Effects of chaotropic salts on global proteome stability in halophilic archaea: Implications for life signatures on Mars

Halophilic archaea thriving in hypersaline environments, such as salt lakes, offer models for putative life in extraterrestrial brines such as those found on Mars. However, little is known about the effect of the chaotropic salts that could be found in such brines, such as MgCl₂, CaCl₂ and (per)chlorate salts, on complex biological samples like cell lysates which could be expected to be more representative of biomarkers left behind putative extraterrestrial life forms. We used intrinsic fluorescence to study the salt dependence of proteomes extracted from five halophilic strains: Haloarcula marismortui, Halobacterium salinarum, Haloferax mediterranei, Halorubrum sodomense and Haloferax volcanii. These strains were isolated from Earth environments with different salt compositions. Among the five strains that were analysed, H. mediterranei stood out as a results of its high dependency on NaCl for its proteome stabilization. Interestingly, the results showed contrasting denaturation responses of the proteomes to chaotropic salts. In particular, the proteomes of strains that are most dependent or tolerant on MgCl₂ for growth exhibited higher tolerance towards chaotropic salts that are abundant in terrestrial and Martian brines. These experiments bridge together global protein properties and environmental adaptation and help guide the search for protein-like biomarkers in extraterrestrial briny environments.     

Phytoplankton nutrient deficiency in lakes of the McMurdo dry valleys, Antarctica

1. The influence of inorganic nitrogen and phosphorus enrichment on phytoplankton photosynthesis was investigated in Lakes Bonney (east and west lobes), Hoare, Fryxell and Vanda, which lie in the ablation valleys adjacent to McMurdo Sound, Antarctica. Bioassay experiments were conducted during the austral summer on phytoplankton populations just beneath the permanent ice cover in all lakes and on populations forming deep-chlorophyll maxima in the east and west lobes of Lake Bonney. 2. Phytoplankton photosynthesis in surface and mid-depth (13 m) samples from both lobes of Lake Bonney were stimulated significantly (P < 0.01) by phosphorus enrichment (2 μM) with further stimulation by simultaneous phosphorus plus NH₄⁺ (20 μM) enrichment. Similar trends were observed in deeper waters (18 m) from the east lobe of Lake Bonney, although they were not statistically significant at P < 0.05. Photosynthesis in this lake was never enhanced by the addition of 20 μM NH₄⁺ alone. Simultaneous addition of phosphorus plus nitrogen stimulated photosynthesis significantly (P < 0.01) in both Lake Hoare and Lake Fryxell. No nutrient response occurred in Lake Vanda, where activity in nutrient-enriched samples was below unamended controls; results from Lake Vanda are suspect owing to excessively long sample storage in the field resulting from logistic constraints. 3. Ambient dissolved inorganic nitrogen (DIN) (NH4⁺+ NO₂^?+ NO₃^?): soluble reactive phosphorus (SRP) ratios partially support results from bioassay experiments indicating strong phosphorus deficiency in Lake Bonney and nitrogen deficiency in Lakes Hoare and Fryxell. DIN : SRP ratios also imply phosphorus deficiency in Lake Vanda, although not as strong as in Lake Bonney. Particulate carbon (PC): particulate nitrogen (PN) ratios all exceed published ratios for balanced phytoplankton growth, indicative of nitrogen deficiency. 4. Vertical nutrient profiles in concert with low advective flux, indicate that new (sensu Dugdale & Goering, 1967) phytoplankton production in these lakes is supported by upward diffusion of nutrients from deep nutrient pools. This contention was tested by computing upward DIN : SRP flux ratios across horizontal planes located immediately beneath each chlorophyll maximum and about 2 m beneath the ice (to examine flux to the phytoplankton immediately below the ice cover). These flux ratios further corroborated nutrient bioassay results and bulk DIN : SRP ratios indicating phosphorus deficiency in Lakes Bonney and Vanda and potential nitrogen deficiency in Lakes Hoare and Fryxell. 5. Neither biochemical reactions nor physical processes appear to be responsible for differences in nutrient deficiency among the study lakes. The differences may instead be related to conditions which existed before or during the evolution of the lakes.     

