Deep and high-efficiency removal of sulfate through a coupling system with sulfate-reducing and sulfur-oxidizing capacity under haloalkaliphilic condition.

Sulfide from anaerobic treatment of high-sulfate wastewater would always have some adverse effects on downstream processes. In this study, a coupling anaerobic/aerobic system was developed and operated under haloalkaliphilic condition to realize deep and high-efficiency removal of sulfate without production of sulfide. A haloalkaliphilic sulfur-oxidizing strain, Thioalkalivibrio versutus SOB306, was responsible for oxidation of sulfide. The anaerobic part was first operated at optimum condition based on a previous study. Then, its effluent with an average sulfide concentration of 674 ± 33 mg·l-1 was further directly treated by a set of 1 l biofilter with SOB306 strain under aerobic condition. Finally, 100% removal rate of sulfide was achieved at aeration rate of 0.75 l·l-1·min-1, ORP of - 392 mV and HRT of 4 h. The average yield of elemental sulfur reached 79.1 ± 1.3% in the filter, and the CROS achieved a conversion rate of sulfate to sulfur beyond 54%. This study for the first time revealed the characteristics and performance of the haloalkaliphilic CROS in deep treatment of high-sulfate wastewater, which paved the way for the development and application of this method in the real world.

Improvement of desulfurizing activity of haloalkaliphilic Thialkalivibrio versutus SOB306 with the expression of Vitreoscilla hemoglobin gene.

OBJECTIVE: To construct efficient transformation and expression system and further improve desulfurizing activity of cells through expression of Vitreoscilla hemoglobin (VHb) in haloalkaliphilic Thialkalivibrio versutus SOB306. RESULTS: We transferred plasmids pKT230 and pBBR-smr into T. versutus SOB306 via a conjugation method. We identified four promoters from among several predicted promoters by scoring for streptomycin resistance, and finally selected tac and p3 based on the efficiency of expression of red fluorescent protein (RFP). Expression of RFP when regulated by tac was more than three times that of p3 in SOB306. Further, we expressed VHb under the control of tac promoter in SOB306. Expression of VHb was verified using CO-difference spectra. The results showed that VHb expression can boost sulfur metabolism, as evidenced by an increase of about 11.7 ± 1.8% in the average rate of thiosulfate removal in the presence of VHb. CONCLUSION: A conjugation transfer and an expression system for Thialkalivibrio, has been developed for the first time and used for expression of VHb to improve desulfurizing activity.

Biocathodes reducing oxygen at high potential select biofilms dominated by Ectothiorhodospiraceae populations harboring a specific association of genes.

Biocathodes polarized at high potential are promising for enhancing Microbial Fuel Cell performances but the microbes and genes involved remain poorly documented. Here, two sets of five oxygen-reducing biocathodes were formed at two potentials (-0.4V and +0.1V vs. saturated calomel electrode) and analyzed combining electrochemical and metagenomic approaches. Slower start-up but higher current densities were observed at high potential and a distinctive peak increasing over time was recorded on cyclic voltamogramms, suggesting the growth of oxygen reducing microbes. 16S pyrotag sequencing showed the enrichment of two operational taxonomic units (OTUs) affiliated to Ectothiorodospiraceae on high potential electrodes with the best performances. Shotgun metagenome sequencing and a newly developed method for the identification of Taxon Specific Gene Annotations (TSGA) revealed Ectothiorhodospiraceae specific genes possibly involved in electron transfer and in autotrophic growth. These results give interesting insights into the genetic features underlying the selection of efficient oxygen reducing microbes on biocathodes.

Arhodomonas sp. strain Seminole and its genetic potential to degrade aromatic compounds under high-salinity conditions.

