Ice-active characteristics of soil bacteria selected by ice-affinity

As an initial screen for microorganisms that produce ice-active macromolecules, ice-affinity was used to select microorganisms from soil consortia originating from three temperate regions. Once selected and subsequently purified to single colonies, these microbes were putatively identified by 16S ribosomal RNA sequencing and assayed for various ice-active properties. Ice-affinity selection appeared to select for bacteria with ice-associating activities: inhibition of ice recrystallization; ice nucleation; ice shaping. Although none of these activities were observed in Paenibacillus amyloliticus C8, others such as Chryseobacterium sp. GL8, demonstrated both ice recrystallization inhibition and ice-shaping activities. Pseudomonas borealis DL7 was classified as a type I ice nucleator, Flavobacterium sp. GL7, was identified as a type III ice nucleator and Acinetobacter radioresistens DL5 demonstrated ice recrystallization inhibition. In all, 19 different culturable bacteria were selected from the thousands of microbes in late-summer collected soil samples. Many of the selected microbes have been previously reported in glacial ice cores or polar sea ice, and of five isolates that were further characterized, four showed ice-associating activities. These results indicate the significant potential of ice-affinity selection even with temperate climate soils, suggesting that sampling in more extreme and remote areas is not required for the isolation of ice-active bacteria.     

Soil microbial communities buffer physiological responses to drought stress in three hardwood species

Trees possess myriad adaptations for coping with drought stress, but the extent to which their drought responses are influenced by interactions with soil microbes is poorly understood. To explore the role of microbes in mediating tree responses to drought stress, we exposed saplings of three species (Acer saccharum, Liriodendron tulipifera, and Quercus alba) to a four week experimental drought in mesocosms. Half of the pots were inoculated with a live soil slurry (i.e., a microbial inoculum derived from soils beneath the canopies of mature A. saccharum, L. tulipifera or Q. alba stands), while the other half of the pots received a sterile soil slurry. Soil microbes ameliorated drought stress in L. tulipifera by minimizing reductions in leaf water potential and by reducing photosynthetic declines. In A. saccharum, soil microbes reduced drought stress by lessening declines in leaf water potential, though these changes did not buffer the trees from declining photosynthetic rates. In Q. alba, soil microbes had no effects on leaf physiological parameters during drought stress. In all species, microbes had no significant effects on dynamic C allocation during drought stress, suggesting that microbial effects on plant physiology were unrelated to source–sink dynamics. Collectively, our results suggest that soil microbes have the potential to alter key parameters that are used to diagnose drought sensitivity (i.e., isohydry or anisohydry). To the extent that our results reflect dynamics occurring in forests, a revised perspective on plant hydraulic strategies that considers root-microbe interactions may lead to improved predictions of forest vulnerability to drought.     

Cadmium stress studies: Media development, enrichment, consortia analysis, and environmental relevance

The effects of Cadmium (Cd) toxicity on bacterial consortia originating from an-aerobic sewage sludge and cultivated under differing enrichment conditions were studied. Cultures were enriched in minimal media developed specifically for Cd stress studies. At inoculation all Cd was soluble in free ion or chelated form. Electron donors and acceptors were varied to obtain each physiological enrichment type. Adaptation leading to higher levels of Cd resistance of the consortia over time was observed under all physiological conditions. Initial and increased Cd tolerances were consistently greatest in multiphysiological enrichments (MPH). Sulfate reducing (SRB), methanogenic (MET), and fermentative (FRM) enrichments had less tolerance however, the level of tolerance to the Cd varied from one inoculation to the next. The Cd remained soluble as free Cd in MPH and FRM conditions and was precipitated significantly in SRB and moderately in MET conditions. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR) amplified 16S rRNA of the SRB, MPH, and FRM enrichments were followed over time. The consortia underwent succession under all physiological conditions when compared with the profile of the inoculum. Microbial population diversity decreased as the consortia were subcultured. The effects of chelators in the MPH medium were also evaluated. The addition of chelators transiently decreased toxicity. Effects of MPH medium on the Cd sorption capacity of soil were evaluated. Microbial growth decreased the amount of Cd left in solution.     