Manganese Reduction by Microbes from Oxic Regions of the Lake Vanda (Antarctica) Water Column

Depth profiles of metals in Lake Vanda, a permanently ice-covered, stratified Antarctic lake, suggest the importance of particulate manganese oxides in the scavenging, transport, and release of metals. Since manganese oxides can be solubilized by manganese-reducing bacteria, microbially mediated manganese reduction was investigated in Lake Vanda. Microbes concentrated from oxic regions of the water column, encompassing a peak of soluble manganese [Mn(II)], reduced synthetic manganese oxides (MnO₂) when incubated aerobically. Pure cultures of manganese-reducing bacteria were readily isolated from waters collected near the oxic Mn(II) peak. Based on phylogenetic analysis of the 16S rRNA gene sequence, most of the isolated manganese reducers belong to the genus Carnobacterium. Cultures of a phylogenetically representative strain of Carnobacterium reduced synthetic MnO₂ in the presence of sodium azide, as was seen in field assays. Unlike anaerobes that utilize manganese oxides as terminal electron acceptors in respiration, isolates of the genus Carnobacterium reduced Mn(IV) via a diffusible compound under oxic conditions. The release of adsorbed trace metals accompanying the solubilization of manganese oxides may provide populations of Carnobacterium with a source of nutrients in this extremely oligotrophic environment.     

Halophilic and salts tolerant protoplast cultures of mangrove plants, Sonneratia alba and Avicennia alba

The effects of sea salts, NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂, on the growth of protoplast cultures of two mangrove species, Sonneratia alba and Avicennia alba, were investigated using 96-well culture plates. Plants of these two species naturally grow at the seaward side of a mangrove forest. Cotyledon protoplasts of S. alba showed halophilic nature to NaCl, KCl, and MgCl₂ at low concentrations (10–50 mM) when cultured in Murashige and Skoog’s (MS) medium containing 0.6 M mannitol. CaCl₂ at a concentration higher than 25 mM was inhibitory to cell growth. On the other hand, in protoplast culture of A. alba suspension cells, which were induced from cotyledon tissues, in the modified amino acid (mAA) medium containing 1.2 M sorbitol, tolerance to NaCl, MgCl₂ and MgSO₄ were observed at a wide range of concentrations up to 400 mM. CaCl₂ was always inhibitory for cell divisions in A. alba, but stimulatory for spherical enlargement of cells. However, no difference in cell enlargement was observed among other salts. Similarity and difference in reactivity to salts between protoplasts and suspension cells from our previous studies were discussed in relation to the site of salt tolerance or halophilic adaptation within mangrove cells. For protoplast cultures, the site(s) for response of S. alba and A. alba are located in the cytoplasm and/or the cell membrane.     

Identification of extremely halophilic archaea associated with adult <Emphasis Type="Italic">Artemia urmiana</Emphasis>

The brine shrimp, Artemia is the dominant macrozooplankton present in many hypersaline environments. Artemia urmiana is the only macroscopic organism in Urmia Salt Lake (Iran), and the high salinity of the lake makes it a suitable environment for halophilic archaea too. Because of common environment for Artemia and extreme halophiles; this investigation is concentrated on studying the relationship between Artemia and halophilic archaea in Urmia Lake. In this study first the procedure of arhaea isolation was done. Then, isolated strains were sub-cultured and DNA was extracted and amplified by PCR using specific primers for amplifying archaeal 16S rRNA. The amplified archeal DNA fragments were purified, and sequenced. 16S rRNA sequences were compared to known sequences using the NCBI BLAST program. Sequences relating to Halorubrum, Haloarcula and Halobacterium species were identified in Urmia Salt Lake water and Artemia adults and the phylogenetic tree of different species was constructed. Only Halorubrum species were present in association with Artemia. They belong to Halobacteriaceae family of archeae which are isolated from different salt lakes in different parts of world and we could show their existence in adult Artemia, another organism living in hypersaline enviroments.     

