Brian E. Jones

Microbial diversity of soda lakes

Abstract Soda lakes are highly alkaline extreme environments that form in closed drainage basins exposed to high evaporation rates. Because of the scarcity of Mg2+ and Ca2+ in the water chemistry, the lakes become enriched in CO32− and Cl−, with pHs in the range 8 to >12. Although there is a clear difference in prokaryotic communities between the hypersaline lakes where NaCl concentrations are >15% w/v and more dilute waters, i.e., NaCl concentrations about 5% w/v, photosynthetic primary production appears to be the basis of all nutrient recycling. In both the aerobic and anaerobic microbial communities the major trophic groups responsible for cycling of carbon and sulfur have in general been identified. Systematic studies have shown that the microbes are alkaliphilic and many represent separate lineages within accepted taxa, while others show no strong relationship to known prokaryotes. Although alkaliphiles are widespread it seems probable that these organisms, especially those unique to the hypersaline lakes, evolved separately within an alkaline environment. Although present-day soda lakes are geologically quite recent, they have probably existed since archaean times, permitting the evolution of independent communities of alkaliphiles since an early period in the Earths history.

Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods

DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley. DNA was also extracted from microbial enrichment cultures of sediment samples. 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons. Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons. Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82%. Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/Aeromonas/Vibrio part of the γ3 subdivision of the Proteobacteria.

Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibericum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis sp. nov. and Thioalkalivibrio denitrificans sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria from soda lakes

Forty-three strains of obligately chemolithoautotrophic sulfur-oxidizing bacteria were isolated from highly alkaline soda lakes in south-east Siberia (Russia) and in Kenya using a specific enrichment procedure at pH 10. The main difference between the novel isolates and known sulfur bacteria was their potential to grow and oxidize sulfur compounds at pH 10 and higher. The isolates fell into two groups that were substantially different from each other physiologically and genetically. Most of the Siberian isolates belonged to the group with a low DNA G+C content (48.0-51.2 mol%). They were characterized by a high growth rate, a low growth yield, a high cytochrome content, and high rates of oxidation of sulfide and thiosulfate. This group included 18 isolates with a DNA homology of more than 40%, and it is described here as a new genus, Thioalkalimicrobium, with two species Thioalkalimicrobium aerophilum (type species) and Thioalkalimicrobium sibericum. The other isolates, mainly from Kenyan soda lakes, fell into a group with a high DNA G+C content (61.0-65.6 mol%). In general, this group was characterized by a low growth rate, a high molar growth yield and low, but relatively equal, rates of oxidation of thiosulfate, sulfide, elemental sulfur and polythionates. The group included 25 isolates with a DNA homology of more than 30%. It was less compact than Thioalkalimicrobium, containing haloalkalophilic, carotenoid-producing, nitrate-reducing and facultatively anaerobic denitrifying strains. These bacteria are proposed to be assigned to a new genus, Thioalkalivibrio, with three species Thioalkalivibrio versutus (type species), Thioalkalivibrio denitrificans and Thioalkalivibrio nitratis. Phylogenetic analysis revealed that both groups belong to the gamma-Proteobacteria. The Thioalkalimicrobium species were closely affiliated with the neutrophilic chemolithoautotrophic sulfur bacteria of the genus Thiomicrospira, forming a new alkaliphilic lineage in this cluster. In contrast, Thioalkalivibrio was not related to any known chemolithoautotrophic taxa, but was distantly associated with anaerobic purple sulfur bacteria of the genus Ectothiorhodospira.

Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries

A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples. Clones expressing a cellulase activity were identified and sequenced. A new cellulase gene was identified. Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake. Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced. These were encoded by novel genes as judged by DNA sequence comparisons. Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples. Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil. A number of new cellulase and lipase/esterase genes were discovered in these libraries. Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment. Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library.

Novel archaeal phylotypes from an East African alkaline saltern

Abstract DNA has been extracted on site from the brines of the final crystallizing pond of an alkaline saltern at Lake Magadi, Kenya. Amplification of 16S rRNA genes followed by cloning, sequencing, and phylogenetic analysis has revealed the presence of two distinct new archaeal lineages. The majority of cloned sequences showed greater than 95% identity to each other, but only 88%–90% similarity to any cultivated haloalkaliphilic Archaea, and form a distinct cluster within the known Haloarchaea. Two cloned genes showed close similarity to each other but only 76% similarity to any known archaeal sequence, and therefore represent a distinct phylotype only distantly related to the euryarchaeotal branch of the Archaea.