Characterization of two diesel fuel degrading microbial consortia enriched from a non acclimated,complex source of microorganisms

Background  

The bioremediation of soils impacted by diesel fuels is very often limited by the lack of indigenous microflora with the required broad substrate specificity. In such cases, the soil inoculation with cultures with the desired catabolic capabilities (bioaugmentation) is an essential option. The use of consortia of microorganisms obtained from rich sources of microbes (e.g., sludges, composts, manure) via enrichment (i.e., serial growth transfers) on the polluting hydrocarbons would provide bioremediation enhancements more robust and reproducible than those achieved with specialized pure cultures or tailored combinations (co-cultures) of them, together with none or minor risks of soil loading with unrelated or pathogenic allocthonous microorganisms.     

Enrichment of microbial cultures able to degrade 1,3-dichloro-2-propanol: a comparison between batch and continuous methods

Microbial cultures able to degrade xenobiotic compounds are the key element for biological treatment of waste effluents and are obtained from enrichment processes. In this study, two common enrichment methods, suspension batch and immobilized continuous, were compared. The main selection factor was the presence of 1,3-dichloro-2-propanol (1,3-DCP) as the single carbon source. Both methods have successfully enriched microbial consortia able to degrade 1,3-DCP. When tested in batch culture, the degradation rates of 1,3-DCP by the two consortia were different, with the consortia obtained by batch enrichment presenting slightly higher rates. A preliminary morphological and biochemical analysis of the predominant colonial types present in each degrading consortia revealed the presence of different constituting strains. Three bacterial isolates capable of degrading 1,3-DCP as single strains were obtained from the batch enrichments. These strains were classified by 16S rRNA analysis as belonging to the Rhizobiaceae group. Degradation rates of 1,3-DCP were lower when single species were used, reaching 45 mg l^-1 d^-1, as compared to 74 mg l^-1 d^-1 of the consortia enriched on the batch method. Mutualistic interactions may explain the better performance of the enriched consortia.     

Microbial characterization of toluene-degrading denitrifying consortia obtained from terrestrial and marine ecosystems

The degradation characteristics of toluene coupled to nitrate reduction were investigated in enrichment culture and the microbial communities of toluene-degrading denitrifying consortia were characterized by denaturing gradient gel electrophoresis (DGGE) technique. Anaerobic nitrate-reducing bacteria were enriched from oil-contaminated soil samples collected from terrestrial (rice field) and marine (tidal flat) ecosystems. Enriched consortia degraded toluene in the presence of nitrate as a terminal electron acceptor. The degradation rate of toluene was affected by the initial substrate concentration and co-existence of other hydrocarbons. The types of toluene-degrading denitrifying consortia depended on the type of ecosystem. The clone RS-7 obtained from the enriched consortium of the rice field was most closely related to a toluene-degrading and denitrifying bacterium, Azoarcus denitrificians (A. tolulyticus sp. nov.). The clone TS-11 detected in the tidal flat enriched consortium was affiliated to Thauera sp. strain S2 (T. aminoaromatica sp. nov.) that was able to degrade toluene under denitrifying conditions. This indicates that environmental factors greatly influence microbial communities obtained from terrestrial (rice field) and marine (tidal flat) ecosystems.     

Methanogenesis facilitated by electric syntrophy via (semi)conductive iron-oxide minerals

Methanogenesis is an essential part of the global carbon cycle and a key bioprocess for sustainable energy. Methanogenesis from organic matter is accomplished by syntrophic interactions among different species of microbes, in which interspecies electron transfer (IET) via diffusive carriers (e.g. hydrogen and formate) is known to be the bottleneck step. We report herein that the supplementation of soil microbes with (semi)conductive iron-oxide minerals creates unique interspecies interactions and facilitates methanogenesis. Methanogenic microbes were enriched from rice paddy field soil with either acetate or ethanol as a substrate in the absence or presence of (semi)conductive iron oxides (haematite or magnetite). We found that the supplementation with either of these iron oxides resulted in the acceleration of methanogenesis in terms of lag time and production rate, while the supplementation with an insulative iron oxide (ferrihydrite) did not. Clone-library analyses of 16S rRNA gene fragments PCR-amplified from the enrichment cultures revealed that the iron-oxide supplementation stimulated the growth of Geobacter spp. Furthermore, the addition of a specific inhibitor for methanogenesis suppressed the growth of Geobacter spp. These results suggest that Geobacter grew under syntrophic association with methanogens, and IET could occur via electric currents through (semi)conductive iron-oxide minerals (termed 'electric syntrophy'). Given the ubiquity of conductive minerals in nature, such energetic interactions may occur widely in soil and sediments and can be used to develop efficient bioenergy processes.     