Ostracods and the Holocene palaeolimnology of Lake Qarun, with special reference to past human–environment interactions in the Faiyum (Egypt)

We present an ostracod record covering the past two millennia from an 8.25-m core taken from Lake Qarun, in the Faiyum Depression of Egypt. The occurrence of ostracod species in the lake is controlled primarily by variations in solute composition, which are in turn related to shifts in catchment land use. At times when the Faiyum Depression supported thriving agriculture, lake water contained Na⁺–Cl^? brine, and Cyprideis torosa dominated the ostracod assemblage. When the Faiyum Depression experienced periods of environmental and economic decline, lake water contained Na⁺–HCO₃ ^? brine, and Limnocythere inopinata dominated. The relative abundance of other ostracod species provides additional information about past conditions in Lake Qarun including salinity and lake level changes. Overall, the ostracod assemblages provide evidence for human influences in the Faiyum, which extend back before instrumental or detailed observational records began.     

The cycling of nutrients in a closed-basin antarctic lake: Lake Vanda

Lake Vanda is a permanently ice covered, meromictic, closed basin lake, located in the Dry Valley region of Southern Victoria Land, Antarctica. A unique feature of the lake water column structure is that the bottom lake waters exist as a natural diffusion cell. The diffusive nature of these waters allows rates of sulfate reduction, nitrification and denitrification to be calculated from nutrient concentration gradients. Calculation reveals that sulfate reduction is by far the most important anoxic process acting to oxidize organic material. In addition, rate calculations reveal that bottom water nutrient profiles are in steady state. One argument in support of this conclusion is that the calculated rate of nitrification balances the flux of ammonia from the anoxic lake waters. The flux of phosphorus from the reducing waters is several times less than would be predicted from the nitrogen and phosphorus content of decomposing lake seston. Solubility calculations show that phosphorus may be actively removed at depth in Lake Vanda by the formation of hydroxyapatite. It is found that estimated rates of nitrogen and phosphorus removal in the bottom lake waters and sediments roughly balance the riverine input flux. This suggests that throughout the lake a nutrient steady state may exist, and that the anoxic zone may be the most important loci for nutrient removal. Finally, the ratio of nitrogen to phosphorus entering Lake Vanda by riverine input is less than the Redfield ratio of 16/1; in contrast to the lake waters which are strongly phosphorus limited at all depths. This curious aspect of the lake's nutrient chemistry is explained by the presence of preformed nitrogen, which has been concentrated in the deep brine due to several episodes of evaporative concentration.     

Microbial diversity and activity in Southern California salterns and bitterns: analogues for remnant ocean worlds

Concurrent osmotic and chaotropic stress make MgCl₂-rich brines extremely inhospitable environments. Understanding the limits of life in these brines is essential to the search for extraterrestrial life on contemporary and relict ocean worlds, like Mars, which could host similar environments. We sequenced environmental 16S rRNA genes and quantified microbial activity across a broad range of salinity and chaotropicity at a Mars-analogue salt harvesting facility in Southern California, where seawater is evaporated in a series of ponds ranging from kosmotropic NaCl brines to highly chaotropic MgCl₂ brines. Within NaCl brines, we observed a proliferation of specialized halophilic Euryarchaeota, which corresponded closely with the dominant taxa found in salterns around the world. These communities were characterized by very slow growth rates and high biomass accumulation. As salinity and chaotropicity increased, we found that the MgCl₂-rich brines eventually exceeded the limits of microbial activity. We found evidence that exogenous genetic material is preserved in these chaotropic brines, producing an unexpected increase in diversity in the presumably sterile MgCl₂-saturated brines. Because of their high potential for biomarker preservation, chaotropic brines could therefore serve as repositories of genetic biomarkers from nearby environments (both on Earth and beyond) making them prime targets for future life-detection missions.     

