Emulsification of hydrocarbons by subsurface bacteria

Summary Biosurfactants have potential for use in enhancement of in situ biorestoration by increasing the bioavailability of contaminants. Microorganisms isolated from biostimulated, contaminated and uncontaminated zones at the site of an aviation fuel spill and hydrocarbon-degrading microorganisms isolated from sites contaminated with unleaded gasoline were examined for their abilities to emulsify petroleum hydrocarbons. Emulsifying ability was quantified by a method involving agitation and visual inspection. Biostimulated-zone microbes and hydrocarbon-degrading microorganisms were the best emulsifiers as compared to contaminated and uncontaminated zone microbes. Biostimulation (nutrient and oxygen addition) may have been the dominant factor which selected for and encouraged growth of emulsifiers; exposure to hydrocarbon was also important. Biostimulated microorganisms were better emulsifiers of aviation fuel (the contaminant hydrocarbon) than of heavier hydrocarbon to which they were not previously exposed. By measuring surface tension changes of culture broths, 11 out of 41 emulsifiers tested were identified as possible biosurfactant producers and two isolates produced large surface tension reductions indicating the high probability of biosurfactant production.     

Assessment of in-situ bioremediation at a refinery waste-contaminated site and an aviation gasoline contaminated site

A combination of geochemical, microbiological and isotopic methods were used to evaluate in-situ bioremediation of petroleum hydrocarbons at one site contaminated with refinery waste and a second site contaminated with aviation gasoline at Alameda Point, California. At each site, geochemical and microbiological characteristics from four locations in the contaminated zone were compared to those from two uncontaminated background locations. At both sites, the geochemical indicators of in-situbiodegradation includeddepleted soil gas and groundwater oxygen, elevated groundwater alkalinity, and elevated soil gas carbon dioxide and methane in the contaminated zone relative to the background. Radiocarbon content of methane and carbon dioxide measured in soil gas at both sites indicated that they were derived from hydrocarbon contaminant degradation. Direct microscopy of soil core samples using cell wall stains and activity stains, revealed elevated microbial numbers and enhanced microbial activities in contaminated areas relative to background areas, corroborating geochemical findings. While microbial plate counts and microcosm studies using soil core samples provided laboratory evidence for the presence of some microbial activity and contaminant degradation abilities, they did not correlate well with either contaminant location, geochemical, isotopic, or direct microscopy data.     

Rapid intrinsic biodegradation of benzene, toluene, and xylenes at the boundary of a gasoline-contaminated plume under natural attenuation

A groundwater plume contaminated with gasoline constituents [mainly benzene, toluene, and xylenes (BTX)] had been treated by pumping and aeration for approximately 10 years, and the treatment strategy was recently changed to monitored natural attenuation (MNA). To gain information on the feasibility of using MNA to control the spread of BTX, chemical and microbiological parameters in groundwater samples obtained inside and outside the contaminated plume were measured over the course of 73 weeks. The depletion of electron acceptors (i.e., dissolved oxygen, nitrate, and sulfate) and increase of soluble iron were observed in the contaminated zone. Laboratory incubation tests revealed that groundwater obtained immediately outside the contaminated zone (the boundary zone) exhibited much higher potential for BTX degradation than those in the contaminated zone and in uncontaminated background zones. The boundary zone was a former contaminated area where BTX were no longer detected. Denaturing gradient gel electrophoresis (DGGE) analysis of polymerase chain reaction (PCR)-amplified bacterial 16S rRNA gene fragments revealed that DGGE profiles for groundwater samples obtained from the contaminated zone were clustered together and distinct from those from uncontaminated zones. In addition, unique bacterial rRNA types were observed in the boundary zone. These results indicate that the boundary zone in the contaminant plumes served as a natural barrier for preventing the BTX contamination from spreading out.     

Dynamics of an oligotrophic bacterial aquifer community during contact with a groundwater plume contaminated with benzene, toluene, ethylbenzene, and xylenes: an in situ mesocosm study

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.     

Dynamics of an Oligotrophic Bacterial Aquifer Community during Contact with a Groundwater Plume Contaminated with Benzene, Toluene, Ethylbenzene, and Xylenes: an In Situ Mesocosm Study

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA- and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.     

