Thomas L. Kieft

Microbial biomass response to a rapid increase in water potential when dry soil is wetted

The proportion of microbial biomass-C released into the soil environment following rapid water potential increase was quantified in two soils using a modified chloroform-fumigation biomass assay. Dry samples were isopiestically equilibrated to −2.8 and − 6.9 M Pa and then wetted to field capacity with either H2O or KCl solutions. The KC1 solutions wetted the soils without altering total soil water potential. The biomass-C released by water potential increase ranged from 17 to 70% of total, depending on the soil, the magnitude of the increase, and the method of calculation. In both soils, a greater proportion of biomass-C was released following a 6.9 MPa than a 2.8 M Pa increase. Biomass-C release was also demonstrated by an increase in soluble organic C in leachates of soils subjected to rapid wetting. Respiration of biomass-C mobilized by water potential increase exceeded respiration of biomass-C made available by preceding desiccation, thereby comprising a significant component of the pulse of respiration observed following wetting of dry soil. Water potential increases associated with the wetting of dry soil may be a major catalyst for soil C turnover.

TEMPORAL DYNAMICS IN SOIL CARBON AND NITROGEN RESOURCES AT A GRASSLAND–SHRUBLAND ECOTONE

Plant communities of large portions of the southwestern United States have changed from grassland to desert shrubland. Previous studies have demonstrated that soil nutrient resources become spatially more heterogeneous and are redistributed into islands of fertility with the shift in vegetation. The research presented here addressed the question of whether soil resources become more temporally heterogeneous as well as more spatially heterogeneous when grassland undergoes desertification to form shrubland. Within adjacent grassland and creosotebush sites, soil profiles were described at three soil pits, and samples were collected for description of nutrient resources within the profile. Relative abundance of plant cover and bare soil was determined within each site using line transects. Surface samples (0-20 cm depth) of bare soil and soil beneath the canopies of grasses and creo- sotebush were collected 17 times during 1992-1994. Soil samples were analyzed for mois- ture, extractable ammonium and nitrate, nitrogen mineralization potential, microbial bio- mass carbon, total organic carbon, microbial respiration, dehydrogenase activity, the ratio of microbial C to total organic C (Cmic/Corg), and the ratio of microbial respiration to biomass carbon (metabolic quotient). The major differences in the structure of soils between sites were the apparent loss of 3-5 cm depth of sandy surface soil at the creosotebush site and an associated increase in calcium carbonate content at a more shallow depth. Soils under plants at both sites had greater total and available nutrient resources, with higher concen- trations under creosotebush than under grasses. Greatest temporal variation in available soil resources was observed in soils under creosotebush. When expressed on the basis of area, available soil resources were higher in the grassland than in the creosotebush shrub- land, primarily due to the difference in plant cover (45% in grassland, 8% in creosotebush shrubland).

Antibacterial action of a novel functionalized chitosan-arginine against gram-negative bacteria

The antimicrobial activity of chitosan and chitosan derivatives has been well established. However, although several mechanisms have been proposed, the exact mode of action is still unclear. Here we report on the investigation of antibacterial activity and the antibacterial mode of action of a novel water-soluble chitosan derivative, arginine-functionalized chitosan, on the Gram-negative bacteria Pseudomonas fluorescens and Escherichia coli. Two different arginine-functionalized chitosans (6% arginine-substituted and 30% arginine-substituted) each strongly inhibited P. fluorescens and E. coli growth. Time-dependent killing efficacy experiments showed that 5000 mg l(-1) of 6%- and 30%-substituted chitosan-arginine killed 2.7 logs and 4.5 logs of P. fluorescens, and 4.8 logs and 4.6 logs of E. coli in 4h, respectively. At low concentrations, the 6%-substituted chitosan-arginine was more effective in inhibiting cell growth even though the 30%-substituted chitosan-arginine appeared to be more effective in permeabilizing the cell membranes of both P. fluorescens and E. coli. Studies using fluorescent probes, 1-N-phenyl-naphthylamine (NPN), nile red (NR) and propidium iodide (PI), and field emission scanning electron microscopy (FESEM) suggest that chitosan-arginines antibacterial activity is, at least in part, due to its interaction with the cell membrane, in which it increases membrane permeability.

Microbiology of vadose zone paleosols in South-Central Washington State

Three unsaturated subsurface paleosols influenced by moisture recharge, including a highly developed calcic paleosol, were studied to investigate the microbiology of paleosols. Two near-surface paleosols, one impacted by moisture recharge and the other beyond the influence of recharge, were also sampled to directly assess the effect of moisture recharge on the activity and composition of the microbial community associated with paleosols. The highly developed paleosol had a higher population of culturable heterotrophs, a greater glucose mineralization potential, a higher microbial diversity based on colony morphology, and a more than 20-fold higher concentration of ATP than the two weakly developed paleosols. The recharged near-surface paleosol, as compared to the near-surface paleosol unaffected by recharge, had a lower population of culturable heterotrophs, smaller mineralization rate constant, and lower richness based on colony morphology. The recharged paleosols contained predominantly gram-negative isolates, whereas the paleosol unaffected by recharge contained predominantly gram-positive isolates. Storage at 4°C of subsurface and near-surface paleosol samples containing high water potential increased the population of culturable aerobic heterotrophs, decreased diversity in colony morphology, and increased first-order rate constants and decreased lag times for glucose mineralization. These results indicate that aerobic heterotrophs are present in deep vadose zone paleosols and that there is potential for stimulation of their in situ growth and activity.