Cold-active halophilic bacteria from the ice-sealed Lake Vida,Antarctica

Lake Vida is a large, permanently ice-covered lake in the Victoria Valley of the McMurdo Dry Valleys, Antarctica and is unique among Dry Valley lakes because it is ice-sealed, with an ice-cover of nearly 19 m. Enrichment cultures of melt-water from Lake Vida 15.9 m ice yielded five pure cultures of aerobic, heterotrophic bacteria. Of these, one strain grew at −8°C and the four others at −4°C. All isolates were either halotolerant or halophilic, with two strains capable of growth at 15% NaCl. Phylogenetic analysis revealed the Lake Vida isolates to be Gammaproteobacteria, related to species of Psychrobacter and Marinobacter. This is the first report of pure cultures of bacteria from Lake Vida, and the isolates displayed a phenotype consistent with life in a cold hypersaline environment.     

Biodegradation of Benzene by Halophilic and Halotolerant Bacteria under Aerobic Conditions

A highly enriched halophilic culture was established with benzene as the sole carbon source by using a brine soil obtained from an oil production facility in Oklahoma. The enrichment completely degraded benzene, toluene, ethylbenzene, and xylenes within 1 to 2 weeks. Also, [¹⁴C]benzene was converted to ¹⁴CO₂, suggesting the culture's ability to mineralize benzene. Community structure analysis revealed that Marinobacter spp. were the dominant members of the enrichment.     

Chemodenitrification in the cryoecosystem of Lake Vida,Victoria Valley,Antarctica

Lake Vida, in the Victoria Valley of East Antarctica, is frozen, yet harbors liquid brine (~20% salt, >6 times seawater) intercalated in the ice below 16 m. The brine has been isolated from the surface for several thousand years. The brine conditions (permanently dark, ?13.4 °C, lack of O₂, and pH of 6.2) and geochemistry are highly unusual. For example, nitrous oxide (N₂O) is present at a concentration among the highest reported for an aquatic environment. Only a minor ¹⁷O anomaly was observed in N₂O, indicating that this gas was predominantly formed in the lake. In contrast, the ¹⁷O anomaly in nitrate () in Lake Vida brine indicates that approximately half or more of the present is derived from atmospheric deposition. Lake Vida brine was incubated in the presence of ¹⁵N‐enriched substrates for 40 days. We did not detect microbial nitrification, dissimilatory reduction of to ammonium (), anaerobic ammonium oxidation, or denitrification of N₂O under the conditions tested. In the presence of ¹⁵N‐enriched nitrite (), both N₂ and N₂O exhibited substantial ¹⁵N enrichments; however, isotopic enrichment declined with time, which is unexpected. Additions of ¹⁵N– alone and in the presence of HgCl₂ and ZnCl₂ to aged brine at ?13 °C resulted in linear increases in the δ¹⁵N of N₂O with time. As HgCl₂ and ZnCl₂ are effective biocides, we interpret N₂O production in the aged brine to be the result of chemodenitrification. With this understanding, we interpret our results from the field incubations as the result of chemodenitrification stimulated by the addition of ¹⁵N‐enriched and ZnCl₂ and determined rates of N₂O and N₂ production of 4.11–41.18 and 0.55–1.75 nmol L^?1 day^?1, respectively. If these rates are representative of natural production, the current concentration of N₂O in Lake Vida could have been reached between 6 and 465 years. Thus, chemodenitrification alone is sufficient to explain the high levels of N₂O present in Lake Vida.     

Comparison of Bacterial Biodiversity and Enzyme Production in Three Hypersaline Lakes; Urmia,Howz-Soltan and Aran-Bidgol

This research is a comparative study on the diversity of halophilic bacteria with hydrolytic activities in three significant hypersaline lakes; Urmia in the northwest and Howz-Soltan and Aran-Bidgol in the central desert in Iran. Isolated strains from these saline lakes were found to be halotolerant, moderately and extremely halophilic bacteria. The bacteria in each saline lake were able to produce different hydrolytic enzymes including amylase, protease, lipase, DNase, inulinase, xylanase, carboxy methyl cellulase, pectinase and pullulanase. 188, 302, 91 halophilic strains were isolated from Urmia Lake, Howz-Soltan and Aran-Bidgol playa, respectively. The numbers of Gram-positive strains were more than Gram-negatives, and among Gram-positive bacteria; spore-forming bacilli were most abundant. Due to the unique physico-chemical conditions of the lake environments, the hydrolytic activities of isolated strains were significantly different. For instance, isolated strains from Howz-Soltan playa did not produce pectinase, DNase, amylase, lipase and inulinase, while the isolates from Aran-Bidgol playa had a great ability to produce pectinase and DNase. The strains from Urmia Lake were also good producers of DNase but failed to show any chitinase activity. The diversity of halophilic bacteria from the mentioned three saline lakes was also determined using PCR-amplified 16S rRNA followed by phylogenetic analysis of the partial 16S rRNA sequences.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0481-9) contains supplementary material, which is available to authorized users.     