Assessment of the microbial potential for nitrate-enhanced bioremediation of a JP-4 fuel-contaminated aquifer

A site that was contaminated with JP-4 jet fuel was characterized microbiologically to assess the feasibility of nitrate-enhanced bioremediation. The results of microcosm studies indicated that the mean pseudo zero-order rate constants for alkylbenzene biodegradation and NO₃ ⁻-N removal were 1.2 and 2.4 mg L⁻¹ per day, respectively. Several alkylbenzenes were removed to a greater extent in samples contaminated with residual JP-4 than in unexposed samples and samples downgradient of the spill; benzene was recalcitrant in all samples. Numbers of total heterotrophs, JP-4-degraders, oligotrophs, total denitrifiers, denitrifiers growing in the presence of JP-4, estimates of cell number by analysis of phospholipid fatty acids, direct counts and aerobic and anaerobic protozoa were determined; however, numbers of microorganisms were not reliable predictors of alkylbenzene biodegradation activity. The presence of aerobic and anaerobic protozoa suggests that protozoa may be active under a variety of different electron acceptor conditions. The results of the characterization study indicated that the site was amenable to nitrate-enhanced bioremediation. Received 12 March 1996/ Accepted in revised form 17 September 1996     

Response of Microorganisms to an Accidental Gasoline Spillage in an Arctic Freshwater Ecosystem

The response of microorganisms to an accidental spillage of 55,000 gallons of leaded gasoline into an Arctic freshwater lake was studied. Shifts in microbial populations were detected after the spillage, reflecting the migration pattern of the gasoline, enrichment for hydrocarbon utilizers, and selection for leaded-gasoline-tolerant microorganisms. Ratios of gasoline-tolerant/utilizing heterotrophs to “total” heterotrophs were found to be a sensitive indicator of the degree of hydrocarbon contamination. Respiration rates were elevated in the highly contaminated area, but did not reflect differences between moderately and lightly contaminated areas. Hydrocarbon biodegradation potential experiments showed that indigenous microorganisms could extensively convert hydrocarbons to CO₂. In situ measurement of gasoline degradation showed that, if untreated, sediment samples retained significant amounts of gasoline hydrocarbons including “volatile components” at the time the lake froze for the winter. Nutrient addition and bacterial inoculation resulted in enhanced biodegradative losses, significantly reducing the amount of residual hydrocarbons. Enhanced biodegradation, however, resulted in the appearance of compounds not detected in the gasoline. Since the contaminated lake serves as a drinking water supply, treatment to enhance microbial removal of much of the remaining gasoline still may be advisable.     

Abundance and Diversity of <Emphasis Type="Italic">n</Emphasis>-Alkane-Degrading Bacteria in a Forest Soil Co-Contaminated with Hydrocarbons and Metals: A Molecular Study on <Emphasis Type="Italic">alkB</Emphasis> Homologous Genes

Unraveling functional genes related to biodegradation of organic compounds has profoundly improved our understanding of biological remediation processes, yet the ecology of such genes is only poorly understood. We used a culture-independent approach to assess the abundance and diversity of bacteria catalyzing the degradation of n-alkanes with a chain length between C₅ and C₁₆ at a forest site co-contaminated with mineral oil hydrocarbons and metals for nearly 60 years. The alkB gene coding for a rubredoxin-dependent alkane monooxygenase enzyme involved in the initial activation step of aerobic aliphatic hydrocarbon metabolism was used as biomarker. Within the area of study, four different zones were evaluated: one highly contaminated, two intermediately contaminated, and a noncontaminated zone. Contaminant concentrations, hydrocarbon profiles, and soil microbial respiration and biomass were studied. Abundance of n-alkane-degrading bacteria was quantified via real-time PCR of alkB, whereas genetic diversity was examined using molecular fingerprints (T-RFLP) and clone libraries. Along the contamination plume, hydrocarbon profiles and increased respiration rates suggested on-going natural attenuation at the site. Gene copy numbers of alkB were similar in contaminated and control areas. However, T-RFLP-based fingerprints suggested lower diversity and evenness of the n-alkane-degrading bacterial community in the highly contaminated zone compared to the other areas; both diversity and evenness were negatively correlated with metal and hydrocarbon concentrations. Phylogenetic analysis of alkB denoted a shift of the hydrocarbon-degrading bacterial community from Gram-positive bacteria in the control zone (most similar to Mycobacterium and Nocardia types) to Gram-negative genotypes in the contaminated zones (Acinetobacter and alkB sequences with little similarity to those of known bacteria). Our results underscore a qualitative rather than a quantitative response of hydrocarbon-degrading bacteria to the contamination at the molecular level.     