Mycorrhizal and rhizomorph dynamics in a loblolly pine forest during 5 years of free-air-CO2-enrichment

Soil fungi couple plant and ecosystem resource demands to pools of soil resources. Research on these organisms is needed to predict how rising atmospheric CO₂ will influence forest ecosystem processes and soil carbon (C) sequestration potential. We examined the influence of free‐air‐CO₂‐enrichment (FACE) on mycorrhizal and extraradical rhizomorph dynamics over a 5‐year period in a loblolly pine forest using minirhizotrons. Standing crop of mycorrhizal root tips varied greatly spatially and through time. Summed across all years, CO₂ enrichment increased mycorrhizal root tip production by 194% in deep soil (15–30 cm) but did not influence mycorrhizal production in shallow soil (0–15 cm). Production and mortality of soil rhizomorph length was 27% and 25% greater in CO₂‐enriched plots compared with controls over a 5‐year period beginning in January of 2000 and running through autumn 2004. Effects of atmospheric CO₂ enrichment on longevity of mycorrhizal root tips and rhizomorphs varied with soil depth (mycorrhizae and rhizomorphs) and with diameter (rhizomorphs). For instance, survival of mycorrhizal tips was reduced in CO₂‐enriched plots in deep soil (15–30 cm depth) but was increased in shallower soil (0–15 cm). Rhizomorph turnover was accelerated in shallow soil but effects on survivorship in deep soil varied according to diameter. A drought in 2002 coupled with loss of leaf area to an ice storm late in 2002 were followed by reductions in rhizomorph and mycorrhizal production, increases in mortality, and decreases in standing crop during 2003 and 2004. These effects tended to be more severe in CO₂‐enriched plots. Positive effects of atmospheric CO₂ enrichment on mycorrhizal fungi, primarily observed in deeper soil, are probably contributing to the prolonged stimulation of NPP by CO₂ enrichment at the Duke FACE study site.     

Freeze-Thaw Tolerance and Clues to the Winter Survival of a Soil Community

Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.     

Direct Cloning from Enrichment Cultures, a Reliable Strategy for Isolation of Complete Operons and Genes from Microbial Consortia

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Δ(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).     

Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia

Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes. We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia. Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes. DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb. The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms. These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB). Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons. Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied. Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit. Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria. In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE).     

Effects of enrichment with phthalate on polycyclic aromatic hydrocarbon biodegradation in contaminated soil

The effect of enrichment with phthalate on the biodegradation of polycyclic aromatic hydrocarbons (PAH) was tested with bioreactor-treated and untreated contaminated soil from a former manufactured gas plant (MGP) site. Soil samples that had been treated in a bioreactor and enriched with phthalate mineralized (14)C-labeled phenanthrene and pyrene to a greater extent than unenriched samples over a 22.5-h incubation, but did not stimulate benzo[a]pyrene mineralization. In contrast to the positive effects on (14)C-labeled phenanthrene and pyrene, no significant differences were found in the extent of biodegradation of native PAH when untreated contaminated soil was incubated with and without phthalate amendment. Denaturing-gradient gel electrophoresis (DGGE) profiles of bacterial 16S rRNA genes from unenriched and phthalate-enriched soil samples were substantially different, and clonal sequences matched to prominent DGGE bands revealed that beta-Proteobacteria related to Ralstonia were most highly enriched by phthalate addition. Quantitative real-time PCR analyses confirmed that, of previously determined PAH-degraders in the bioreactor, only Ralstonia-type organisms increased in response to enrichment, accounting for 89% of the additional bacterial 16S rRNA genes resulting from phthalate enrichment. These findings indicate that phthalate amendment of this particular PAH-contaminated soil did not significantly enrich for organisms associated with high molecular weight PAH degradation or have any significant effect on overall degradation of native PAH in the soil.     

