Alkaliphiles — from an industrial point of view

Abstract: Organisms with pH optima for growth in excess of pH 9 are defined as alkaliphiles (or sometimes alkalophiles). Alkaliphiles contain prokaryotes, eukaryotes, and archaea. It is clear that many different taxa are represented among the alkaliphiles, and some of them are proposed as new taxa. Alkaliphiles can b isolated from normal environments such as garden soil, although counts of the alkaliphiles are higher in alkaline environments. Alkaliphiles have made a great impact in industrial applications. Biological detergents contain alkaline enzymes, such as alkaline cellulases and/or alkaline proteases that have been produced from alkaliphiles. The current proportion of total world enzyme production destined for the laundry detergents market exceeds 30%. Another important application is the industrial production of cyclodextrin with alkaline cyclomaltodextrin glucanotransferase. This enzyme reduced the production cost and paved the way for its use in large quantities in foodstuffs, chemicals and pharmaceuticals. Besides these applications, there are other possible applications in food and waste treatment industries.     

嗜碱性细菌在酶制剂生产中的应用

嗜碱性菌是一类生长于碱性环境中的特殊微生物。自发现以来引起人们广泛重视,并在许多方面得到了应用。阐述了嗜碱性菌在酶制剂方面的应用和发展。     

Alkaliphilic bacteria: applications in industrial biotechnology

Alkaliphiles are interesting groups of extremophilic organisms that thrive at pH of 9.0 and above. Many of their products, in particular enzymes, have found widespread applications in industry, primarily in the detergent and laundry industries. While the enzymes have been a runaway success from the industrial point of view, many more products have been reported from alkaliphiles such as antibiotics and carotenoids. Less known are their potential for degradation of xenobiotics. They also play a key role in biogeocycling of important inorganic compounds. This review provides an insight into the huge diversity of alkaliphilic bacteria, the varied products obtained from them, and the need for further investigations on these interesting bacteria.     

Cyclodextrin glucanotransferase production by cell biocatalysts of alkaliphilic bacilli

Cells of obligated alkaliphiles Bacillus pseudalcaliphilus 20RF and Bacillus pseudalcaliphilus 8SB isolated from Bulgarian habitats, producers of cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19), were immobilized by three different techniques: on two types of polysulphone membranes; entrapped in agar-gel beads containing magnetite and by nano-particles of silanized magnetite covalently bound on the cell surface. The biocatalysts obtained demonstrated the opportunity for a significantly enhanced CGTase production compared to free cells for a long period of time (10 days semicontinuous cultivation) without impact on their mechanical stability. The cell membrane-biocatalysts exhibited the highest enzyme activity after 240 h repeated batch cultivation and retained 1.3–2.3-fold increase of the CGTase yield compared to free cells at the end of the process. Membrane biocatalysts were applied for a direct cyclodextrin (CD) production. The results obtained demonstrated the possibility of starch conversion into cyclodextrins by immobilized cells without using of crude or purified enzyme. The membrane biocatalysts of both obligated alkaliphiles formed mainly β- and γ-CDs after 6 h enzyme reaction at pH 9.0 of the reaction mixture. Under these conditions, the quantity of γ-CDs was a relative high, to 35–37% of the total CD amount.     

Facultative alkaliphiles lack fatty acid desaturase activity and lose the ability to grow at near-neutral pH when supplemented with an unsaturated fatty acid.

Two obligate alkaliphiles were found to have high levels of fatty acid desaturase, whereas two facultative alkaliphiles had no detectable activity. Supplementation of the growth medium of one facultative strain with palmitoleic acid, but not palmitic acid, at pH 7.5 inhibited growth. The obligate strain outgrows the facultative strain in a chemostat at a very high pH, whereas the converse is true at a pH of 7.5, and the two strains grow equally well at pH 9.0. Thus, the obligate strain is compromised at a near-neutral pH but is better adapted than a related facultative alkaliphile to an extremely alkaline pH.     

Microbial Thiocyanate Utilization under Highly Alkaline Conditions

Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS⁻) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO⁻) to ammonia and CO₂. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a “cyanate pathway” in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.     

Microbial alkaline proteases: from a bioindustrial viewpoint

Alkaline proteases are of considerable interest in view of their activity and stability at alkaline pH. This review describes the proteases that can resist extreme alkaline environments produced by a wide range of alkalophilic microorganisms. Different isolation methods are discussed which enable the screening and selection of promising organisms for industrial production. Further, strain improvement using mutagenesis and/or recombinant DNA technology can be applied to augment the efficiency of the producer strain to a commercial status. The various nutritional and environmental parameters affecting the production of alkaline proteases are delineated. The purification and properties of these proteases is discussed, and the use of alkaline proteases in diverse industrial applications is highlighted.     