Microbial Abundance and Activities in Relation to Water Potential in the Vadose Zones of Arid and Semiarid Sites

Numbers and activities of microorganisms were measured in the vadose zones of three arid and semiarid areas of the western United States, and the influence of water availability was determined. These low-moisture environments have vadose zones that are commonly hundreds of meters thick. The specific sampling locations chosen were on or near U.S. Department of Energy facilities: the Nevada Test Site (NTS), the Idaho National Engineering Laboratory (INEL), and the Hanford Site (HS) in southcentral Washington State. Most of the sampling locations were uncontaminated, but geologically representative of nearby locations with storage and/or leakage of waste compounds in the vadose zone. Lithologies of samples included volcanic tuff, basalt, glaciofluvial and fluvial sediments, and paleosols (buried soils). Samples were collected aseptically, either by drilling bore-holes (INEL and HS), or by excavation within tunnels (NTS) and outcrop faces (paleosols near the HS). Total numbers of microorganisms were counted using direct microscopy, and numbers of culturable microorganisms were determined using plate-count methods. Desiccation-tolerant microorganisms were quantified by plate counts performed after 24 h desiccation of the samples. Mineralization of 14C-labeled glucose and acetate was quantified in samples at their ambient moisture contents, in dried samples, and in moistened samples, to test the hypothesis that water limits microbial activities in vadose zones. Total numbers of microorganisms ranged from log 4.5 to 7.1 cells g-1 dry wt. Culturable counts ranged from log <2 to 6.7 CFU g-1 dry wt, with the highest densities occurring in paleosol (buried soil) samples. Culturable cells appeared to be desiccation-tolerant in nearly all samples that had detectable viable heterotrophs. Water limited mineralization in some, but not all samples, suggesting that an inorganic nutrient or other factor may limit microbial activities in some vadose zone environments.

Subsurface Microbial Diversity in Deep-Granitic-Fracture Water in Colorado

ABSTRACT A microbial community analysis using 16S rRNA gene sequencing was performed on borehole water and a granite rock core from Henderson Mine, a >1,000-meter-deep molybdenum mine near Empire, CO. Chemical analysis of borehole water at two separate depths (1,044 m and 1,004 m below the mine entrance) suggests that a sharp chemical gradient exists, likely from the mixing of two distinct subsurface fluids, one metal rich and one relatively dilute; this has created unique niches for microorganisms. The microbial community analyzed from filtered, oxic borehole water indicated an abundance of sequences from iron-oxidizing bacteria (Gallionella spp.) and was compared to the community from the same borehole after 2 weeks of being plugged with an expandable packer. Statistical analyses with UniFrac revealed a significant shift in community structure following the addition of the packer. Phospholipid fatty acid (PLFA) analysis suggested that Nitrosomonadales dominated the oxic borehole, while PLFAs indicative of anaerobic bacteria were most abundant in the samples from the plugged borehole. Microbial sequences were represented primarily by Firmicutes, Proteobacteria, and a lineage of sequences which did not group with any identified bacterial division; phylogenetic analyses confirmed the presence of a novel candidate division. This “Henderson candidate division” dominated the clone libraries from the dilute anoxic fluids. Sequences obtained from the granitic rock core (1,740 m below the surface) were represented by the divisions Proteobacteria (primarily the family Ralstoniaceae) and Firmicutes. Sequences grouping within Ralstoniaceae were also found in the clone libraries from metal-rich fluids yet were absent in more dilute fluids. Lineage-specific comparisons, combined with phylogenetic statistical analyses, show that geochemical variance has an important effect on microbial community structure in deep, subsurface systems.

The Distribution of Microbial Taxa in the Subsurface Water of the Kalahari Shield, South Africa

Microbial communities within deep subsurface environments were analyzed by 16S rRNA gene cloning. Clone libraries from 27 borehole fluid, 7 mining-contaminated, and 5 rock samples were compared. Borehole fluids derived from deep fractures were populated by microbial communities with low diversity with an average of 11 and 5 bacterial and archaeal OTUs respectively. Low taxa richness was likely driven by limited biogeochemical reactions available for growth and not extreme parameters such as pH and temperature. Novel taxa of Firmicutes were discovered, commonly found in warm, slightly alkaline, anoxic fracture fluids. Highly divergent lineages of Archaea, unique to South African deep subsurface fracture fluids, are also described. Clone library clustering analyses based on LIBSHUFF phylogenetic relatedness revealed distinct groups of samples corresponding with sample source and geochemistry.

