from a salt mine.

A Gram-stain-positive, coccoid-shaped, halophilic bacterium, strain SR-1(T), was isolated from a salt crystal obtained from a mine located in Tarija, Bolivia. The strain was investigated using a polyphasic approach. The optimum conditions for growth of strain SR-1(T) were reached at 5% (w/v) NaCl, pH 7.6 and 37-40 °C. The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The isoprenoid quinone was MK-7. The major cellular fatty acids of strain SR-1(T) were anteiso-C(15:0), anteiso-C(17:0) and iso-C(16:0). The DNA G+C content of strain SR-1(T) was 48.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed a close relationship between strain SR-1(T) and Marinococcus halophilus JCM 2479(T) (99.7% 16S rRNA gene sequence similarity), Marinococcus halotolerans KCTC 19045(T) (99.4%) and Marinococcus luteus KCTC 13214(T) (99.8%). However, strain SR-1(T) also showed low levels of DNA-DNA relatedness with these reference strains (47, 61 and 58%, respectively). On the basis of phenotypic differences and DNA-DNA hybridization results, strain SR-1(T) is considered to represent a novel species of the genus Marinococcus, for which the name Marinococcus tarijensis sp. nov. is proposed. The type strain is SR-1(T) ( =LMG 26930(T) =CECT 8130(T)).

Polyester production by halophilic and halotolerant bacterial strains obtained from mangrove soil samples located in Northern Vietnam.

This research article reports halophilic and halotolerant bacteria isolated from mangrove forests located in Northern Vietnam. Several of these bacteria were able to synthesize polyhydroxyalkanoates (PHAs). PHAs are polyesters stored by microorganisms under the presence of considerable amounts of a carbon source and deficiency of other essential nutrient such as nitrogen or phosphorous. Mangrove forests in Northern Vietnam are saline coastal habitats that have not been microbiologically studied. Mangrove ecosystems are, in general, rich in organic matter, but deficient in nutrients such as nitrogen and phosphorus. We have found about 100 microorganisms that have adapted to mangrove forests by accumulating PHAs. The production of polyesters might therefore be an integral part of the carbon cycle in mangrove forests. Three of the strains (ND153, ND97, and QN194) isolated from the Vietnamese forests were identified as Bacillus species, while other five strains (QN187, ND199, ND218, ND240, and QN271) were phylogenetically close related to the α-proteobacterium Yangia pacifica. These strains were found to accumulate PHAs in noticeable amounts. Polymer inclusions and chemical structure were studied by transmission electron microscopy and proton nuclear magnetic resonance (NMR) spectroscopy analyses, respectively. Strains ND153, ND97, QN194, QN187, ND240, and QN271 synthesized poly(3-hydroxybutyrate) (PHB) from glucose, whereas strains ND199 and ND218 synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from this carbohydrate. With the exception of strain QN194, the strains accumulated PHBV when a combination of glucose and propionate was included in the culture medium. The polymer yields and cell growth reached by one Bacillus isolate, strain ND153, and one Gram-negative bacterium, strain QN271, were high and worth to be researched further. For experiments performed in shake flasks, strain ND153 reached a maximum PHBV yield of 71 wt% and a cell dry weight (CDW) of 3.6 g/L while strain QN271 attained a maximum PHB yield of 48 wt% and a CDW of 5.1 g/L. Both strain ND153 and strain QN271 may only represent a case in point that exemplifies of the potential that mangrove forests possess for the discovery of novel halophilic and halotolerant microorganisms able to synthesize different types of biopolyesters.

Evolutionary patterns of carbohydrate transport and metabolism in Halomonas boliviensis as derived from its genome sequence: influences on polyester production.