Nonfermentative Thermoalkaliphilic Growth Is Restricted to Alkaline Environments

Caldalkalibacillus thermarum strain TA2.A1 grew in pH-controlled batch culture containing a fermentable growth substrate (i.e., sucrose) from pH 7.5 to 10.0 with no significant change in the specific growth rate, suggesting that this bacterium was a facultative alkaliphile. However, when strain TA2.A1 was grown on a nonfermentable carbon source, such as succinate or malate, no growth was observed until the external pH was >9.0, suggesting that this bacterium was an obligate alkaliphile. Succinate transport and sucrose transport by strain TA2.A1 showed pH profiles similar to that of growth on these carbon sources, and the molar growth yield on sucrose was higher at pH 9.5 than at pH 7.5, despite the increased energy demands on the cell for intracellular pH regulation. Succinate transport, succinate-dependent oxygen consumption, and succinate dehydrogenase and F₁F_o-ATPase specific activities were all significantly lower in cultures of strain TA2.A1 grown at pH 7.5 than in those cultured at pH 9.5. No significant ATP synthesis via the F₁F_o-ATP synthase was detected until the external pH was >8.5. On the basis of these results, we propose that nonfermentative thermoalkaliphilic growth is specialized to function at high pH values, but not at pH values near neutral pH.Alkaliphilic microorganisms have been isolated from a diverse range of environments and have traditionally been classified into two distinct groups based on their pH profile for growth (8). Bacteria that grow across a broad pH range from 7.0 to 11.0 have been classified as facultative alkaliphiles (e.g., Bacillus pseudofirmus OF4) (28), and those that are able to grow only above pH 9.0 have been classified as obligate alkaliphiles (e.g., Bacillus alcalophilus) (4). The reasons why obligate alkaliphiles fail to grow below pH 9.0 remain speculative.While the classification of alkaliphilic bacteria based on pH profiles for growth has gained universal acceptance, it does not consider the nature of the carbon source that is used to grow the cells, and for aerobic alkaliphiles, this may have important consequences. For example, growth on succinate in aerobic bacteria is strictly coupled to oxidative phosphorylation and ATP is produced in the cell via the membrane-bound F₁F_o-ATP synthase. Growth on fermentable carbon sources, such as glucose, allows the cells to bypass this machinery, as ATP can be produced via substrate-level phosphorylation and incomplete oxidation of glucose to acetate can occur.A thermoalkaliphilic bacterium, Bacillus sp. strain TA2.A1, capable of optimal aerobic growth at a temperature of 65°C at pH 9.5 was isolated from an alkaline thermal bore at Mt. Te Aroha, New Zealand (19). The 16S rRNA gene sequence of strain TA2.A1, compared with those available in the EMBL database, shows 99.5% similarity to Caldalkalibacillus thermarum strain HA6^T, an aerobic, heterotrophic, thermophilic bacterium isolated from an alkaline hot spring in China (30). On the basis of the similarity of its phenotypic and genotypic characteristics to those of strain HA6^T, we assign strain TA2.A1 to the genus and species Caldalkalibacillus thermarum. C. thermarum strain TA2.A1 grows on sucrose, common C₄-dicarboxylates, glutamate, pyruvate, and trehalose; however, glucose and fructose fail to support growth (19). We originally described strain TA2.A1 as a facultative alkaliphile based on its pH profile for growth on glutamate or sucrose (18-20); however, both are substrates whose metabolism is not strictly coupled to oxidative phosphorylation.In this communication, we determine the pH profile for growth of C. thermarum strain TA2.A1 on nonfermentable (i.e., succinate and malate) and fermentable carbon sources (i.e., sucrose) using pH-controlled batch culture and demonstrate that strain TA2.A1 was unable to grow below pH 9.0 in pH-controlled batch culture on succinate but grew from pH 7.5 to 10 on sucrose. The physiological and biochemical bases for this phenomenon were investigated.     

Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell''s pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways.     

Microbial diversity of soda lakes

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. Received: January 22, 1998 / Accepted: February 16, 1998     

Rapid access to genes of biotechnologically useful enzymes by partial genome sequencing: the thermoalkaliphile Anaerobranca gottschalkii

Anaerobranca gottschalkii strain LBS3 T is an extremophile living at high temperature (up to 65 degrees C) and in alkaline environments (up to pH 10.5). An assembly of 696 DNA contigs representing about 96% of the 2.26-Mbp genome of A. gottschalkii has been generated with a low-sequence-coverage shotgun-sequencing strategy. The chosen sequencing strategy provided rapid and economical access to genes encoding key enzymes of the mono- and polysaccharide metabolism, without dilution of spare resources for extensive sequencing of genes lacking potential economical value. Five of these amylolytic enzymes of considerable commercial interest for biotechnological applications have been expressed and characterized in more detail after identification of their genes in the partial genome sequence: type I pullulanase, cyclodextrin glycosyltransferase (CGTase), two alpha-amylases (AmyA and AmyB), and an alpha-1,4-glucan-branching enzyme.     

