Brian K. Kinkle

A Chemoautotrophically Based Cave Ecosystem

Microbial mats discovered in a ground-water ecosystem in southern Romania contain chemoautotrophic bacteria that fix inorganic carbon, using hydrogen sulfide as an energy source. Analysis of stable carbon and nitrogen isotopes showed that this chemoautotrophic production is the food base for 48 species of cave-adapted terrestrial and aquatic invertebrates, 33 of which are endemic to this ecosystem. This is the only cave ecosystem known to be supported by in situ autotrophic production, and it contains the only terrestrial community known to be chemoautotrophically based.

Mycobacterium Diversity and Pyrene Mineralization in Petroleum-Contaminated Soils

ABSTRACT Degradative strains of fast-growing Mycobacteriumspp. are commonly isolated from polycyclic aromatic hydrocarbon (PAH)-contaminated soils. Little is known, however, about the ecology and diversity of indigenous populations of these fast-growing mycobacteria in contaminated environments. In the present study 16S rRNA genes were PCR amplified usingMycobacterium-specific primers and separated by temperature gradient gel electrophoresis (TGGE), and prominent bands were sequenced to compare the indigenous Mycobacteriumcommunity structures in four pairs of soil samples taken from heavily contaminated and less contaminated areas at four different sites. Overall, TGGE profiles obtained from heavily contaminated soils were less diverse than those from less contaminated soils. This decrease in diversity may be due to toxicity, since significantly fewerMycobacterium phylotypes were detected in soils determined to be toxic by the Microtox assay than in nontoxic soils. Sequencing and phylogenetic analysis of prominent TGGE bands indicated that novel strains dominated the soil Mycobacteriumcommunity. Mineralization studies using [14C]pyrene added to four petroleum-contaminated soils, with and without the addition of the known pyrene degrader Mycobacterium sp. strain RJGII-135, indicated that inoculation increased the level of degradation in three of the four soils. Mineralization results obtained from a sterilized soil inoculated with strain RJGII-135 suggested that competition with indigenous microorganisms may be a significant factor affecting biodegradation of PAHs. Pyrene-amended soils, with and without inoculation with strain RJGII-135, experienced both increases and decreases in the population sizes of the inoculated strain and indigenous Mycobacterium populations during incubation.

Degradation of acid orange 7 in an aerobic biofilm.

A stable microbial biofilm community capable of completely mineralizing the azo dye acid orange 7 (AO7) was established in a laboratory scale rotating drum bioreactor (RDBR) using waste liquor from a sewage treatment plant. A broad range of environmental conditions including pH (5.8-8.2), nitrification (0.0-4.0 mM nitrite), and aeration (0.2-6.2 mg O2 l(-1)) were evaluated for their effects on the biodegradation of AO7. Furthermore the biofilm maintained its biodegradative ability for over a year while the effects of these environmental conditions were evaluated. Reduction of the azo bond followed by degradation of the resulting aromatic amine appears to be the mechanism by which this dye is biodegraded. Complete loss of color, sulfanilic acid, and chemical oxygen demand (COD) indicate that AO7 is mineralized. To our knowledge this is the first reported occurrence of a sulfonated phenylazonaphthol dye being completely mineralized under aerobic conditions. Two bacterial strains (ICX and SAD4i) originally isolated from the RDBR were able to mineralize, in co-culture, up to 90% of added AO7. During mineralization of AO7, strain ICX reduces the azo bond under aerobic conditions and consumes the resulting cleavage product 1-amino-2-naphthol. Strain SAD4i consumes the other cleavage product, sulfanilic acid. The ability of the RDBR biofilm to aerobically mineralize an azo dye without exogenous carbon and nitrogen sources suggests that this approach could be used to remediate industrial wastewater contaminated with spent dye.

