Characterization of potential stress responses in ancient Siberian permafrost psychroactive bacteria

Past studies of cold-acclimated bacteria have focused primarily on organisms not capable of sub-zero growth. Siberian permafrost isolates Exiguobacterium sp. 255-15 and Psychrobacter sp. 273-4, which grow at subzero temperatures, were used to study cold-acclimated physiology. Changes in membrane composition and exopolysaccharides were defined as a function of growth at 24, 4 and -2.5 degrees C in the presence and absence of 5% NaCl. As expected, there was a decrease in fatty acid saturation and chain length at the colder temperatures and a further decrease in the degree of saturation at higher osmolarity. A shift in carbon source utilization and antibiotic resistance occurred at 4 versus 24 degrees C growth, perhaps due to changes in the membrane transport. Some carbon substrates were used uniquely at 4 degrees C and, in general, increased antibiotic sensitivity was observed at 4 degrees C. All the permafrost strains tested were resistant to long-term freezing (1 year) and were not particularly unique in their UVC tolerance. Most of the tested isolates had moderate ice nucleation activity, and particularly interesting was the fact that the Gram-positive Exiguobacterium showed some soluble ice nucleation activity. In general the features measured suggest that the Siberian organisms have adapted to the conditions of long-term freezing at least for the temperatures of the Kolyma region which are -10 to -12 degrees C where intracellular water is likely not frozen.     

Bioremediation of hydrocarbon-contaminated polar soils

Bioremediation is increasingly viewed as an appropriate remediation technology for hydrocarbon-contaminated polar soils. As for all soils, the successful application of bioremediation depends on appropriate biodegradative microbes and environmental conditions in situ. Laboratory studies have confirmed that hydrocarbon-degrading bacteria typically assigned to the genera Rhodococcus, Sphingomonas or Pseudomonas are present in contaminated polar soils. However, as indicated by the persistence of spilled hydrocarbons, environmental conditions in situ are suboptimal for biodegradation in polar soils. Therefore, it is likely that ex situ bioremediation will be the method of choice for ameliorating and controlling the factors limiting microbial activity, i.e. low and fluctuating soil temperatures, low levels of nutrients, and possible alkalinity and low moisture. Care must be taken when adding nutrients to the coarse-textured, low-moisture soils prevalent in continental Antarctica and the high Arctic because excess levels can inhibit hydrocarbon biodegradation by decreasing soil water potentials. Bioremediation experiments conducted on site in the Arctic indicate that land farming and biopiles may be useful approaches for bioremediation of polar soils.     

Optimization of procedures for the recovery of heterotrophic bacteria from marine sediments

A study was undertaken of the various factors affecting the recovery of heterotrophic bacteria from marine sediments. The dilution medium and culture medium were found to be of great importance in the recovery of heterotrophic colony forming units (CFU). Statistical analysis of the total viable counts obtained under the test conditions showed that artificial seawater (ASW) without further supplementation was equal to or superior to ASW plus 0.1% peptone or ASW plus 0.1% peptone and 0.1% glycerol. The addition of a surfactive agent, on the other hand, resulted in 95% inhibition of the recoverable CFU. The elapsed time (up to 12 hr) between recovery of a sedimentary core and completion of plating procedures was found to have little effect provided the sedimentary sample was removed from the core, placed in ASW, and stored in a refrigerator until actual plating occurred. It was further noted that lower organic nutrient concentrations, approximately one-tenth of those generally in use, resulted in significantly higher total viable counts. Finally, replicate contiguous sampling at three depths in a core resulted in no significant changes in the number of CFU from the surface samples, indicating a greater surface homogeneity than that previously suspected. The same pattern was not true, however, for samples obtained a lower positions in the core, thus indicating pockets of microbial concentration.     

Optimization of procedures for the recovery of heterotrophic bacteria from marine sediments

A study was undertaken of the various factors affecting the recovery of heterotrophic bacteria from marine sediments. The dilution medium and culture medium were found to be of great importance in the recovery of heterotrophic colony forming units (CFU). Statistical analysis of the total viable counts obtained under the test conditions showed that artificial seawater (ASW) without further supplementation was equal to or superior to ASW plus 0.1% peptone or ASW plus 0.1% peptone and 0.1% glycerol. The addition of a surfactive agent, on the other hand, resulted in 95% inhibition of the recoverable CFU. The elapsed time (up to 12 hr) between recovery of a sedimentary core and completion of plating procedures was found to have little effect provided the sedimentary sample was removed from the core, placed in ASW, and stored in a refrigerator until actual plating occurred. It was further noted that lower organic nutrient concentrations, approximately one-tenth of those generally in use, resulted in significantly higher total viable counts. Finally, replicate contiguous sampling at three depths in a core resulted in no significant changes in the number of CFU from the surface samples, indicating a greater surface homogeneity than that previously suspected. The same pattern was not true, however, for samples obtained a lower positions in the core, thus indicating pockets of microbial concentration. Person to whom correspondence and request for reprints should be sent.     