Physiological and molecular characterization of a microbial community established in unsaturated, petroleum-contaminated soil

The microbial communities established in soil samples from an unsaturated, petroleum-contaminated zone and from an adjacent uncontaminated site were characterized by physiological and molecular approaches. Possible electron acceptors such as sulfate and nitrate had been completely depleted in these soil samples. Slurries of these soil samples were incubated in bottles in the presence of hydrocarbon indicators (benzene, toluene, xylene and decane), and the degradation of these compounds was examined. Supplementation with electron acceptors stimulated hydrocarbon degradation, although the stimulatory effect was small in the contaminated soil. The initial degradation rates in the contaminated soil under fermentative/methanogenic conditions were comparable to those under aerobic conditions. The microbial populations in the original soil samples were analysed by cloning and sequencing of polymerase chain reaction (PCR)-amplified bacterial and archaeal 16S rRNA gene fragments, showing that the sequences retrieved from these soils were substantially different. For instance, Epsilonproteobacteria, Gammaproteobacteria, Crenarchaeota and Methanosarcinales could only be detected at significant levels in the contaminated soil. Denaturing gradient gel electrophoresis (DGGE) analyses of 16S rRNA gene fragments amplified by PCR from the incubated soil-slurry samples showed that supplementation of the electron acceptors resulted in a shift in the major populations, while the DGGE profiles after incubating the contaminated soil under the fermentative/methanogenic conditions were not substantially changed. These results suggest that petroleum contamination of the unsaturated zone resulted in the establishment of a fermentative/methanogenic community with substantial hydrocarbon-degrading potential.     

Natural attenuation of diesel-oil contamination in a subantarctic soil (Crozet Island)

An accidental contamination occurred in subantarctic Crozet Island between July and November 1997 near the "Alfred Faure" scientific station (51°51'E-46°25'S). More than 20,000 l of diesel fuel was spilled in the soil in the vicinity of the power station. In order to evaluate the efficiency of the expected bioattenuation process, the contaminated area was sampled in December 1999 for bacterial and chemical analysis. All samples were analysed for total bacteria, heterotrophic viable assemblages and hydrocarbon-utilising microflora. The results of this first analytical survey clearly show a significant response of subantarctic microbial soil communities to the hydrocarbon contamination. Significant increases of total, heterotrophic and hydrocarbon-utilising micro-organisms occurred in the more contaminated zone (from less than 5᎒⁴ MPN g^-1 wet soil to more than 10⁸ MPN g^-1 wet soil for hydrocarbon-degrading micro-organisms). The very high numbers of hydrocarbon-degrading micro-organisms present in the more contaminated zone are clearly linked to early bioattenuation activities. Chemical results provided some clear indications that spilled fuel was still well preserved from chemical and biological weathering 1 year after the spill, as light aliphatics and aromatics were present in all oiled samples and little differences were usually observed between samples.     

