Richard F. Unz

Kinetics of Perchlorate- and Chlorate-Respiring Bacteria

ABSTRACT Ten chlorate-respiring bacteria were isolated from wastewater and a perchlorate-degrading bioreactor. Eight of the isolates were able to degrade perchlorate, and all isolates used oxygen and chlorate as terminal electron acceptors. The growth kinetics of two perchlorate-degrading isolates, designated “Dechlorosoma” sp. strains KJ and PDX, were examined with acetate as the electron donor in batch tests. The maximum observed aerobic growth rates of KJ and PDX (0.27 and 0.28 h−1, respectively) were only slightly higher than the anoxic growth rates obtained by these isolates during growth with chlorate (0.26 and 0.21 h−1, respectively). The maximum observed growth rates of the two non-perchlorate-utilizing isolates (PDA and PDB) were much higher under aerobic conditions (0.64 and 0.41 h−1, respectively) than under anoxic (chlorate-reducing) conditions (0.18 and 0.21 h−1, respectively). The maximum growth rates of PDX on perchlorate and chlorate were identical (0.21 h−1) and exceeded that of strain KJ on perchlorate (0.14 h−1). Growth of one isolate (PDX) was more rapid on acetate than on lactate. There were substantial differences in the half-saturation constants measured for anoxic growth of isolates on acetate with excess perchlorate (470 mg/liter for KJ and 45 mg/liter for PDX). Biomass yields (grams of cells per gram of acetate) for strain KJ were not statistically different in the presence of the electron acceptors oxygen (0.46 ± 0.07 [n = 7]), chlorate (0.44 ± 0.05 [n = 7]), and perchlorate (0.50 ± 0.08 [n = 7]). These studies provide evidence that facultative microorganisms with the capability for perchlorate and chlorate respiration exist, that not all chlorate-respiring microorganisms are capable of anoxic growth on perchlorate, and that isolates have dissimilar growth kinetics using different electron donors and acceptors.

Biological Perchlorate Reduction in High-Salinity Solutions

Perchlorate (ClO4-) has been detected in numerous ground and surface waters, and has recently been added to the drinking water Candidate Contaminant List in the United States. Perchlorate can be removed from drinking water using ion exchange, but this results in the production of highly saline (7-12%) perchlorate-contaminated brines. Perchlorate-degrading microbial enrichments capable of growth in highly saline water were obtained by screening six salt water environments including marine and lake surface waters, salt marshes, subtidal sediments, and a biofilm/sludge from a seawater filter. Perchlorate reduction was obtained in three of these samples (seawater, saline lake water, and biofilm/sludge) at a salinity of 3%. The salinity range of two of these cultures was extended through serial transfers into media having higher salt concentrations (3-7%). Growth rates were measured over a salinity range of 1-15%. The maximum growth rate measured for the saline lake-water enrichment was 0.060+/-0.003 d(-1) (doubling time of 11.6+/-0.8 d) at a salinity of 5%. Growth rates decreased to 0.037+/-0.002 d(-1) at a salinity of 11%, and no growth was observed at salinities of 13 or 15%. These results demonstrate for the first time that biological perchlorate reduction is possible in solutions having a salinity typical of ion exchange brines.

Persistence of Perchlorate and the Relative Numbers of Perchlorate- and Chlorate-Respiring Microorganisms in Natural Waters, Soils, and Wastewater

Cell numbers of perchlorate (PRM)- and chlorate (CRM)-reducing microorganisms and the persistence of perchlorate were determined in samples of soils, natural waters, and wastewater incubated under laboratory conditions. Complete perchlorate reduction in raw wastewater and creek water was achieved in 4 to 7 days and 8 to 29 days, respectively, depending on the individual growth substrate (acetate, lactate, citric acid, or molasses) employed. Perchlorate persisted in most mixed cultures developed with 2 g of “pristine” soil, but declined in mixed cultures developed with 100 g of soil. Less than seven days were required to completely reduce perchlorate in cultures started with 10 g of a perchlorate-contaminated soil obtained from a site in Texas. The concentration of PRM was estimated using a 5-tube most probable number (MPN) procedure. To account for discrepancies due to differences in the total number of bacteria (per mass of sample) in the samples, difficulty in removing bacteria from soil samples, and the lack of an unequivocal method to measure total viable cells in these different systems, we normalized our MPN results on the basis of 106 or 109 total bacteria counted using acridine orange direct counts (AODC). There were more PRM in wastewater samples on a per-cell basis (15 to 350 PRM/106-AODC) than in water samples (0.02 to 0.4 PRM/106-AODC). There were also more PRM in soils from sites exhibiting direct evidence of perchlorate contamination (100 to 200 PRM/109-AODC) than from other sites (nondetectable to 0.77 PRM/109-AODC). These results demonstrate that perchlorate-reducing bacteria are present at perchlorate-contaminated sites, and that perchlorate can be degraded by these microorganisms through the addition of different electron donors, such as acetate and lactate.