青藏高原察尔汗盐湖地区可培养中度嗜盐菌的群落结构与多样性

【目的】研究青海察尔汗盐湖地区的可培养中度嗜盐菌的群落结构及多样性。【方法】采用多种选择性培养基进行中度嗜盐菌的分离、培养;通过16S r RNA基因序列扩增、测定,根据序列信息,进行系统进化树构建、群落结构组成分析及多样性指数计算。【结果】从察尔汗盐湖卤水及湖泥中分离到中度嗜盐菌421株,合并重复菌株后共83株中度嗜盐菌。菌株16S rRNA基因序列信息显示,4株中度嗜盐菌为潜在的新分类单元。83株嗜盐细菌分布于3个门的6个科16个属。其中,Bacillus属、Oceanobacillus属和Halomonas属为优势属。多样性结果显示,水样中的菌株多样性高于泥样,而泥样中的菌株优势度高于水样。【结论】察尔汗盐湖中度嗜盐菌具有丰富的遗传多样性,种群种类丰富,优势菌群集中,该盐湖地区存在可分离培养的中度嗜盐菌的疑似新物种。     

Bacterial chitin utilisation at extremely haloalkaline conditions

Chitin is produced in large amounts in hypersaline habitats with neutral pH due to the high biomass production of brine shrimp Artemia. Recently, a high abundance of Artemia was also noticed in hypersaline soda lakes in the Kulunda Steppe (Altai, Russia), which prompted us to survey the possibility of microbial chitin utilization at extremely haloalkaline conditions in soda brines. Most active chitin utilisation-supporting microbial growth was found at anaerobic conditions at pH 10 and up to 3.5?M total Na⁺. At aerobic conditions, the degradation of chitin was slower, mostly incomplete and active at <2?M total Na⁺, although very slow partial degradation was possible up to 4?M Na⁺. Anaerobic enrichments at pH 10 yielded two different groups of obligately haloalkaliphilic fermentative anaerobes, exclusively specialized to utilise insoluble chitin as the only growth substrate. One group was represented by a single strain growing at moderate salinity, and another comprised multiple isolates growing up to 3.5?M Na⁺. These groups represent two novel bacterial phyla not closely related to any other cultured bacteria. Aerobic enrichments from the lake sediments were dominated by several obligately haloalkaliphilic members of the genus Marinimicrobium in the Gammaproteobacteria. They were less specialised than the anaerobes and grew with chitin and its monomer and oligomers at a pH of 10 up to 2.5?M Na⁺. Furthermore, several strains of haloalkaliphilic Gram-positive chitinolytics belonging to bacilli and actinobacteria were isolated from soda lake sediments and surrounding soda soils. In general, the results indicate the presence of an active and diverse haloalkaliphilic chitinolytic microbial community in hypersaline soda habitats.     

A comparison and optimization of methods and factors affecting the transformation of Escherichia coli

DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl₂–CaCl₂, CaCl₂ and Hanahan''s methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan''s method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl₂ method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria–Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109′s competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria.     

Effects of mineral salts on the growth,sporulation and virulence of Esteya vermicola,an endoparasitic fungus of the pinewood nematode,Bursaphelenchus xylophilus

Esteya vermicola, an endoparasitic fungus of pinewood nematode, exhibits great potential as a biological agent against nematodes. In this study, various mineral supplements, such as chloride salts (KCl, CaCl₂, MgCl₂, FeCl₂, and FeCl₃) and calcium salts (CaCl₂, CaCO₃, and CaSO₄) were evaluated for their ability to enhance the growth, sporulation and virulence of E. vermicola. Of the cations tested, CaCl₂ provided the greatest enhancement of growth speed and sporulation. Of the anions tested, CaCO₃ produced the highest proportion of lunate conidia, and CaCl₂ produced the highest adhesive rate and mortality against the nematode, Bursaphelenchus xylophilus. The optimum concentration of CaCl₂ for optimization of sporulation and virulence was 0.4–0.6%. In conclusion, CaCl₂ is highly effective in enhancing growth, sporulation and virulence of Esteya vermicola.     