Exploring the emergence of a biojet fuel supply chain in Brazil: An agent‐based modeling approach

The aviation industry accounts for more than 2% of global CO₂ emissions. Biojet fuel is expected to make an essential contribution to the decarbonization of the aviation sector. Brazil is seen as a key player in developing sustainable aviation biofuels owing to its long‐standing experience with biofuels. Nevertheless, a clear understanding of what policies may be conducive to the emergence of a biojet fuel supply chain is lacking. We extended a spatially explicit agent‐based model to explore the emergence of a biojet fuel supply chain from the existing sugarcane–ethanol supply chain. The model accounts for new policies (feed‐in tariff and capital investment subsidy) and new considerations into the decision making about production and investment in processing capacity. We found that in a tax‐free gasoline regime, a feed‐in tariff above 3 R$/L stimulates the production of biojet fuel. At higher levels of gasoline taxation (i.e., 2.46 R$/L), however, any feed‐in tariff is insufficient to ensure the production of biojet fuel. Thus, at these levels of gasoline taxation, it is needed to introduce regulations on the production of biojet fuel to ensure its production. Given the current debate about the future direction of the biofuel policy in Brazil, we recommend further research into the effect of market mechanisms based on greenhouse gas emissions on the emergence of a Brazilian biojet fuel supply chain.     

Pyrene and Phenanthrene Influence on Soil Microbial Populations

Two studies were conducted to evaluate microbial populations in polycyclic aromatic hydrocarbon-contaminated soil. Captina silt loam was freshly exposed to (1) 0 or 2000?mg pyrene/kg and sampled after 10- and 61-wk incubation and (2) 0 or 505?mg pyrene + 445?mg phenanthrene/kg and sampled after a 21-wk incubation. Microbial numbers were determined by plate-count techniques. Isolated bacteria, selected degraders, and wholesoil extracts were analyzed by fatty acid methyl ester analysis (FAME). In the pyrene experiment, pyrene did not affect total bacterial or fungal numbers, but pyrene degraders increased from undetectable levels to 7.09 log₁₀ degraders/g in the contaminated soil. The FAME analysis of bacterial isolates detected no pyrene effect, but wholesoil FAME indicated an increase in the contaminated soil of a fatty acid characteristic of protozoa and a major fatty acid detected in isolated degraders. In the pyrene + phenanthrene experiment, the contaminants had no impact on bacterial, fungal, or actinomycete numbers but increased degrader numbers. No effect of pyrene + phenanthrene was detected by isolate FAME, but whole-soil FAME indicated an effect similar to that in the pyrene experiment. The results indicate that pyrene, although not impacting microbial numbers, may have altered the soil microbial composition and that Captina silt loam can develop an effective degrader population under tested conditions.     

Effects of Heavy Metal Pollution on Oak Leaf Microorganisms

During the growing season, comparisons were made of the leaf surface microflora of (i) two groups of mature oak trees, one in the vicinity of a smelting complex contaminated by heavy metals and the other at a relatively uncontaminated site, and (ii) two groups of oak saplings at the uncontaminated site, one of which was sprayed with zinc, lead, and cadmium to simulate the heavy metal pollution from the smelter without the complicating effects of other pollutants. Total viable counts of bacteria, yeasts, and filamentous fungi (isolated by leaf washing) were generally little affected by the spraying treatment, whereas polluted leaves of mature trees supported fewer bacteria compared with leaves of mature trees at the uncontaminated site. Numbers of pigmented yeasts were lower on polluted oaks and on metal-dosed saplings compared with their respective controls. Polluted leaves of mature trees supported both greater numbers of Aureobasidium pullulans and Cladosporium spp. and a greater percentage of metal-tolerant fungi compared with oak leaves at the uncontaminated site. There were no significant overall differences in the degree of mycelial growth between the two groups of saplings or the mature trees.     

Greenhouse gases emission from soil contaminated with automobile industry residue in Brazil

Solid waste of the automobile industry containing large amounts of heavy metals might affect the emission of greenhouse gases (GHG) when applied to the soil. Accumulation of inorganic chemical elements in the environment generally occurs due to human activity (industry, agriculture, mining and waste landfills). Residues from human activities may release heavy metals to the soil solution, causing toxicity to plants and other soil organisms. Heavy metals may also be adsorbed to clay minerals and/or complexed by the soil organic matter, becoming a potential source of pollutants. Not much is known about the behavior of solid wastes in tropical soil as regarded as source of greenhouse gases (GHG). The emission of GHG (CO₂, CH₄ and N₂O) was evaluated in incubated soil samples collected in an area contaminated with a solid residue from an automobile industry. Samples were randomly collected at 0 to 0.2 m (a mix of soil and residue), 0.2 to 0.4 m (only residue) and 0.4 to 0.6 m (only soil). A contiguous uncontaminated area, cultivated with sugarcane, was also sampled following the same protocol. Canonical Discriminant Analysis and Principal Component Analysis were applied to the data to evaluate the GHG emission rates. Emission rates of GHG were greater in the samples from the contaminated than the sugarcane area, particularly high during the first days of incubation. CO₂ emissions were greater in samples collected at the upper layer for both areas, while CH₄ and N₂O emissions were similar in all samples. The emission rates of CH₄ were the most efficient variables to differentiate contaminated and uncontaminated areas.     