The nutrition of Thiothrix, type 021N, Beggiatoa and Leucothrix strains

Abstract Filamentous sulfur bacteria (Thiothrix, Beggiatoa and type 021N) and Leucothrix sp. were studied to determine their carbon and nitrogen requirements and growth response to nutrient concentration and salinity. Two Thiothrix strains required both a reduced sulfur compound and organic carbon source for growth. Others grew heterotrophically on organic acids or fructose-related sugars. Thiothrix isolates differed in regard to salinity tolerance. Type 021N bacteria utilized various sugars, amino acids and organic acids as sole carbon sources. Amino acids, but not nitrate, satisfied the nitrogen requirements for most strains. The Beggiatoa isolate utilized only simple organic acids and alcohols, and was inhibited by 0.3% concentrations of various salts. Strains of wastewater Leucothrix utilized various carbon and nitrogen compounds for growth. Low levels of lactate, ammonium and phosphate supported maximum growth of Thiothrix, Beggiatoa and type 021, but not Leucothrix strains. The optimum pH for growth initiation of the majority of bacteria was pH 7.0–7.5.

Isolation and characterization of filamentous bacteria present in bulking activated sludge

SummarySeveral types of filamentous microorganisms were observed and identified in samples of poorly settling (bulking) activated sludge. The major types encountered and the frequency (percentage) of appearance in the total of all treatment plants sampled were: Eikelboom type 0041 (60), type 1701 (45), Haliscomenobacter hydrossis (35), type 021N (30), Thiothrix spp. (20), and Sphaerotilus natans (20). Isolation techniques and culture media were developed and used to recover 42 axenic strains of filamentous bacteria from sludge samples collected. The isolates were identified as strains of Thiothrix, Beggiatoa, S. natans, and Eikelboom types 021N, 1701, 0041, and 0803. Nutritional and differential characterization of the bacteria was important to the differentiation of groups which could not be easily distinguished on the basis of morphology. Although certain treatment plant operating parameters (organic loading) seemed associated with the presence of specific filamentous organism types, possible interaction among factors precluded definite establishment of a cause and effect relationship for most of the treatment plant characteristics and organisms observed.

Acidiphilium angustum sp. nov., Acidiphilium facilis sp. nov., and Acidiphilium rubrum sp. nov. : acidophilic heterotrophic bacteria isolated from acidic coal mine drainage

Acidophilic heterotrophic bacteria recovered from samples of water and sediment collected from acidic mine drainage streams were compared nutritionally, genetically, and morphologically. All 37 bacterial strains examined were rod shaped, motile, gram negative, and strictly aerobic, utilized citric acid and Tween 80 as sole carbon sources, and were unable to grow at or above pH 6.0. The ultrastructure of representative strains was not markedly different from that of gram-negative bacteria. Differences among the strains were evident in cell size (4.2 by 0.6 to 1.2 by 0.6 p.m), pigmentation (when present), and nutritional faculties (the carbon sources suitable for growth of individual strains ranged from 8 to 20 of the 32 compounds tested). The guanine-plus-cytosine base composition of eight typical strains ranged from 63 to 68 mol%. All of the strains exhibited primary characteristics of the recently described genus Acidiphilium; however, important differences between our strains and the type species Acidiphilium cryptum suggested that new Acidiphilium species should be described. No significant deoxyribonucleic acid-deoxyribonucleic acid homology was found between five acidophilic heterotrophic strains and A. cryptum. Furthermore, no significant deoxyribonucleic acid-deoxyribonucleic acid homology was evident between the acidophilic heterotrophs and six Thiobacillus species. The bacteria which we studied could be divided into three groups based on genetic and nutritional characteristics. We propose the following three new species: Acidiphilium rubrum (type strain, strain OP [= ATCC 35905]), Acidiphilium angustum (type strain, strain KLB [= ATCC 35903]), and Acidiphilium facilis (type strain, strain PW2 [= ATCC 35904]).