BACKGROUND: Halomonas boliviensis is a halophilic bacterium that is included in the γ-Proteobacteria sub-group, and is able to assimilate different types of carbohydrates. H. boliviensis is also able to produce poly(3-hydroxybutyrate) (PHB) in high yields using glucose as the carbon precursor. Accumulation of PHB by microorganisms is induced by excess of intracellular NADH.The genome sequences and organization in microorganisms should be the result of evolution and adaptation influenced by mutation, gene duplication, horizontal gen transfer (HGT) and recombination. Furthermore, the nearly neutral theory of evolution sustains that genetic modification of DNA could be neutral or selected, albeit most mutations should be at the border between neutrality and selection, i.e. slightly deleterious base substitutions in DNA are followed by a slightly advantageous substitutions. RESULTS: This article reports the genome sequence of H. boliviensis. The chromosome size of H. boliviensis was 4 119 979 bp, and contained 3 863 genes. A total of 160 genes of H. boliviensis were related to carbohydrate transport and metabolism, and were organized as: 70 genes for metabolism of carbohydrates; 47 genes for ABC transport systems and 43 genes for TRAP-type C4-dicarboxylate transport systems. Protein sequences of H. boliviensis related to carbohydrate transport and metabolism were selected from clusters of orthologous proteins (COGs). Similar proteins derived from the genome sequences of other 41 archaea and 59 bacteria were used as reference. We found that most of the 160 genes in H. boliviensis, c.a. 44%, were obtained from other bacteria by horizontal gene transfer, while 13% of the genes were acquired from haloarchaea and thermophilic archaea, only 34% of the genes evolved among Proteobacteria and the remaining genes encoded proteins that did not cluster with any of the proteins obtained from the reference strains. Furthermore, the diversity of the enzymes derived from these genes led to polymorphism in glycolysis and gluconeogenesis. We found further that an optimum ratio of glucose and sucrose in the culture medium of H. boliviensis favored cell growth and PHB production. CONCLUSIONS: Results obtained in this article depict that most genetic modifications and enzyme polymorphism in the genome of H. boliviensis were mainly influenced by HGT rather than nearly neutral mutations. Molecular adaptation and evolution experienced by H. boliviensis were also a response to environmental conditions such as the type and amount of carbohydrates in its ecological niche. Consequently, the genome evolution of H. boliviensis showed to be strongly influenced by the type of microorganisms, genetic interaction among microbial species and its environment. Such trend should also be experienced by other prokaryotes. A system for PHB production by H. boliviensis that takes into account the evolutionary adaptation of this bacterium to the assimilation of combinations of carbohydrates suggests the feasibility of a bioprocess economically viable and environmentally friendly.

High productivity of ectoines by Halomonas boliviensis using a combined two-step fed-batch culture and milking process.

A process comprising two-step fed-batch cultivation has been investigated for the production of ectoines using the halophilic bacterium Halomonas boliviensis DSM 15516(T). The first cultivation was performed under optimal conditions for cell growth and resulted in cell mass concentration of about 41 g l(-1) after 24 h of cultivation. During the second cultivation at higher salt concentration, accumulation of ectoines increased while cell mass decreased with increasing salt concentration. Maximum productivity of total ectoines reached was 10 g l(-1) d(-1) with ectoine concentration of 6 g l(-1) and hydroxyectoine concentration of 8 g l(-1) after 9 h of cultivation at 18.5% NaCl, which is among the highest reported so far. H. boliviensis cells were further recycled for the production process after releasing the ectoines. About 75% of the accumulated ectoines were released by subjecting the cells to hypoosmotic shock. On subsequent reincubation in a medium containing higher salt concentration the cells were able to re-synthesize the ectoines resulting in a global productivity of 11.1 g l(-1) d(-1), and ectoine and hydroxyectoine productivities of 9.1 g l(-1) d(-1) and 2.0 g l(-1) d(-1), respectively.

Halomonas andesensis sp. nov., a moderate halophile isolated from the saline lake Laguna Colorada in Bolivia.