Mercury-resistant bacteria from permafrost sediments and prospects for their use in comparative studies of mercury resistance determinants

Mercury-resistant bacteria were isolated from permafrost sediments of Kolyma lowland and Canada existing over five thousand to two million years. Their content was shown to vary within the range 0.001-2.9% and to depend on the amount of mercury in sampling sites (coefficient of correlation 0.75). A collection of mercury-resistant bacterial strains was created. In this collection, various representatives of both Gram-positive bacteria (Bacillus, Exiguobacterium, Micrococcus, Arthrobacter) and Gram-negative bacteria (Pseudomonas, Acinetobacter, Plesiomonas, Myxobacteriales) were identified. Most resistant bacteria were found to contain determinants homologous to mer-operons of contemporary bacteria. The isolated strains of paleobacteria are proposed to be used for a comparative structural study of contemporary and ancient plasmids and transposons carrying mercury resistance determinants.     

Characterization of the microbial diversity in a permafrost sample from the Canadian high Arctic using culture-dependent and culture-independent methods

A combination of culture-dependent and culture-independent methodologies (Bacteria and Archaea 16S rRNA gene clone library analyses) was used to determine the microbial diversity present within a geographically distinct high Arctic permafrost sample. Culturable Bacteria isolates, identified by 16S rRNA gene sequencing, belonged to the phyla Firmicutes, Actinobacteria and Proteobacteria with spore-forming Firmicutes being the most abundant; the majority of the isolates (19/23) were psychrotolerant, some (11/23) were halotolerant, and three isolates grew at -5 degrees C. A Bacteria 16S rRNA gene library containing 101 clones was composed of 42 phylotypes related to diverse phylogenetic groups including the Actinobacteria, Proteobacteria, Firmicutes, Cytophaga - Flavobacteria - Bacteroides, Planctomyces and Gemmatimonadetes; the bacterial 16S rRNA gene phylotypes were dominated by Actinobacteria- and Proteobacteria-related sequences. An Archaea 16S rRNA gene clone library containing 56 clones was made up of 11 phylotypes and contained sequences related to both of the major Archaea domains (Euryarchaeota and Crenarchaeota); the majority of sequences in the Archaea library were related to halophilic Archaea. Characterization of the microbial diversity existing within permafrost environments is important as it will lead to a better understanding of how microorganisms function and survive in such extreme cryoenvironments.     

Comparative study of enrichment methods for the isolation of autotrophic nitrifying bacteria from soil, estuarine and marine sediments

Abstract A comparative study has been undertaken to determine the efficiency of methods for the enrichment and isolation of autotrophic nitrifying bacteria from soils and estuarine and marine sediments. Chemostat enrichments proved to be the most efficient means of isolating autotrophic NH⁺₄ oxidisers whereas NO⁻₂ oxidising bacteria were never successfully enriched by this method. In contrast, gel enrichment and traditional batch culture enrichments of nitrifying bacteria were comparatively time consuming procedures and the degree of enrichment obtained for NH⁺₄ oxidising bacteria never approached that obtained with continuous culture enrichments. Gel enrichments, however, because they have continuous physicochemical gradients provide qualitative advantages in that morphologically distinct types of nitrifying bacteria can be isolated from the same gel.     