Dietzia natronolimnaios sp. nov., a new member of the genus Dietzia isolated from an East African soda lake

Abstract Two novel alkaliphilic aerobic organotrophic bacteria have been isolated from a moderately saline and alkaline East African soda lake. The new isolates grow at pH values between 6 and 10, with a pH optimum for growth of 9.0, and at a salt concentration between 0% and 10% (w/v). Phylogenetic analysis based on 16S rDNA sequence shows that these isolates are very closely related (99.6% similarity) and are members of the monospecific genus Dietzia (98.8% and 98.7% similarity). DNA/DNA hybridization revealed a relatedness of 83% between the two isolates, but only 8% between them and the type strain Dietzia maris. The G + C content as measured by thermal denaturation is 66.1 mol%. Phenotypic comparisons between D. maris and one isolate showed that they share very similar morphological and chemotaxonomic properties, but differ significantly in carbon source utilization profiles and halotolerance in alkaline medium. We propose a second species of this genus which we name Dietzia natronolimnaios (type strain 15LN1 = CBS 107.95).

Alkaliphiles: Diversity and Identification

Micro-organisms that grow in hostile or extreme environments are currently a popular subject for study. The fascination with so-called ‘extremophiles’ reflects a perceived microbial biotechnological importance and a desire to investigate possible early conditions on Earth and the origins of life. Extremes of pH represent a particularly hostile regime for microbial life, especially when combined with extremes of salinity or temperature.This review deals with some of the aspects of micro-organisms that inhabit environments in the alkaline range of the pH spectrum.

Microbial Biogeography of Six Salt Lakes in Inner Mongolia, China, and a Salt Lake in Argentina

ABSTRACT We used cultivation-independent methods to investigate the prokaryotic biogeography of the water column in six salt lakes in Inner Mongolia, China, and a salt lake in Argentina. These lakes had different salt compositions and pH values and were at variable geographic distances, on both local and intercontinental scales, which allowed us to explore the microbial community composition within the context of both contemporary environmental conditions and geographic distance. Fourteen 16S rRNA gene clone libraries were constructed, and over 200 16S rRNA gene sequences were obtained. These sequences were used to construct biotic similarity matrices, which were used in combination with environmental similarity matrices and a distance matrix in the Mantel test to discover which factors significantly influenced biotic similarity. We showed that archaeal biogeography was influenced by contemporary environmental factors alone (Na+, CO32−, and HCO3− ion concentrations; pH; and temperature). Bacterial biogeography was influenced both by contemporary environmental factors (Na+, Mg2+, and HCO3− ion concentrations and pH) and by geographic distance.

A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification of cellulase genes from these cultures

Samples of sediments and surrounding soda soils (SS) from the extremely saline and alkaline lakes of the Wadi el Natrun in the Libyan Desert, Egypt, were obtained in October 2000. Anaerobic enrichment cultures were grown from these samples, DNA isolated, and the bacterial diversity assessed by 16S rRNA gene clone analysis. Clones derived from lake sediments (LS) most closely matched Clostridium spp., Natronoincola histidinovorans, Halocella cellulolytica, Bacillus spp., and the Cytophaga–Flexibacter–Bacteroides group. Similar clones were identified in the SS, but Bacillus spp. predominated. Many of the clones were most closely related to organisms already identified in alkaline or saline environments. Two genomic DNA libraries were made from the pooled LS enrichments and a single SS-enrichment sample. A novel cellulase activity was identified and characterized in each. The lake cellulase ORF encoded a protein of 1,118 amino acids; BLASTP analysis showed it was most closely related to an endoglucanase from Xanthomonas campestris. The soil-cellulase ORF encoded a protein of 634 amino acids that was most closely related to an endoglucanase from Fibrobacter succinogenes.

Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley

Abstract A number of novel alkaliphilic organotrophic bacteria have been isolated from several saline and alkaline East African soda lakes. The new isolates grow at pH values between 7.0 and 11.0, with pH optima for growth between 9.0 and 10.0. Growth occurs at total salts concentration between 0% and 20% (w/v) with optimum at 0%–7% (w/v). Phylogenetic analyses based on 16S rDNA sequence comparison indicate that these isolates are related (>96% similarity) to members of the Halomonadaceae within the γ-3 subdivision of the Proteobacteria. These analyses indicate that existing species within the Halomonadaceae fell within three main groups, one group comprising the type species of Halomonas, Halomonas elongata, and a number of other known species including one soda lake isolate. A second group constituting most of the remaining known species of Halomonas and related Chromohalobacter spp. includes 3 soda lake isolates with high DNA–DNA homologies. The third group included Halomonas halodenitrificans, Halomonas desiderata, Halomonas cupida, and 13 soda lake isolates. Phenotypic comparisons indicated that the majority of soda lake strains shared similar morphological, phenotypic, and chemotaxonomic properties to known strains of Halomonas but grew under alkaline conditions. The 3 soda lake isolates with high DNA–DNA homologies were, however, significantly different in antibiotic sensitivity pattern and in the utilization of several substrates, were unable to reduce nitrite, and showed low DNA–DNA homologies with known halomonads in the same group. We propose that these isolates comprise a new species of the genus Halomonas that we name Halomonas magadii sp. nov. The type strain is strain 21 MI (NCIMB 13595).