Hybridization Analysis of Microbial DNA from Fuel Oil–Contaminated and Noncontaminated Soil

Abstract The enrichment of several genes (xylE, nahAcd, todC1C2BA, tmoABCDE, alkB) that encode enzymes responsible for key steps in the degradation of hydrocarbons, and one gene specific to rRNA group I of the genus Pseudomonas, was studied in DNA extracted from a fuel oil–contaminated field site, and in laboratory microcosms (with the exception of alkB). Toluene, ethylbenzene, xylene, and naphthalene concentrations were related to the extent of hybridization of the genes in the field studies. Significant differences were observed in the extent of hybridization of some of the genes between contaminated and noncontaminated samples. In the microcosm studies, gasoline at rates ranging from 0.5 mg to 125 mg gasoline/g of soil as applied to soils, and the changes in hybridization intensity of these genes monitored with time. The lower threshold of gene enrichment of these genes in response to gasoline addition was below 0.5 mg/g soil. Small increases were observed at the 0.5-mg exposure level, but hybridization intensity quickly decreased to levels below detection 6–8 days after addition of the gasoline. A dose-response effect was observed from treatments with gasoline concentrations ranging from 0.5 to 35 mg/g soil. Inhibition by toxic components in gasoline was observed at 75 and 125 mg/g soil levels. Hybridization of the Pseudomonas group 1 probe to field DNA was not significantly enriched in the contaminated field site, although these sequences were enriched in the microcosm studies. Among the genes tested, xylE was the most sensitive indicator of low levels of fuel oil contamination. Received: 23 July 1996; Accepted 9 October 1996     

Effect of ethanol on the biodegradation of gasoline in an unsaturated tropical soil

Leaking underground storage tanks are a worldwide problem nowadays, therefore gasoline degradation and distribution in the subsurface have been widely studied. Brazil is the only country to currently use ethanol as fuel and as an oxygenate additive to gasoline, in amounts varying from 20 to 26% v/v. Consequently, both gasoline and ethanol may contaminate the environment when spills and leaks occur. Some authors suggest that despite the high degradability of gasoline, its degradation in the aquifer is hindered by the preferential biodegradation of ethanol, which would delay gasoline degradation; however, processes in the unsaturated zone are far less understood. The present study concentrates on degradation and distribution processes of ethanol-amended gasoline in tropical soil under unsaturated conditions. A multi-parametrical approach was adopted to assess the effects of ethanol on the fate of gasoline. Undisturbed natural soil was used to evaluate biodegradation processes while tank experiments determined differences in infiltration, distribution and retention of gasoline in unsaturated artificial porous medium. Physical, chemical and microbiological results suggest that ethanol enhances BTEX retention in soil, boosts microbial activity but delays BTEX biodegradation.     

Contrasts between subsurface microbial communities and their metabolic adaptation to polycyclic aromatic hydrocarbons at a forested and an urban coal-tar disposal site