A moderately halophilic, motile, Gram-negative, rod-shaped bacterium, strain LC6(T), was isolated from a water sample of lake Laguna Colorada in the Bolivian Andes. The major cellular fatty acids were C(18 : 1)omega7c, iso-C(16 : 1)omega7c 2-OH, C(16 : 0) and C(12 : 0) 3-OH. The respiratory ubiquinones found in strain LC6(T) were Q-9 (97 %) and Q-8 (3 %). Strain LC6(T) was aerobic, heterotrophic, and able to utilize various carbohydrates and other substrates as carbon source. The G+C content of the genomic DNA of strain LC6(T) was 52.5 mol%. The organism was able to grow at pH 6.0-11.0 (optimum, pH 7.0-8.0), at 4-45 degrees C (optimum, 30-35 degrees C) and in the presence of 0.5-20 % (w/v) NaCl (optimum, 1-3 %, w/v). Based on 16S rRNA gene sequence analysis, strain LC6(T) was most closely related to Halomonas hydrothermalis DSM 15725(T) and Halomonas venusta DSM 4743(T) (98.8 % similarity), followed by Halomonas aquamarina DSM 30161(T), Halomonas axialensis DSM 15723(T) and Halomonas meridiana DSM 5425(T) (98.4 %). However, levels of DNA-DNA relatedness between strain LC6(T) and the above type strains were low (<31 %). Strain LC6(T) resembled recognized Halomonas species with respect to various physiological, biochemical and nutritional characteristics. Combined phenotypic data and DNA-DNA hybridization data supported the conclusion that strain LC6(T) represents a novel species of the genus Halomonas, for which the name Halomonas andesensis is proposed. The type strain is LC6(T) (=CCUG 54844(T)=LMG 24243(T)=DSM 19434(T)).

Synthesis and production of polyhydroxyalkanoates by halophiles: current potential and future prospects.

Biodegradable materials with plastic or elastomeric properties are in great demand for a variety of applications. Polyhydroxyalkanoates (PHAs), polyesters synthesized by microorganisms, possess such desired features. Industrial production of PHAs is currently achieved using recombinant Escherichia coli. Nevertheless, recent research on halophiles, salt requiring microorganisms, has shown a remarkable potential for biotechnological production of PHAs. The halophilic archaeon Haloferax mediterranei accumulates a co-polymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in large amounts using glucose, starch, and hydrolyzed whey as carbon sources. Chemical composition and molecular weight of PHAs produced by H. mediterranei can be modified depending on the substrate utilized as precursor. Phylogenetic studies on haloarchaeal enzymes able to polymerize the components of PHAs (i.e., PHA synthases) reveal a novel cluster, with a close relationship with PHA polymerases of bacteria and archaea found in marine-related niches. On the other hand, sequences of PHA synthases of two halophilic bacteria are more closely affiliated to synthases of Proteobacteria. Several bacterial species of the family Halomonadaceae accumulate PHAs. Halomonas boliviensis reached PHA yields and volumetric productivities close to the highest reported so far. Furthermore, H. boliviensis and other Halomonas species are able to co-produce PHA and osmolytes, i.e., ectoines and hydroxyectoine, in one process.

A process for the production of ectoine and poly(3-hydroxybutyrate) by Halomonas boliviensis.

The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co-production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concentrations showed the highest level of intracellular accumulation of ectoine (0.74 g L(-1)) at 10-15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L(-1)) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L(-1), respectively, were obtained at 5-7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed-the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH (4) (+) , and PO (4) (3-) . In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L(-1) h(-1) and ectoine concentration, content, and volumetric productivity of 4.3 g L(-1), 7.2 wt.%, and 2.8 g L(-1) day(-1), respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and productivity to 3.4 g L(-1) day(-1).

Poly(3-hydroxybutyrate) production by Halomonas boliviensis in fed-batch culture.