Environmental effects on CO2 efflux from riparian tundra in the northern foothills of the Brooks Range,Alaska, USA

Summary Carbon dioxide efflux and soil microenvironmental factors were measured diurnally in Carex aquatilus-and Eriophorum angustifolium-dominated riparian tundra communities to determine the relative importance of soil environmental factors controlling ecosystem carbon dioxide exchange with the atmosphere. Measurements were made weekly between 18 June and 24 July 1990. Diurnal patterns in carbon dioxide efflux were best explained by changes in soil temperature, while seasonal changes in efflux were correlated with changes in depth to water table, depth to frozen soil and soil moisture. Carbon dioxide efflux rates were lowest early in the growing season when high water tables and low soil temperatures limited microbial and root activity. Individual rainfall events that raised the water table were found to strongly reduce carbon dioxide efflux. As the growing season progressed, rainfall was low and depth to water table and soil temperatures increased. In response, carbon dioxide efflux increased strongly, attaining rates late in the season of approximately 10 g CO₂ m^–2 day^–1. These rates are as high as maxima recorded for other arctic sites. A mathematical model is developed which demonstrates that soil temperature and depth to water table may be used as efficient predictors of ecosystem CO₂ efflux in this habitat. In parallel with the field measurements of CO₂ efflux, microbial respiration was studied in the laboratory as a function of temperature and water content. Estimates of microbial respiration per square meter under field conditions were made by adjusting for potential respiring soil volume as water table changed and using measured soil temperatures. The results indicate that the effect of these factors on microbial respiration may explain a large part of the diurnal and seasonal variation observed in CO₂ efflux. As in coastal tundra sites, environmental changes that alter water table depth in riparian tundra communities will have large effects on ecosystem CO₂ efflux and carbon balance.     

Application of pretreatments for the isolation of bioactive actinomycetes from marine sediments

Summary Actinomycetes were isolated from marine sediments collected in Sandy Hook Bay, New Jersey. A variety of pretreatments, including heat and phenol, were employed to improve the recovery of these microorganisms. In addition, plating of sediment samples on chitin agar, and filtration through cellulose membrane filters were also utilized. These pretreatments eliminated or severely curtailed the growth of contaminating microorganisms thereby facilitating the isolation of actinomycetes. A total of 120 isolates was obtained, of which 19 displayed significant antimicrobial activity. Most of the activity was directed against gram-positive bacteria, but inhibition of gram-negative species and a yeast were also evident.     

A rapid screening method for the isolation of metal-accumulating microorganisms

Summary An agar plate screening method was developed for the rapid isolation of heavy metal-accumulating microorganisms and preliminary estimation of their biosorption capacity. The test is based on the visulaization and interpretation of the metal distribution between agar and colonies by chemical preciptitation with hydrogen sulphide or ammonium sulphide. The heavy metals silver, thallium, lead, copper, nickel and cadmium have been tested successfully. The efficiency of the method is demonstrated for isolating silver-accumulating bacterian and estimating silver biosorption capacity.     

Study on the potential of cold-active lipases from psychrotrophic fungi for detergent formulation

Lipases from psychrotrophic fungal isolates BPF4 and BPF6 identified as Penicilium canesense and Pseudogymnoascus roseus respectively were characterized for their compatibility towards laundry detergent. BPF4 and BPF6 lipases showed maximum activity at pH 11 and 9 respectively and at 40?°C. The residual activities at 20?°C and 4?°C of BPF4 lipase were 35% and 20% and of BPF6 lipase were 70% and 20?°C respectively. Both the enzymes were stable at 4?°C, 20?°C and 40?°C for 2?h losing at the most 20% of activities. Both the enzymes were metalloenzymes with activity enhancement by nearly threefold by Ca²⁺. Contrary to BPF6 lipase, BPF4 enzyme was not stimulated by EDTA nor inhibited, rather stimulated by SDS and Triton X-100 by 125% and 330% respectively. Both the lipases showed minor to moderate inhibition by NaClO₃ and H₂O₂, and exhibited nearly 90% residual activity after 1?h of incubation in selected detergent brands thus indicating potential for their inclusion in detergent formulation thereby facilitating cold-washing as a step towards mitigation of climate change.     

The fungus Penicillium citrinum Thom 1910 VKM FW-800 isolated from ancient permafrost sediments as a producer of the ergot alkaloids agroclavine-1 and epoxyagroclavine-1

The study of the secondary metabolites of the relict strain Penicillium citrinum VKM FW-800 isolated from ancient Arctic permafrost sediments showed that this fungus produces agroclavine-1 and epoxyagroclavine-1, which are rare ergot alkaloids with the 5R,10S configuration of the tetracyclic ergoline ring system. The production of the alkaloids by the fungus showed a biphasic behavior, being intense in the phase of active growth and slowing down in the adaptive lag phase and in the stationary growth phase. The addition of zinc ions to the incubation medium led to a fivefold increase in the yield of the alkaloids. The alkaloids-producing Penicillium fungi isolated from different regions exhibited the same tendencies of growth and alkaloid production.     