Acidic Cave-Wall Biofilms Located in the Frasassi Gorge, Italy

Acidic biofilms present on cave walls in the sulfidic region of the Frasassi Gorge, Italy, were investigated to determine their microbial composition and their potential role in cave formation and ecosystem functioning. All biofilm samples examined had pH values < 1.0. Scanning electron microscopy of the biofilms revealed the presence of various filaments and rods associated in large clusters with mineral crystals. Qualitative energy-dispersive x-ray analysis was used to determine that the crystals present on the cave walls, associated with the microbial biofilm, were composed of calcium and barium sulfate. Ribosomal RNA-based methods to determine the microbial composition of these biofilms revealed the presence of at least two strains of potential acidophilic, sulfur-oxidizing bacteria, belonging to the genera Thiobacillus and Sulfobacillus. An acid-producing strain of Thiobacillus sp. also was obtained in pure culture. Stable isotope ratio analysis of carbon and nitrogen showed that the wall biofilms are isotopically light, suggesting that in situ chemoautotrophic activity plays an important role in this subsurface ecosystem.Acidic biofilms present on cave walls in the sulfidic region of the Frasassi Gorge, Italy, were investigated to determine their microbial composition and their potential role in cave formation and ecosystem functioning. All biofilm samples examined had pH values < 1.0. Scanning electron microscopy of the biofilms revealed the presence of various filaments and rods associated in large clusters with mineral crystals. Qualitative energy-dispersive x-ray analysis was used to determine that the crystals present on the cave walls, associated with the microbial biofilm, were composed of calcium and barium sulfate. Ribosomal RNA-based methods to determine the microbial composition of these biofilms revealed the presence of at least two strains of potential acidophilic, sulfur-oxidizing bacteria, belonging to the genera Thiobacillus and Sulfobacillus. An acid-producing strain of Thiobacillus sp. also was obtained in pure culture. Stable isotope ratio analysis of carbon and nitrogen showed that the wall biofilms...

Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX

Sphingomonas sp strain 1CX was isolated from a wastewater treatment plant and is capable of aerobically degrading a suite of azo dyes, using them as a sole source of carbon and nitrogen. All azo dyes known to be decolorized by strain 1CX (Orange II, Acid Orange 8, Acid Orange 10, Acid Red 4, and Acid Red 88) have in their structure either 1-amino-2-naphthol or 2-amino-1-naphthol. In addition, an analysis of the structures of the dyes degraded suggests that there are certain positions and types of substituents on the azo dye which determine if degradation will occur. Growth and dye decolorization occurs only aerobically and does not occur under fermentative or denitrification conditions. The mechanism by which 1CX decolorizes azo dyes appears to be through reductive cleavage of the azo bond. In the case of Orange II, the initial degradation products were sulfanilic acid and 1-amino-2-naphthol. Sulfanilic acid, however, was not used by 1CX as a growth substrate. The addition of glucose or inorganic nitrogen inhibited growth and decoloration of azo dyes by 1CX. Attempts to grow the organism on chemically defined media containing several different amino acids and sugars as sources of nitrogen and carbon were not successful. Phylogenetic analysis of Sphingomonas sp strain 1CX shows it to be related to, but distinct from, other azo dye-decolorizing Sphingomonas spp strains isolated previously from the same wastewater treatment facility.

Pyrite Framboids as Biomarkers for Iron-Sulfur Systems

The role of microorganisms in the formation of pyrite framboids has been suggested, but remains unresolved. We identified subsurface habitats from a sulfidic aquifer present in carbonate rock in Romania, where abundant pyrite framboids were found associated with microbial mats. Three types of subsurface microbial communities were studied: submerged mats overlaying clay-rich sediments, floating mats developed at the water-gas (sulfide/carbon dioxide) interface, and detrital FeS-rich sediments associated with floating microbial mats. All three habitats are anoxic, low temperature, low salinity, saturated with bicarbonate, and contain hydrogen sulfide, thiosulphate, and elemental sulfur. Sulfur stable isotope fractionation suggests that pyrite framboids from the floating microbial mats have a biogenic origin. Pyrite framboids characteristics, such as nucleation, growth, and spheroidicity, depend on conditions within the microbial mats and the inheritance of shape during the replacement of spheroidal greigite. We hypothesize that biogenic pyrite is formed in these microbial communities via a sulfide/ferrous iron pyritization mechanism. Some of the signatures of this type of biogenic pyritization are a similarity in size distribution between FeS spheroids and FeS2 framboids, the presence of microorganisms using sulfide and ferrous iron as a source of energy, reversed iron sulfide bilayers, a narrow size distribution of the pyrite framboids, a small numbers of large microcrystallites within the framboids, a S-fractionation up to 11.14 between H2S and FeS and up to 16.24 between H2S and FeS2. These signatures are relevant for the study of the biologically controlled pyritization and thus they are valuable tools for geomicrobiology, paleo-microbiology and exobiology.