High poly(3-hydroxybutyrate) (PHB) content and volumetric productivity were achieved by fed-batch culture of Halomonas boliviensis using a defined medium. Initial shake flask cultivations in a minimal medium revealed that the growth of H. boliviensis was supported only when the medium was supplemented with aspartic acid, glycine, or glutamine. Addition of 0.1% (w/v) glutamine in the medium resulted in the highest cell dry weight (CDW; 3.9 g l(-1)). Glutamine was replaced by the less expensive monosodium glutamate (MSG) in the medium without any notable change in the final cell density. Effect of initial concentrations of NH(4)Cl and K(2)HPO(4) on cell growth and PHB accumulation by H. boliviensis was then analyzed using a fed-batch fermentation system. The best conditions for PHB production by H. boliviensis were attained using 0.4% (w/v) NH(4)Cl and 0.22% (w/v) K(2)HPO(4) and adding MSG intermittently to the fermentor. Poly(3-hydroxybutyrate) content and CDW reached 90 wt.% and 23 g l(-1), respectively, after 18 h of cultivation. In order to increase CDW and PHB content, MSG, NH(4)Cl, and K(2)HPO(4) were initially fed to the fermentor to maintain their concentrations at 2%, 0.4%, and 0.22% (w/v), respectively, and subsequently their feed was suppressed. This resulted in a CDW of 44 g l(-1), PHB content of 81 wt.%, and PHB volumetric productivity of 1.1 g l(-1) h(-1).

Optimizing conditions for poly(beta-hydroxybutyrate) production by Halomonas boliviensis LC1 in batch culture with sucrose as carbon source.

Halomonas boliviensis LC1 is able to accumulate poly(beta-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l(-1) CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l(-1) and 14 g l(-1), respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l(-1) in 17 h but reduced the PHB content in the cells to 52 wt.%.

Nesterenkonia aethiopica sp. nov., an alkaliphilic, moderate halophile isolated from an Ethiopian soda lake.

Strain DSM 17733(T), isolated from the shore of Lake Abjata in Ethiopia, is a heterotrophic, alkaliphilic, moderately halophilic, Gram-positive, strictly aerobic, non-motile,non-endospore-forming bacterium. The organism grows optimally at 30-37 degrees C, pH 9 and 3 % (w/v) NaCl. Analysis of the cell wall showed the presence of murein of the type L-lys-gly-L-Glu, variation A4alpha. The G + C content of the genomic DNA was 69.0 mol%. Sequence analysis of 16S rRNA gene sequence of strain DSM 17733(T) placed the isolate in the genus Nesterenkonia. DNA-DNA hybridization of DSM 17733(T) with those organisms with the closest phylogenetic affiliation, i.e. Nesterenkonia halobia, Nesterenkonia lacusekhoensis and Nesterenkonia xinjiangensis, gave relatedness values of 48.5 %, 63.7 % (repetition, 57.2 %) and 35.7 % (repetition, 29.3 %), respectively. On the basis of both phenotypic and phylogenetic criteria and the low levels of DNA-DNA relatedness with the phylogenetically closest species N. xinjiangensis and N. halobia, it is proposed that the isolate be classified in a novel species, Nesterenkonia aethiopica sp. nov. The type strain is DSM 17733(T) (=CCUG 48939(T)).

A toxaphene-degrading bacterium related to Enterobacter cloacae, strain D1 isolated from aged contaminated soil in Nicaragua.

Enterobacter sp. strain D1 is a facultative anaerobic, Gram-negative heterotrophic bacterium isolated from toxaphene-contaminated soil. This organism was identified and characterized through phylogenetic and taxonomic studies. Based on 16S rDNA analysis, the strain D1 was clustered closely with the species Enterobacter cloacae subsp. dissolvens (LMG 2683) and E. cloacae (ATCC 13047T). Strain D1 resembled these E. cloacae strains with respect to various biochemical and nutritional characteristics, but also exhibited differences. Moreover, strain D1 is able to grow and survive with toxaphene supplied in the medium in the range 3-96 mg/L. Amongst the chemical components of toxaphene, octachlorocamphenes, nonachlorobornanes and decachlorobornanes were seen to be rapidly metabolized, although levels of hexachlorocamphenes and heptachlorobornanes were found to be slowly degraded, and subsequently accumulated during the last stage of the cultivation.