Influence of metals and metal speciation on the growth of filamentous bacteria

Abstract Calcium, copper, nickel and zinc were examined for their effect on the growth of axenic filamentous bacteria which included strains of Thiothrix , type 021N and type 1701. All organisms grew best at the upper range of calcium concentrations tested. Heavy metals were also less toxic to Thiothrix strain A1 at higher calcium concentrations. Copper was more inhibitory than either nickel or zinc and copper-nickel and copper-zinc mixtures appeared to act synergistically in suppressing the development of Thiothrix strain A1. In contrast, nickel toxicity could be reduced by the addition of zinc to the medium. Metal toxicity could also be diminished by the chelating agent, EDTA; therefore, free metal ions were most likely responsible for the observed toxic effects. A computer model, MINEQL, was used to predict metal speciation and to determine the potential for various media components to control speciation of the metals. The suitability of the computer model was determined by comparison of predicted and analyzed free metal ion concentrations.

Concurrent denitrification and oxygen uptake in microbial films

Abstract Mixed microbial films were grown on the inner walls of a tubular reactor with recirculation of the reactor contents and continuous flow-through of nutrient solution. The loss of total oxidized nitrogen was correlated to the film population, the nitrite concentration and the dissolved oxygen concentration in the reactor. When film population was greater than 0.5 × 10 9 cells cm −2 , reactor dissolved oxygen concentration greater than 1 mg l −1 had little effect on the nitrate loss. Nitrate loss declined for film populations greater than 2 × 10 9 cells cm −2 . Models based on Monod and zero-order microbial kinetics were calibrated using these data and a nonlinear least squares method. There was little difference in the residual errors with these methods.

Observations on the formation of wastewater zoogloeae

Abstract Finger-like bacterial zoogloea formation was observed microscopically in wet mount preparations of activated sludge flocs. Elongated zoogloeae were shown by time lapse cinephotomicrography to emerge from flocs as a result of the unidirectional movement and multiplication of bacteria which synthesized an exocellular gelatinous substance. Seventy per cent of the finger-like zoogloeae measured extended at a rate of between 5.1 and 15.0 μm h−1. Certain bacteria moved freely within zoogloeae and the mean generation time of cells in three zooglocae ranged from 1.5 to 1.6 h. The development of finger-like zoogloeae may have been stimulated by an aerotactic response by the etiologic bacteria. The gelatinous nature of the zoogloeal matrix is evidenced by the slow movement of bacteria in the colony.

CHEMICAL DIAGENESIS OF IRON AND MANGANESE IN CONSTRUCTED WETLANDS RECEIVING ACIDIC MINE DRAINAGE

ABSTRACT A field/laboratory investigation was undertaken concerning the chemical diagenesis of iron and manganese in the sediments and interstitial waters of two constructed wetlands in Pennsylvania receiving chemically dissimilar mine waters. The Hartzfeld wetland located in Luthersburg receives a drainage of pH 6 and low metal loading rates. The Frenchville site receives a strong drainage of pH 3 and high metal loading. Redox potential measurements revealed distinct zones of oxidized and reduced sediments. Sediment characterization indicated that metal precipitation in the oxidized zone was the dominant metal removal process occurring within each wetland. Sediment equilibration experiments suggested that the activity of iron was controlled by the solubility of goethite at Hartzfeld and by cation exchange at Frenchville. The activity of manganese at both sites appeared to be controlled by cation exchange rather than by mineral solubility. Concentration-depth profiles showed an abundance of metal oxides and relatively low concentrations of dissolved metals in the oxidized zone. The reduced zone was characterized by decreased metal oxides and increased dissolved iron and manganese. Diagenetic redistribution of oxidized iron and manganese within the sediments demonstrates the potential for precipitation of pyrite, siderite, and rhodochrosite in the reduced zone, although reaction kinetics may be too slow to permit appreciable accumulation of these minerals.