High performance degradation of azo dye Acid Orange 7 and sulfanilic acid in a laboratory scale reactor after seeding with cultured bacterial strains.

Bacterial strains 1CX and SAD4i--previously isolated from the mixed liquor of a municipal sewage treatment plant--are capable of degrading the azo dye Acid Orange 7 (AO7) and sulfanilic acid, respectively. A rotating drum bioreactor (RDBR), operating under continuous flow and nutrient conditions designed to simulate the effluent from a dye manufacturing plant, was seeded with strains 1CX and SAD4i, forming a biofilm capable of degrading AO7 and sulfanilic acid. In addition, an RDBR containing a pre-existing biofilm capable of degrading AO7, but not sulfanilic acid, was seeded with strain SAD4i alone. Strain SAD4i was incorporated into the existing biofilm and degraded the sulfanilic acid resulting from the degradation of AO7 by indigenous members of the biofilm. The ability to seed a bioreactor with bacterial strains capable of degrading azo dyes, and resulting by-products, in a mixed microbial community suggests that this process could have commercial applications.

Ecological Assessment and Geological Significance of Microbial Communities from Cesspool Cave, Virginia

Microbial mats from hydrogen sulfide-rich waters and cave-wall biofilms were investigated from Cesspool Cave, Virginia, to determine community composition and potential geomicrobiological functioning of acid-producing bacteria. Rates of microbial mat chemoautotrophic productivity were estimated using [ 14 C]-bicarbonate incorporations and microbial heterotrophy was determined using [ 14 C]-leucine incubations. Chemoautotrophic fixation was measured at 30.4 - 12.0 ng C mg dry wt -1 h -1 , whereas heterotrophic productivity was significantly less at 0.17 - 0.02 ng C mg dry wt -1 h -1 . The carbon to nitrogen ratios of the microbial mats averaged 13.5, indicating that the mats are not a high quality food source for higher trophic levels. Ribosomal RNA-based methods were used to examine bacterial diversity in the microbial mats, revealing the presence of at least five strains of bacteria. The identity of some of the strains could be resolved to the genus Thiothrix and the Flexibacter - Cytophaga - Bacteriodes...

Biodegradation of carbazole by Ralstonia sp. RJGII.123 isolated from a hydrocarbon contaminated soil

The use of microorganisms for bioremediation of contaminated soils may be enhanced with an understanding of the pathways involved in their degradation of hazardous compounds. Ralstonia sp. strain RJGII.123 was isolated from soil located at a former coal gasification plant, based on its ability to mineralize carbazole, a three-ring N-heterocyclic pollutant. Experiments were carried out with strain RJGHII.123 and 14C-carbazole (2 mg/L and 500 mg/L) as the sole organic carbon source. At 15 days, 80% of the 2 mg/L carbazole was recovered as CO2, and <1% remained as undegraded carbazole, while 24% of the 500 mg/L carbazole was recovered as CO2 and approximately 70% remained as undegraded carbazole. Several stable intermediates were formed during this time. These intermediates were separated by high performance liquid chromatography (HPLC) and were characterized using high resolution mass spectroscopy (HR-MS) and gas chromatography - mass spectroscopy (GC-MS). At least 10 ring cleavage products of carbazole degradation were identified; four of these were confirmed as anthranilic acid, indole-2-carboxylic acid, indole-3-carboxylic acid, and (1H)-4-quinolinone by comparison with standards. These data indicate that strain RJGII.123 shares aspects of carbazole degradation with previously described Pseudomonas spp., and may be useful in facilitating the bioremediation of NHA from contaminated soils.

Productivity-Diversity Relationships from Chemolithoautotrophically Based Sulfidic Karst Systems

INTRODUCTION Microorganisms in most natural systems have typically been considered ecologically important in the cycling of carbon through the ‘microbial loop’ and decomposer niches (e.g., Azam et al., 1993; Bachofen et al., 1998). The discovery of diverse communities at deep-sea hydrothermal vents and cold seeps brought to light the importance of microorganisms as significant contributors to ecosystem autotrophic processes (e.g., Deming & Baross, 1993; Sassen et al., 1993). Indeed, chemolithoautotrophy (e.g., metabolic pathways where energy is gained from inorganic compounds