Differential bioleaching of copper by mesophilic and moderately thermophilic acidophilic consortium enriched from same copper mine water sample

Three acidophilic enrichment consortium were developed from mine water sample of copper mine site at Khetri, India were compared for their copper leaching efficiency. Out of these one was mesophilic (35 degrees C) and two were moderately thermophilic (50 degrees C). Consortia were named as mesophilic acidophilic chemolithotrophic consortia (MACC), thermophilic acidophilic chemolithotrophic consortia (TACC), and Sulfobacillus acidophilic consortia (SAC). Copper extraction ability of both the thermophilic consortia (77-78% extraction) was almost double to that of mesophilic consortia (40% extraction) at 10% pulp density after 55 days. Both the thermophilic consortia were equally effective in leaching of other metals like Ni, Co, Zn, Mn. After 55 days, the percentage of extractions of copper by TACC was 76, 74, 67, 48 and 45 at 5%, 10%, 15%, 20% and 30% pulp density, respectively. Total number of bacteria was maximum at 5% pulp density which decreases with increase in pulp density. Sulfobacillus-like bacteria were seen in the Sulfobacillus enrichment cultures. Moderately thermophilic consortia proved to be better in leaching performance than the mesophilic counterpart.     

Characterization of a Defined 2,3,5,6-Tetrachlorobiphenyl-ortho-Dechlorinating Microbial Community by Comparative Sequence Analysis of Genes Coding for 16S rRNA

Defined microbial communities were developed by combining selective enrichment with molecular monitoring of total community genes coding for 16S rRNAs (16S rDNAs) to identify potential polychlorinated biphenyl (PCB)-dechlorinating anaerobes that ortho dechlorinate 2,3,5,6-tetrachlorobiphenyl. In enrichment cultures that contained a defined estuarine medium, three fatty acids, and sterile sediment, a Clostridium sp. was predominant in the absence of added PCB, but undescribed species in the δ subgroup of the class Proteobacteria, the low-G+C gram-positive subgroup, the Thermotogales subgroup, and a single species with sequence similarity to the deeply branching species Dehalococcoides ethenogenes were more predominant during active dechlorination of the PCB. Species with high sequence similarities to Methanomicrobiales and Methanosarcinales archaeal subgroups were predominant in both dechlorinating and nondechlorinating enrichment cultures. Deletion of sediment from PCB-dechlorinating enrichment cultures reduced the rate of dechlorination and the diversity of the community. Substitution of sodium acetate for the mixture of three fatty acids increased the rate of dechlorination, further reduced the community diversity, and caused a shift in the predominant species that included restriction fragment length polymorphism patterns not previously detected. Although PCB-dechlorinating cultures were methanogenic, inhibition of methanogenesis and elimination of the archaeal community by addition of bromoethanesulfonic acid only slightly inhibited dechlorination, indicating that the archaea were not required for ortho dechlorination of the congener. Deletion of Clostridium spp. from the community profile by addition of vancomycin only slightly reduced dechlorination. However, addition of sodium molybdate, an inhibitor of sulfate reduction, inhibited dechlorination and deleted selected species from the community profiles of the class Bacteria. With the exception of one 16S rDNA sequence that had the highest sequence similarity to the obligate perchloroethylene-dechlorinating Dehalococcoides, the 16S rDNA sequences associated with PCB ortho dechlorination had high sequence similarities to the δ, low-G+C gram-positive, and Thermotogales subgroups, which all include sulfur-, sulfate-, and/or iron(III)-respiring bacterial species.The extensive industrial use of polychlorinated biphenyls (PCBs) during the 20th century has resulted in the release of an estimated several million pounds of PCBs into the environment (2). Due to the hydrophobicity and chemical stability of these compounds, PCBs ultimately accumulate in subsurface anaerobic sediments, where reductive dechlorination by anaerobic microorganisms is proposed to be an essential step in PCB degradation and detoxification (6). Although anaerobic reductive dechlorination has been documented in the environment and in the laboratory, attempts to identify and isolate anaerobic PCB-dechlorinating microbes by classical enrichment and isolation techniques have been unsuccessful (for a review, see reference 2). Isolation of anaerobic PCB-dechlorinating microbes has been hindered in part by the inability to maintain and sequentially transfer dechlorinating consortia in defined medium. May et al. (24) were the first to demonstrate that single colonies could be obtained by plating highly enriched PCB-dechlorinating enrichment cultures on agar-solidified media. Although two of the colonies exhibited para dechlorination activity when transferred back to liquid enrichment medium, the colonies contained a mixed community of microorganisms and dechlorination required the addition of sediment to the medium. More recently, highly enriched PCB-ortho-dechlorinating enrichment cultures were developed from Baltimore Harbor sediments in minimal media that contained sediments and a single congener (3) or Aroclor 1260 (37). These were the first confirmed reports of sustained ortho dechlorination of PCBs throughout sequential transfers in medium with estuarine sediments. Finally, Cutter et al. demonstrated that a consortium of PCB-ortho-dechlorinating anaerobes from Baltimore Harbor could be sequentially transferred and maintained in minimal medium without the addition of sterile sediment (9). With the ability to maintain PCB dechlorination in a completely defined medium, highly enriched PCB-dechlorinating consortia could be developed by sequential transfers in medium that contained the minimal growth requirements for dechlorinating species.The current study identifies putative PCB-dechlorinating anaerobes in ortho-dechlorinating enrichment cultures by a comprehensive approach that combines traditional selective enrichment techniques with molecular monitoring (SEMM). Microbial consortia enriched for PCB ortho dechlorination in minimal medium were analyzed by comparative sequence analysis of genes coding for 16S rRNA (16S rDNA) amplified from total community DNAs. Protocols were developed for chromosomal DNA extraction from sediment, 16S rDNA amplification by PCR, cloning of partial 16S rDNA PCR fragments, screening by restriction fragment length polymorphism (RFLP) analysis, and DNA sequencing for comparative sequence analysis. By utilizing these techniques, shifts in the microbial community were monitored as the cultures were further enriched for PCB-dechlorinating anaerobes by elimination of undefined medium components (i.e., sediment), changes in carbon source, and addition of selective physiological inhibitors. The results presented herein demonstrate the applicability of the SEMM approach for the selection and monitoring of highly defined PCB-dechlorinating microbial consortia.     