Observations pertaining to the origin and ecology of microorganisms recovered from the deep subsurface of Taylorsville Basin, Virginia

To understand the conditions under which microorganisms exist in deep hydrocarbon reservoirs, sidewall cores were collected from a natural gas‐bearing formation, 2800 m below the surface in Taylorsville Basin, Virginia. Data from chemical and microbial tracers and controls indicate that the interiors of some sidewall cores contained microorganisms indigenous to the rock formation. The cultured microorganisms were composed primarily of saline‐tolerant, thermophilic fermenting, Fe(III)‐reducing, and sulfate‐reducing bacteria (1 to 104 cells/g). The physiological capabilities of the cultured microorganisms are compatible with the temperature (76°C), pressure (32 MPa), and salinity (≈0.8 wt.% NaCl equivalent) in the sampled interval. The petrological data indicated that the strata contain intercrystalline pores of micrometer size, that occur between late diagenetic cement in siltstone and within cross‐cutting, mineralized fractures in shale. These pores made up only 0.04% of the rock volume, were mostly gas‐f...

The origin and age of biogeochemical trends in deep fracture water of the Witwatersrand Basin, South Africa

Water residing within crustal fractures encountered during mining at depths greater than 500 meters in the Witwatersrand basin of South Africa represents a mixture of paleo-meteoric water and 2.0–2.3 Ga hydrothermal fluid. The hydrothermal fluid is highly saline, contains abiogenic CH 4 and hydrocarbon, occasionally N 2 , originally formed at ∼ 250–300°C and during cooling isotopically exchanged O and H with minerals and accrued H 2 , 4 He and other radiogenic gases. The paleo-meteoric water ranges in age from ∼ 10 Ka to > 1.5 Ma, is of low salinity, falls along the global meteoric water line (GMWL) and is CO 2 and atmospheric noble gas-rich. The hydrothermal fluid, which should be completely sterile, has probably been mixing with paleo-meteoric water for at least the past ∼100 Myr, a process which inoculates previously sterile environments at depths > 2.0 to 2.5 km. Free energy flux calculations suggest that sulfate reduction is the dominant electron acceptor microbial process for the high salinity fracture water and that it is 10 7 times that normally required for cell maintenance in lab cultures. Flux calculations also indicate that the potential bioavailable chemical energy increases with salinity, but because the fluence of bioavailable C, N and P also increase with salinity, the environment remains energy-limited. The 4 He concentrations and theoretical calculations indicate that the H 2 that is sustaining the subsurface microbial communities (e.g. H 2 -utilizing SRB and methanogens) is produced by water radiolysis at a rate of ∼1 nM yr −1 . Microbial CH 4 mixes with abiogenic CH 4 to produce the observed isotopic signatures and indicates that the rate of methanogenesis diminishes with depth from ∼ 100 at < 1 kmbls, to < 0.01 nM yr −1 at > 3 kmbls. Microbial Fe(III) reduction is limited due to the elevated pH. The δ13C of dissolved inorganic carbon is consistent with heterotrophy rather than autotrophy dominating the deeper, more saline environments. One potential source of the organic carbon may be microfilms present on the mineral surfaces.

Grazing and plant-canopy effects on semiarid soil microbial biomass and respiration

The major objectives of this study were to determine the influence of grazing on the soil microbial biomass and activity in semiarid grassland and shrubland areas and to quantify the canopy effect (the differences in soil microbial biomass and activities between soils under plant canopies and soils in the open between plants). We also quantified changes in microbial biomass and activity during seasonal transition from dry to moist conditions. Chronosequences of sites withdrawn from grazing for 0, 11, and 16 years were sampled in a grassland (Bouteloua spp.) area and a shrubland (Atriplex canescens) area on and near the Sevilleta National Wildlife Reguge in central New Mexico, USA. Samples were obtained from beneath the canopies of plants (Yucca glauca in the grassland and A. canescens in the shrubland) and from open soils; they were collected three times during the spring and summer of a single growing season. Organic C, soil microbial biomass C, and basal respiration rates (collectively called the “soil C triangle”) were measured. We also calculated the microbial: organic C ratio and the metabolic quotient (ratio of respiration to microbial C) as measures of soil organic C stability and turnover. Although we had hypothesized that individual values of the soil C triangle would increase and that the ratios would decrease with time since grazing, differences in microbial parameters between sites located along the chronosequences were generally not significant. Grazing did not have a consistion effect on organic C, microbial C, and basal respiration in our chronosequences. The microbial: organic C ratio and the metabolic quotient generally increased with time since grazing on the shrubland chronosequence. The microbial: organic C ratio decreased with time since grazing and the metabolic quotient increased with time since grazing on the grassland chronosequence. The canopy effect was observed at all sites in nearly all parameters including organic C, microbial C, basal respiration, the microbial: organic C ratio, and the metabolic quotient which were predominantly higher in soils under the canopies of plants than in the open at all sites. Microbial biomass and activity did not increase during the experiment, even though the availability of moisture increased dramatically. The canopy effects were approximately equal on the shrubland and grassland sites. The microbial: organic C ratios and the metabolic quotients were generally higher in the shrubland soils than in the grassland soils.