Biological production of monoethanolamine by engineered Pseudomonas putida S12.

Pseudomonas putida S12 was engineered for the production of monoethanolamine (MEA) from glucose via the decarboxylation of the central metabolite L-serine, which is catalyzed by the enzyme L-serine decarboxylase (SDC). The host was first evaluated for its tolerance towards MEA as well as its endogenous ability to degrade this alkanolamine. Growth inhibition was observed at MEA concentrations above 100 mM, but growth was never completely arrested even at 750 mM of MEA. P. putida S12 was able to catabolize MEA in the absence of ammonia, but deletion of the eutBC genes that encode ethanolamine ammonia-lyase (EAL) enzyme sufficed to eliminate this capacity. For the biological production of MEA, the sdc genes from Arabidopsis thaliana (full-length and a truncated version) and Volvox carteri were expressed in P. putida S12. From 20 mM of glucose, negligible amounts of MEA were produced by P. putida S12 ΔeutBC expressing the sdc genes from A. thaliana and V. carteri. However, 0.07 mmol of MEA was obtained per g of cell dry weight of P. putida S12 ΔeutBC expressing the truncated variant of the A. thaliana SDC. When the medium was supplemented with L-serine (30 mM), MEA production increased to 1.25 mmol MEA g⁻¹ CDW, demonstrating that L-serine availability was limiting MEA production.

Bacterial diversity and activity along a salinity gradient in soda lakes of the Kulunda Steppe (Altai, Russia).

Here we describe the diversity and activity of sulfate reducing bacteria along a salinity gradient in four different soda lakes from the Kulunda Steppe (South East Siberia, Russia). For this purpose, a combination of culture-dependent and independent techniques was applied. The general bacterial and SRB diversity were analyzed by denaturing gradient gel electrophoresis (DGGE) targeting the 16S rDNA gene. DNA was used to detect the microbial populations that were present in the soda lake sediments, whereas ribosomal RNA was used as a template to obtain information on those that were active. Individual DGGE bands were sequenced and a phylogenetic analysis was performed. In addition, the overall activity of SRB was obtained by measuring the sulfate reduction rates (SRR) and their abundance was estimated by serial dilution. Our results showed the presence of minor, but highly active microbial populations, mostly represented by members of the Proteobacteria. Remarkably high SRR were measured at hypersaline conditions (200 g L(-1)). A relatively high viable count indicated that sulfate reducing bacteria could be highly active in hypersaline soda lakes. Furthermore, the increase of sodium carbonate/bicarbonate seemed to affect the composition of the microbial community in soda lakes, but not the rate of sulfate reduction.

Diversity, activity, and abundance of sulfate-reducing bacteria in saline and hypersaline soda lakes.

Soda lakes are naturally occurring highly alkaline and saline environments. Although the sulfur cycle is one of the most active element cycles in these lakes, little is known about the sulfate-reducing bacteria (SRB). In this study we investigated the diversity, activity, and abundance of SRB in sediment samples and enrichment cultures from a range of (hyper)saline soda lakes of the Kulunda Steppe in southeastern Siberia in Russia. For this purpose, a polyphasic approach was used, including denaturing gradient gel electrophoresis of dsr gene fragments, sulfate reduction rate measurements, serial dilutions, and quantitative real-time PCR (qPCR). Comparative sequence analysis revealed the presence of several novel clusters of SRB, mostly affiliated with members of the order Desulfovibrionales and family Desulfobacteraceae. We detected sulfate reducers and observed substantial sulfate reducing rates (between 12 and 423 micromol/dm(3) day(-1)) for most lakes, even at a salinity of 475 g/liter. Enrichments were obtained at salt saturating conditions (4 M Na(+)), using H(2) or volatile fatty acids as electron donors, and an extremely halophilic SRB, strain ASO3-1, was isolated. Furthermore, a high dsr gene copy number of 10(8) cells per ml was detected in a hypersaline lake by qPCR. Our results indicate the presence of diverse and active SRB communities in these extreme ecosystems.

Sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake with an unprecedented high sulfide content.

Culture-dependent and -independent techniques were used to study the diversity of chemolithoautotrophic sulfur-oxidizing bacteria in Soap Lake (Washington State), a meromictic, haloalkaline lake containing an unprecedentedly high sulfide concentration in the anoxic monimolimnion. Both approaches revealed the dominance of bacteria belonging to the genus Thioalkalimicrobium, which are common inhabitants of soda lakes. A dense population of Thioalkalimicrobium (up to 10(7) cells/ml) was found at the chemocline, which is characterized by a steep oxygen-sulfide gradient. Twelve Thioalkalimicrobium strains exhibiting three different phenotypes were isolated in pure culture from various locations in Soap Lake. The isolates fell into two groups according to 16S rRNA gene sequence analysis. One of the groups was closely related to T. cyclicum, which was isolated from Mono Lake (California), a transiently meromictic, haloalkaline lake. The second group, consisting of four isolates, was phylogenetically and phenotypically distinct from known Thioalkalimicrobium species and unique to Soap Lake. It represented a new species, for which we suggest the name Thioalkalimicrobium microaerophilum sp. nov.

Genetic diversity and biogeography of haloalkaliphilic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio.

A group of 85 isolates of haloalkaliphilic obligately chemolithoautotrophic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio were recently obtained from soda lakes in Mongolia, Kenya, California, Egypt and Siberia. They have been analyzed by repetitive extragenic palindromic (rep)-PCR genomic fingerprinting technique with BOX- and (GTG)5-primer set. Cluster analysis was performed using combined fingerprint profiles and a dendrogram similarity value (r) of 0.8 was used to define the same genotype. Fifty-six genotypes were found among the isolates, revealing a high genetic diversity. The strains can be divided into two major clusters, including isolates from the Asiatic (Siberia and Mongolia) and the African (Kenya and Egypt) continents, respectively. The majority (85.9%) of the genotypes were found in only one area, suggesting an endemic character of the Thioalkalivibrio strains. Furthermore, a correlation between fingerprint clustering, geographic origin and the characteristics of the lake of origin was found.