黄土高原石油污染土壤微生物群落结构及其代谢特征

针对污染胁迫下土壤微生物群落变化和代谢变异等问题,基于平板稀释法和Biolog微平板分析方法,研究了陕北黄土高原石油污染土壤微生物群落结构、代谢特征及其功能多样性。结果表明,不同类群的土壤微生物对石油污染胁迫的响应不同,污染土壤细菌和真菌数量高出清洁土壤1个数量级,而污染土壤的放线菌数量极显著减少(P0.01);污染土壤和清洁土壤微生物对糖类和多聚物类碳源较易利用,污染土壤微生物总体上代谢碳源的种类和活性均低于清洁土壤。微生物群落主成分分析(PCA)表明,石油污染土壤和清洁土壤的微生物群落存在显著差异(P0.01),起分异作用的碳源主要为糖类,其次是羧酸类和氨基酸类;随着土壤石油含量增加,典型变量值变异(离散)增大,土壤微生物群落结构稳定性降低。微生物群落多样性分析表明,Shannon丰富度指数(H)、McIntosh均一度指数(U)和Simpson优势度指数(1/D)均达到极显著差异(P0.01),污染土壤微生物群落H和U低于清洁土壤,但是一定浓度的石油污染可以刺激土壤微生物群落中优势种群的生长,1/D增高。研究结果为陕北黄土高原石油污染区土壤微生物修复提供理论基础。     

Freeze-thaw tolerance and clues to the winter survival of a soil community

Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.     

Seasonal variation in seed bank composition and its interaction with nutrient enrichment in the Everglades wetlands

In the Florida Everglades, nutrient enrichment from agricultural outflow and the change in hydrology have collectively contributed to the expansion of cattails (Typha spp.). To assess the effectiveness of prescribed fire in controlling cattails and to predict vegetation dynamics after the fire, it is important to understand the seasonal variation of the soil seed bank and how the seed bank is affected by nutrient enrichment and fire. This paper investigates the effects of season, nutrient enrichment, and fire on soil seed bank species composition, richness, and density along a nutrient gradient in Water Conservation Area 2A (WCA 2A) of the Florida Everglades. Species richness was significantly affected by nutrient enrichment and season but not their interaction. Total seed density, however, was significantly affected by the interaction between nutrient enrichment and season. Yet, at species level, the relationship between seed density, nutrient enrichment and season varied. The highest seed density of cattail occurred in summer at highly enriched sites, but that of sawgrass occurred in fall regardless of enrichment; the seed density of water lily was very low regardless of season and nutrient enrichment, and the highest Amarathus seed density occurred at highly enriched sites year round. Moreover, germination timing differed greatly among species. While cattail seeds had a short incubation period and started to germinate 2–3 days after initiation of the germination assay, sawgrass seeds generally started to germinate 4 weeks later. Further, both the prescribed summer fire at the highly enriched site and the natural winter fire at the moderately enriched site reduced the seed density of cattail but not of sawgrass. Our results suggest that fire application for vegetation recovery in WCA 2A would benefit from explicitly considering seasonal dynamics of the seed bank.     