Isolation and identification of moderately halophilic bacteria producing hydrolytic enzymes from the largest hypersaline playa in Iran

Iran has many hypersaline environments, both the permanent and seasonal ones. One of the seasonal hypersaline lakes in the central desert zone is Aran-Bidgol Lake in which microbial diversity has not been characterized, thus the potential usage of this microbial community in biotechnology remained unknown. In this study, screening the halophilic hydrolytic enzyme-producing bacteria from different areas of this lake led to isolation of 61 gram-positive and 22 gram-negative moderately halophilic bacteria. These bacterial isolates were shown to produce a wide variety of hydrolytic enzymes including DNase, inulinase, amylase, lipase, pectinase, protease, chitinase, pullulanase, cellulase, and xylanase. The most common enzymes were DNase and inulinase in gram-positive bacteria, lipase in gram-negative bacteria, and pullulanase and cellulase in gram-positive cocci. Interestingly, combined hydrolytic activates were observed in some isolates. According to their phenotypic characteristics and comparative partial 16S rRNA sequence analysis, the moderately halophilic strains belonged to the genera Halobacillus, Thalassobacillus, Bacillus, Salinicoccus, Idiomarina, Salicola, and Halomonas.     

Isolation and Characterization of Strains CVO and FWKO B, Two Novel Nitrate-Reducing, Sulfide-Oxidizing Bacteria Isolated from Oil Field Brine

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O₂). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO₂ as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO₂. Both strains grow at temperatures between 5 and 40°C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.     

Formation and Regeneration of Protoplasts of the Actinorhizal Nitrogen-Fixing Actinomycete Frankia

Procedures for forming and regenerating protoplasts of four Frankia strains are described. Cells obtained from growth medium containing 0.1% glycine were digested with lysozyme (250 μg/ml) in a medium containing 0.5 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂. Protoplasts were formed during 15 to 120 min of digestion at 25°C. Optimum conditions for protoplast regeneration involved placing protoplasts on a layer of complex growth medium containing 0.3 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂ which was overlaid with a layer of 0.8% low-melting-point agarose containing 0.5 M sucrose, 5.0 mM MgCl₂, and 5.0 mM CaCl₂. The maximum regeneration efficiency was 36.9% for strain CpI1, 1.3% for strain ACN1^AG, 27% for strain EAN1pec, and 20% for strain EuI1c.     

Growth Potential of Halophilic Bacteria Isolated from Solar Salt Environments: Carbon Sources and Salt Requirements

Eighteen strains of extremely halophilic bacteria and three strains of moderately halophilic bacteria were isolated from four different solar salt environments. Growth tests on carbohydrates, low-molecular-weight carboxylic acids, and complex medium demonstrated that the moderate halophiles and strains of the extreme halophiles Haloarcula and Halococcus grew on most of the substrates tested. Among the Halobacterium isolates were several metabolic groups: strains that grew on a broad range of substrates and strains that were essentially confined to either amino acid (peptone) or carbohydrate oxidation. One strain (WS-4) only grew well on pyruvate and acetate. Most strains of extreme halophiles grew by anaerobic fermentation and possibly by nitrate reduction. Tests of growth potential in natural saltern brines demonstrated that none of the halobacteria grew well in brines which harbor the densest populations of these bacteria in solar salterns. All grew best in brines which were unsaturated with NaCl. The high concentrations of Na⁺ and Mg²⁺ found in saltern crystallizer brines limited bacterial growth, but the concentrations of K⁺ found in these brines had little effect. MgSO₄ was relatively more inhibitory to the extreme halophiles than was MgCl₂, but the reverse was true for the moderate halophiles.