Strategies for isolation of in vivo expressed genes from bacteria

The discovery and characterization of genes specifically induced in vivo upon infection and/or at a specific stage of the infection will be the next phase in studying bacterial virulence at the molecular level. Genes isolated are most likely to encode virulence-associated factors or products essential for survival, bacterial cell division and multiplication in situ. Identification of these genes is expected to provide new means to prevent infection, new targets for, antimicrobial therapy, as well as new insights into the infection process. Analysis of genes and their sequences initially discovered as in vivo induced may now be revealed by functional and comparative genomics. The new field of virulence genomics and their clustering as pathogenicity islands makes feasible their in-depth analysis. Application of new technologies such as in vivo expression technologies, signature-tagged mutagenesis, differential fluorescence induction, differential display using polymerase chain reaction coupled to bacterial genomics is expected to provide a strong basis for studying in vivo induced genes, and a better understanding of bacterial pathogenicity in vivo. This review presents technologies for characterization of genes expressed in vivo.     

Rare actinomycete bacteria from the shallow water sediments of the Trondheim fjord, Norway: isolation, diversity and biological activity

Actinomycete bacteria produce a wide variety of secondary metabolites with diverse biological activities, some of which have been developed for human medicine. Rare actinomycetes are promising sources in search for new drugs, and their potential for producing biologically active molecules is poorly studied. In this work, we have investigated the diversity of actinomycetes in the shallow water sediments of the Trondheim fjord (Norway). Due to the use of different selective isolation methods, an unexpected variety of actinomycete genera was isolated. Although the predominant genera were clearly Streptomyces and Micromonospora, representatives of Actinocorallia, Actinomadura, Knoellia, Glycomyces, Nocardia, Nocardiopsis, Nonomuraea, Pseudonocardia, Rhodococcus and Streptosporangium genera were isolated as well. To our knowledge, this is the first report describing isolation of Knoellia and Glycomyces species from the marine environment. 35 selected actinomycete isolates were characterized by 16S rDNA sequencing, and were shown to represent strains from 11 different genera. In addition, these isolates were tested for antimicrobial activity and the presence of polyketide synthase and non-ribosomal peptide synthetase genes. This study confirms the significant biodiversity of actinobacteria in the Norwegian marine habitats, and their potential for producing biologically active compounds.     

Soil salinity: A serious environmental issue and plant growth promoting bacteria as one of the tools for its alleviation

Salinity is one of the most brutal environmental factors limiting the productivity of crop plants because most of the crop plants are sensitive to salinity caused by high concentrations of salts in the soil, and the area of land affected by it is increasing day by day. For all important crops, average yields are only a fraction – somewhere between 20% and 50% of record yields; these losses are mostly due to drought and high soil salinity, environmental conditions which will worsen in many regions because of global climate change. A wide range of adaptations and mitigation strategies are required to cope with such impacts. Efficient resource management and crop/livestock improvement for evolving better breeds can help to overcome salinity stress. However, such strategies being long drawn and cost intensive, there is a need to develop simple and low cost biological methods for salinity stress management, which can be used on short term basis. Microorganisms could play a significant role in this respect, if we exploit their unique properties such as tolerance to saline conditions, genetic diversity, synthesis of compatible solutes, production of plant growth promoting hormones, bio-control potential, and their interaction with crop plants.     

Electropositively charged filters for the recovery of yeasts and bacteria from beverages

The ability of electropositively charged filters to recover yeasts and lactic acid bacteria from a variety of beverages was evaluated. Filtration through 'Zeta plus', grade O5S, filters recovered nearly all of the yeast contaminants from table wines, sherry and port. Recovery of yeasts from cream liqueurs and egg-based beverages was also good but it was not possible to filter drinks containing orange juice, even through filters with nominal pore sizes of 2 to 10 micron. Lactic acid bacteria proved more difficult to recover than yeasts, even though smaller pore-sized filters (1 to 4 micron) were employed. However, a sufficiently high percentage of bacteria were recovered to justify use of these filters for quality assurance. The advantage of concentrating contaminants by using charged filters, and the influence of product composition on the efficiency of microbial adsorption are discussed. The growth of wine-spoiling yeasts and lactic acid bacteria were not inhibited by water- or ethanol-soluble extracts of the filter material.