Microbial alkaline proteases: From a bioindustrial viewpoint

Two major types of environment provide habitats for the most xerophilic organisms known: foods preserved by some form of dehydration or enhanced sugar levels, and hypersaline sites where water availability is limited by a high concentration of salts (usually NaCl). These environments are essentially microbial habitats, with high-sugar foods being dominated by xerophilic (sometimes called osmophilic) filamentous fungi and yeasts, some of which are capable of growth at a water activity (a(w)) of 0.61, the lowest a(w) value for growth recorded to date. By contrast, high-salt environments are almost exclusively populated by prokaryotes, notably the haloarchaea, capable of growing in saturated NaCl (a(w) 0.75). Different strategies are employed for combating the osmotic stress imposed by high levels of solutes in the environment. Eukaryotes and most prokaryotes synthesize or accumulate organic so-called 'compatible solutes' (osmolytes) that have counterbalancing osmotic potential. A restricted range of bacteria and the haloarchaea counterbalance osmotic stress imposed by NaCl by accumulating equivalent amounts of KCl. Haloarchaea become entrapped and survive for long periods inside halite (NaCl) crystals. They are also found in ancient subterranean halite (NaCl) deposits, leading to speculation about survival over geological time periods.

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Life at low water activity.

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 CO3
2− 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 Earth's history.

Microbial diversity of soda lakes.

Summary: One hundred and nineteen strains of alkaliphilic and alkalitolerant, aerobic endospore-forming bacteria were examined for 47 physiological and biochemical characters, and DNA base composition. Numerical analysis (S
J and S
SM/UPGMA clustering) revealed 11 clusters that comprised three or more strains. Most of the phena were further characterized by analysis of carbohydrate utilization profiles using the API 50CH system, but strains of two taxa could not be cultured by this method. DNA reassociation studies showed that nine of the phena were homogeneous, but strains of phenon 4 and phenon 8 were each subdivided into two DNA hydridization groups. The strains could therefore be classified into 13 taxa plus a number of unassigned single-membered clusters. Two taxa were equated with Bacillus cohnii and B. alcalophilus and nine of the remainder are proposed as new species with the following names: B. agaradhaerens sp. nov., B. clarkii sp. nov., B. clausii sp. nov., B. gibsonii sp. nov., B. halmapalus sp. nov., B. halodurans comb, nov., B. horikoshii sp. nov., B. pseudalcalophilus sp. nov. and B. pseudofirmus sp. nov. Two taxa were insufficiently distinct to allow confident identification and these have therefore not been proposed as new species.

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Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species

We analyzed the variation with depth in the composition of members of the domain Bacteria in samples from alkaline, hypersaline, and currently meromictic Mono Lake in California. DNA samples were collected from the mixolimnion (2 m), the base of the oxycline (17.5 m), the upper chemocline (23 m), and the monimolimnion (35 m). Composition was assessed by sequencing randomly selected cloned fragments of 16S rRNA genes retrieved from the DNA samples. Most of the 212 sequences retrieved from the samples fell into five major lineages of the domain Bacteria: α- and γ-Proteobacteria (6 and 10%, respectively), Cytophaga-Flexibacter-Bacteroides (19%), high-G+C-content gram-positive organisms (Actinobacteria; 25%), and low-G+C-content gram-positive organisms (Bacillus and Clostridium; 19%). Twelve percent were identified as chloroplasts. The remaining 9% represented β- and δ-Proteobacteria, Verrucomicrobiales, and candidate divisions. Mixolimnion and oxycline samples had low microbial diversity, with only 9 and 12 distinct phylotypes, respectively, whereas chemocline and monimolimnion samples were more diverse, containing 27 and 25 phylotypes, respectively. The compositions of microbial assemblages from the mixolimnion and oxycline were not significantly different from each other (P = 0.314 and 0.877), but they were significantly different from those of chemocline and monimolimnion assemblages (P < 0.001), and the compositions of chemocline and monimolimnion assemblages were not significantly different from each other (P = 0.006 and 0.124). The populations of sequences retrieved from the mixolimnion and oxycline samples were dominated by sequences related to high-G+C-content gram-positive bacteria (49 and 63%, respectively) distributed in only three distinct phylotypes, while the population of sequences retrieved from the monimolimnion sample was dominated (52%) by sequences related to low-G+C-content gram-positive bacteria distributed in 12 distinct phylotypes. Twelve and 28% of the sequences retrieved from the chemocline sample were also found in the mixolimnion and monimolimnion samples, respectively. None of the sequences retrieved from the monimolimnion sample were found in the mixolimnion or oxycline samples. Elevated diversity in anoxic bottom water samples relative to oxic surface water samples suggests a greater opportunity for niche differentiation in bottom versus surface waters of this lake.

Depth distribution of microbial diversity in Mono Lake, a meromictic soda lake in California

Organisms with pH optima for growth in excess of pH8, usually between 9 and 10, are properly defined as alkaliphiles (or sometimes alkalophiles). Most of the organisms described to date as growing under very alkaline conditions are prokaryotes, comprising a heterogeneous collection of eubacteria (including cyanobacteria) with a few examples of archaebacteria. With the exception of the alkaliphilic archaebacteria (which are also halophilic), few of the isolates have been comprehensively classified. However, it is clearly that may different taxa are represented amongst the alkaliphiles, and that some of these at least are likely to be new taxa. Alkaliphiles can be isolated from ‘normal’ environments such as garde soil, presumably because there are transient alkaline conditions generated in such environments by biological activity, but a relatively restricted range of types results from the exploration of such environments (often Bacillus spp.). Naturally-occurring stable alkaline environments such as eutrophic soda (Na2CO3) lakes harbour a much wider range of types. A similarity wide range of types may exist in oligotrophic Ca(OH)2-dominated ground water in certain parts of the world. Alkaliphilic microorganisms have already made a large impact in the application of biotechnology for the manufacture of mass market consumer products. “Biological detergents” contain enzymes that have usually been obtained from alkaliphilic or alkalitolerant bacteria. The current proportion of total world enzyme production destined for the laundry detergents market comfortably exceeds 25%, and there are other possible applications in food and waste treatment industies. To date, most commercially available enzymes are derived from a few bacterial taxa (notably Bacillus spp.) and we have been very conservative in our choice of environments in which to look for new products. It is reasonable to suppose that soda lakes (and other alkaline environments) will yield a wealth of new organisms with useful commercial properties.

Alkaliphiles: Ecology, diversity and applications

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.

Alkaliphiles: Diversity and Identification

A novel haloalkaliphilic archaeon was isolated from Lake Magadi, a Kenyan alkaline soda lake. Cells of the organism contain large gas vacuoles in the stationary phase of growth, and colonies produced by these archaea are bright pink in appearance. The major polar lipids of these organisms are C20C20 and C20C25 derivatives of phosphatidylglycerol phosphate and phosphatidylglycerol, and the organisms contain an unidentified phospholipid as a minor component. The G+C content of the DNA is 62.7 mol%. The name Natronobacterium vacuolata sp. nov. is proposed. The type strain is designated NCIMB 13189.

Wanjiru E. Mwatha

Papers

Alkaliphiles: Ecology, diversity and applications

Alkaliphiles: Diversity and Identification

Natronobacterium vacuolata sp. nov., a Haloalkaliphilic Archaeon Isolated from Lake Magadi, Kenya