Optimization of extracellular thermotolerant alkaline protease produced by marine <Emphasis Type="Italic">Roseobacter</Emphasis> sp. (MMD040)

Marine endosymbiontic Roseobacter sp. (MMD040), which produced high yields of protease, was isolated from marine sponge Fasciospongia cavernosa, collected from the peninsular coast of India. Maximum production of enzyme was obtained in Luria-Bertani broth. Catabolite repression was observed when the medium was supplemented with readily available carbon sources. The optimum temperature and pH for the enzyme production was 37 degrees C and 7.0, respectively. The enzyme exhibited maximum activity in pH range of 6-9 with an optimum pH of 8.0 and retained nearly 92.5% activity at pH 9.0. The enzyme was stable at 40 degrees C and showed 89% activity at 50 degrees C. Based on the present findings, the enzyme was characterized as thermotolerant alkaline protease, which can be developed for industrial applications.     

Characterization of an Invertase with pH Tolerance and Truncation of Its N-Terminal to Shift Optimum Activity toward Neutral pH

Most invertases identified to date have optimal activity at acidic pH, and are intolerant to neutral or alkaline environments. Here, an acid invertase named uninv2 is described. Uninv2 contained 586 amino acids, with a 100 amino acids N-terminal domain, a catalytic domain and a C-terminal domain. With sucrose as the substrate, uninv2 activity was optimal at pH 4.5 and at 45°C. Removal of N-terminal domain of uninv2 has shifted the optimum pH to 6.0 while retaining its optimum temperaure at 45°C. Both uninv2 and the truncated enzyme retained highly stable at neutral pH at 37°C, and they were stable at their optimum pH at 4°C for as long as 30 days. These characteristics make them far superior to invertase from Saccharomyces cerevisiae, which is mostly used as industrial enzyme.     

Acidophilic Green Alga Pseudochlorella sp. YKT1 Accumulates High Amount of Lipid Droplets under a Nitrogen-Depleted Condition at a Low-pH

Microalgal storage lipids are considered to be a promising source for next-generation biofuel feedstock. However, microalgal biodiesel is not yet economically feasible due to the high cost of production. One of the reasons for this is that the use of a low-cost open pond system is currently limited because of the unavoidable contamination with undesirable organisms. Extremophiles have an advantage in culturing in an open pond system because they grow in extreme environments toxic to other organisms. In this study, we isolated the acidophilic green alga Pseudochlorella sp. YKT1 from sulfuric acid mine drainage in Nagano Prefecture, Japan. The vegetative cells of YKT1 display the morphological characteristics of Trebouxiophyceae and molecular phylogenetic analyses indicated it to be most closely related to Pseudochlorella pringsheimii. The optimal pH and temperature for the growth of YKT1 are pH 3.0–5.0 and a temperature 20–25°C, respectively. Further, YKT1 is able to grow at pH 2.0 and at 32°C, which corresponds to the usual water temperature in the outdoors in summer in many countries. YKT1 accumulates a large amount of storage lipids (∼30% of dry weigh) under a nitrogen-depleted condition at low-pH (pH 3.0). These results show that acidophilic green algae will be useful for industrial applications by acidic open culture systems.     

Identification and characterization of a highly alkaline and thermotolerant novel xylanase from Streptomyces sp.

Xylanases constitute an important industrial enzyme, which hydrolyzes the polysaccharide xylan. In this work, a novel Streptomyces strain producing cellulase-free xylanase was isolated from the soil samples collected from the mangrove forest of Kadalundi, Kerala, India. The strain produced unique enzyme, which exhibited optimal activity at pH 9.0 and tolerance up to pH 12.0. Media engineering was carried out to improve the enzyme production, which showed best enzyme production at 30°C, medium pH 9.0 and incubation time of 48 h. Enzyme was highly thermo-tolerant up to 70°C and alkaline tolerant. Partial gene amplification as well as partial purification of enzyme was carried out to characterize the enzyme. The unique features of the enzyme make it an ideal candidate for industrial application for paper and pulp industry.     

Specific alpha-cyclodextrin production by a novel thermostable cyclodextrin glycosyltransferase

Summary A novel bacterial isolate, Bacillus amyloliquefaciens strain AL 35, produced high yields of a cyclodextrin glycosyltransferase (CGT'ase) when grown in a submerged culture. The stability of CGT'ase to high temperature and alkaline pH enabled processing for cyclodextrin production to be carried out at 60° C and pH 9.0. Crude culture filtrates containing the CGT'ase could convert gelatinized starch substrates to predominatly -cyclodextrins (up to 95% of the total cyclodextrin yields).     

Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m³ fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4–30°C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70–75°C. The enzyme was almost thermostable (91–92%) at pH 6.5 and 9.0 after 41 h preincubation at 55°C and lost only 20–33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0. Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55°C and pH 6.5. Correspondence to: J. Gomes     

Production and characterization of a thermostable alpha-amylase from Nocardiopsis sp. endophyte of yam bean

Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications.     

Cloning of a Phenol Oxidase Gene from Acremonium murorum and Its Expression in Aspergillus awamori

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60°C) and is fully stable for at least 1 h at 60°C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.