Arhodomonas sp. strain Seminole was isolated from a crude oil-impacted brine soil and shown to degrade benzene, toluene, phenol, 4-hydroxybenzoic acid (4-HBA), protocatechuic acid (PCA), and phenylacetic acid (PAA) as the sole sources of carbon at high salinity. Seminole is a member of the genus Arhodomonas in the class Gammaproteobacteria, sharing 96% 16S rRNA gene sequence similarity with Arhodomonas aquaeolei HA-1. Analysis of the genome predicted a number of catabolic genes for the metabolism of benzene, toluene, 4-HBA, and PAA. The predicted pathways were corroborated by identification of enzymes present in the cytosolic proteomes of cells grown on aromatic compounds using liquid chromatography-mass spectrometry. Genome analysis predicted a cluster of 19 genes necessary for the breakdown of benzene or toluene to acetyl coenzyme A (acetyl-CoA) and pyruvate. Of these, 12 enzymes were identified in the proteome of toluene-grown cells compared to lactate-grown cells. Genomic analysis predicted 11 genes required for 4-HBA degradation to form the tricarboxylic acid (TCA) cycle intermediates. Of these, proteomic analysis of 4-HBA-grown cells identified 6 key enzymes involved in the 4-HBA degradation pathway. Similarly, 15 genes needed for the degradation of PAA to the TCA cycle intermediates were predicted. Of these, 9 enzymes of the PAA degradation pathway were identified only in PAA-grown cells and not in lactate-grown cells. Overall, we were able to reconstruct catabolic steps for the breakdown of a variety of aromatic compounds in an extreme halophile, strain Seminole. Such knowledge is important for understanding the role of Arhodomonas spp. in the natural attenuation of hydrocarbon-impacted hypersaline environments.

Aerobic carboxydotrophy under extremely haloalkaline conditions in Alkalispirillum/Alkalilimnicola strains isolated from soda lakes.

Aerobic enrichments from soda lake sediments with CO as the only substrate resulted in the isolation of five bacterial strains capable of autotrophic growth with CO at extremely high pH and salinity. The strains belonged to the Alkalispirillum/Alkalilimnicola cluster in the Gammaproteobacteria, where the ability to oxidize CO, but not growth with CO, has been demonstrated previously. The growth with CO was possible only at an oxygen concentration below 5 % and CO concentration below 20 % in the gas phase. The isolates were also capable of growth with formate but not with H(2). The carboxydotrophic growth occurred within a narrow pH range from 8 to 10.5 (optimum at 9.5) and a broad salt concentration from 0.3 to 3.5 M total Na(+) (optimum at 1.0 M). Cells grown on CO had high respiration activity with CO and formate, while the cells grown on formate actively oxidized formate alone. In CO-grown cells, CO-dehydrogenase (CODH) activity was detectable both in soluble and membrane fractions, while the NAD-independent formate dehydrogenase (FDH) resided solely in membranes. The results of total protein profiling and the failure to detect CODH with conventional primers for the coxL gene indicated that the CO-oxidizing enzyme in haloalkaliphilic isolates might differ from the classical aerobic CODH complex. A single cbbL gene encoding the RuBisCO large subunit was detected in all strains, suggesting the presence of the Calvin cycle of inorganic carbon fixation. Overall, these results demonstrated the possibility of aerobic carboxydotrophy under extremely haloalkaline conditions.

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.

Growth kinetics of haloalkaliphilic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15 in continuous culture.

The chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15, isolated from a soda lake in Kenya, was grown in a continuous culture, with thiosulfate or polysulfide as growth-limiting energy source and oxygen as electron acceptor, at pH 10 and at pH 0.6, 2 M and 4 M total sodium. The end product of the sulfur-compound oxidation was sulfate. Elemental sulfur and a cell-bound, polysulfide-like compound appeared as intermediates during substrate oxidation. In the thiosulfate-limited culture, the biomass yields and maximum specific growth rates decreased two and three times, respectively, with increasing sodium concentration. The apparent affinity constant measured for thiosulfate and polysulfide was in the micromolar range (Ks = 6 +/- 3 microM). The maintenance requirement (ms = 8 +/- 5 mmol S2O3(2)/g dry weight h(-1)) was in the range of values found for other autotrophic sulfur-oxidizing bacteria. The organism had a comparable maximum specific rate of oxygen uptake with thiosulfate, polysulfide, and sulfide, while elemental sulfur was oxidized at a lower rate. Glycine betaine was the main organic compatible solute. The respiration rates with different species of polysulfides (Sn2-) were tested. All polysulfide species were completely oxidized at high rates to sulfate. Overall data demonstrated efficient growth and sulfur compounds oxidation of haloalkaliphilic chemolithoautotrophic bacteria from soda lakes.