The abundance and distribution of microorganisms and their potential for mineralizing polycyclic aromatic hydrocarbons (PAHs) were measured in subsurface sediment samples at two geographically separate buried coal-tar sites. At a relatively undisturbed forested site in the northeastern United States, metabolic adaptation to the PAHs was evident: Radiolabeled naphthalene and phenanthrene were converted to ¹⁴CO₂ in core material from inside but not outside a plume of groundwater contamination. However, at the urban site in the midwestern United States these PAHs were mineralized in sediments from both contaminated and uncontaminated boreholes. Thus, clear qualitative evidence showing an adaptational response by the subsurface microbial community was not obtained at the urban site. Instead, subtler clues suggesting metabolic adaptation by subsurface microorganisms from the urban site were discerned by comparing lag periods and extents of ¹⁴CO₂ production from radiolabeled PAHs added to samples from contaminated and uncontaminated boreholes. Despite slightly higher PAH mineralization activity in contaminated borehole samples, p-hydroxybenzoate was mineralized equally in all samples from the urban site regardless of location. No striking trends in the abundances of actinomycetes, fungi, and either viable or total bacteria were encountered. However, colonies of the soil bacterium, Bacillus mycoides, were detected on enumeration plates of several samples from unsaturated and saturated zones in both urban boreholes. Furthermore, other common soil bacteria, Myxococcus xanthus and Chromobacterium violaceum, were identified in samples from the uncontaminated urban borehole. The occurrence of bacteria usually restricted to surface soil, combined with the observation of fragments of building materials in many of the core samples, suggested that past excavation and backfilling operations may have caused mixing of surface soil with subsurface materials at the urban site. We speculate that this mixing, as well as non-coal-tar-derived sources of PAHs, contributed to the PAH-mineralizing activity present in the sediment samples from the uncontaminated urban borehole.     

Distribution of protozoa in subsurface sediments of a pristine groundwater study site in oklahoma

Sediment core samples were obtained at a groundwater study site in Oklahoma in January and June 1985. Most-probable-number estimates showed that protozoan numbers declined steeply with depth in subsoil. Flagellates and amoebae dominated the protozoan population, which declined to a most probable number of 28 . g (dry weight) in a clay loam layer at the bottom of the unsaturated zone. Samples from a texturally variable interface zone between 3 and 4 m down also were variable in their content of protozoa. Four contiguous clay loam samples in a single core from this zone contained variable numbers of amoebae ranging from 0.2 to 44 . g (dry weight). However, a sandy clay loam layer at the bottom of the core contained a mixture of flagellates and amoebae with a combined population density of 67 . g (dry weight). A slow-growing filose amoeba was isolated from interface zone samples and was tentatively classified in a new family in the order Aconchulinida. Protozoa were not detected in the saturated zone except in a very permeable gravelly, loamy sand layer at a depth of approximately 7.5 m. Low numbers (4 to 6 . g [dry weight]) of surface-type flagellates and amoebae, as well as the filose amoeba seen in the interface zone, were observed in this layer. Acid-treated and untreated samples contained equivalent numbers of protozoa, showing that the majority of protozoa in the layer at 7.5 m and the interface zone samples were encysted. Increased numbers of bacteria also were found in the layer at 7.5 m, indicating that it was biologically more active than other saturated-zone layers. Cyanobacteria grew in illuminated samples from this layer, suggesting that it may be connected hydrologically to a nearby river.     

Rumen Microbial Ecology in Mule Deer

Mule deer rumen microbial populations from animals in the natural habitat in Utah and from captive deer fed various rations were studied. The microorganisms were characterized on the basis of morphology and Gram reaction. Rumen samples contained 13 identifiable types of bacteria and one genus of ciliate protozoa (Entodinium). Highest rumen bacterial populations were produced on rations containing barley. No differences in proportions of ruminal bacteria in the various morphological groups could be detected when animals were fed either natural browse plants or alfalfa hay. The total numbers of bacteria were similar for animals feeding on controlled diets of browse or hay and those in the natural habitat. Numbers of some bacterial types were directly related to ciliate protozoal numbers, whereas others were inversely related. Highest rumen ciliate protozoal populations were observed on rations containing barley. No differences in protozoal populations were noted between diets containing only browse or hay. Seasonal variations were noted in ciliate protozoal numbers from deer feeding in the natural habitat. The total number of ciliate protozoa decreased in the fall and winter and remained low until spring. There were indications that salt in the deer diet favorably affected rumen ciliate protozoa. Rather than revealing direct deer management applications, this study serves to stimulate and illuminate new approaches to research in range and wildlife nutrition.