Emerging strategies for engineering microbial communities

From biosynthesis to bioremediation, microbes have been engineered to address a variety of biotechnological applications. A promising direction in these endeavors is harnessing the power of designer microbial consortia that consist of multiple populations with well-defined interactions. Consortia can accomplish tasks that are difficult or potentially impossible to achieve using monocultures. Despite their potential, the rules underlying microbial community maintenance and function (i.e. the task the consortium is engineered to carry out) are not well defined, though rapid progress is being made. This limited understanding is in part due to the greater challenges associated with increased complexity when dealing with multi-population interactions. Here, we review key features and design strategies that emerge from the analysis of both natural and engineered microbial communities. These strategies can provide new insights into natural consortia and expand the toolbox available to engineers working to develop novel synthetic consortia.     

Experimental formation of sea ice: importance of water circulation and wave action for incorporation of phytoplankton and bacteria

The efficiency of physical concentration mechanisms for enrichment of algae and bacteria in newly formed sea-ice was investigated under defined conditions in the laboratory. Sea-ice formation was simulated in a 3,000 l tank under different patterns of water movement. When ice formed in an artificially generated current pattern, algal cells were substantially enriched within the ice matrix. Enrichment factors for chlorophyll a calculated from the ratio between the concentrations in ice and underlying water reached values of up to 53. Repeated mixing of ice crystals into the water column, as well as flow of water through the new ice layer, contributed to the enrichment of algae in the ice. Wave action during ice formation revealed lower phytoplankton enrichment factors of up to 9. Mixing of floating ice crystals with underlying water and pumping of water into the ice matrix by periodical expansion and compression of the slush ice layer were responsible for the wave-induced enrichment of algal cells. Physical enrichment of bacteria within the ice was negligible. Bacterial biomass within new ice was enhanced only when the concentration of algae was high. At low algal biomass, bacteria experienced substantial losses in the ice, most likely due to brine drainage, which were not observed for the microalgae. Bacterial cells are therefore not scavenged by ice crystals and the observed enrichment and sustainment of bacterial biomass within newly formed ice depend on their attachment to cells or aggregates of algae. Division rates of bacteria changed only slightly during ice formation. Received: 21 October 1997 / Accepted: 9 April 1998     

Microbial community and function of enrichment cultures with methane and toluene

The interaction effect of co-existence of toluene and CH₄ on community and activity of methanotrophs and toluene-degrading bacteria was characterized in three consortia enriched with CH₄ and toluene (MT), toluene (T), and CH₄ (M), respectively, in this study. The CH₄ oxidation activity in the enrichment culture of MT was significantly lower than that of M at the end of the experiment (P?=?0.001). The toluene degradation rate could be enhanced by continuous addition of CH₄ and toluene in the initial days, but it was inhibited in the later days. Phylogenetic analysis of 16S rRNA genes showed that Proteobacteria and Bacteroidetes were dominant in the three enriched consortia, but the community of methanotrophs and toluene-degrading bacteria was significantly affected by the co-existence of CH₄ and toluene. Both Methylosinus (91.8 %) and Methylocystis (8.2 %) were detected in the enrichment culture of MT, while only Methylocystis species were detected in M. The toluene-degrading bacteria including Burkholderia, Flavobacteria, Microbacterium, and Azoarcus were all detected in the enrichment culture of T. However, only Azoarcus was found in the enrichment culture of MT. Significantly higher contents of extracellular polymeric substances polysaccharose and protein in the enrichment culture of MT than that of T and M suggested that a higher environmental stress occurred in the enrichment culture of MT.