Larry Baresi

Caldicellulosiruptor owensensis sp. nov., an anaerobic, extremely thermophilic, xylanolytic bacterium.

An anaerobic, extremely thermophilic, xylanolytic, non-spore-forming bacterium was isolated from a sediment sample taken from Owens Lake, California, and designated strain OLT (T = type strain). Strain OLT had a Gramnegative reaction and occurred as short rods which sometimes formed long chains containing a few coccoid cells. It grew at 50-80 degrees C, with an optimum at 75 degrees C. The pH range for growth was 5.5-9.0 with an optimum at about pH 7.5. When grown on glucose at optimal conditions, its doubling time was 7.3 h. In addition to glucose, the isolate utilized sucrose, xylose, fructose, ribose, xylan, starch, pectin and cellulose. Yeast extract stimulated growth on carbohydrates but was not obligately required. The end products from glucose fermentation were lactate, acetate, ethanol, H2 and CO2. The G + C content of strain OLT was 36.6 mol%. The 16S rDNA sequence analysis indicated that strain OLT was a member of the subdivision containing Gram-positive bacteria with DNA G + C content of less than 55 mol% and clustered with members of the genus Caldicellulosiruptor. Because strain OLT is phylogenetically and phenotypically different from other members of this genus, it is proposed to designate this isolate Caldicellulosiruptor owensensis sp. nov. Strain OLT is the type strain (= ATCC 700167T).

Haloanaerobium alcaliphilum sp. nov., an Anaerobic Moderate Halophile from the Sediments of Great Salt Lake, Utah

A strictly anaerobic, moderately halophilic, gram-negative, rod-shaped bacterium was isolated from Great Salt Lake, Utah, sediments and designated GSLST (T = type strain). Strain GSLST grew optimally at pH 6.7 to 7.0 but had a very broad pH range for growth (pH 5.8 to 10.0). The optimum temperature for growth was 37 degrees C, and no growth occurred at 15 or 55 degrees C. The optimum salt concentration for growth was 10%. Strain GSLST required yeast extract and Trypticase peptone to ferment carbohydrates, pyruvate, and glycine betaine. Strain GSLST was resistant to penicillin, D-cycloserine, tetracycline, and streptomycin. The G + C content of this isolate was 31 mol%. The fermentation products from glucose utilization were acetate, butyrate, lactate, CO2, and H2, and in addition strain GSLST fermented glycine betaine to acetate and trimethylamine. All of these traits distinguish this organism from all previously described halophilic anaerobes. The 16S rRNA gene sequence of strain GSLST was found to be similar to, but also significantly different from, the 16S rRNA sequences of Haloanaerobium salsugo and Haloanaerobium praevalens. Therefore, strain GSLST (= DSM 8275T) is described as a new species, Haloanaerobium alcaliphilum.

Salinivibrio costicola subsp. vallismortis subsp. nov., a halotolerant facultative anaerobe from Death Valley, and emended description of Salinivibrio costicola

Strain DVT, a halotolerant, Gram-negative, facultatively anaerobic bacterium, was isolated from a hypersaline pond located in Death Valley, California. The cells were non-spore-forming, motile, curved rods (1.0-1.8 x 0.5-0.6 microns) and occurred singly, in pairs or rarely in chains. Strain DVT was oxidase-, catalase-, Voges-Proskauer-, amylase-, gelatinase- and lipase-positive and indole-negative. Nitrate, sulfate and fumarate were not used as electron acceptors. Carbohydrates served as energy sources both aerobically and anaerobically. Strain DVT grew optimally at 37 degrees C (temperature range 20-50 degrees C) with 2.5% NaCl (NaCl range 0-12.5%) and pH 7.3 (pH range of 5.5-8.5) in a glucose/yeast extract medium with a doubling time of 20 min (aerobically) or 41 min (anaerobically). The end products of glucose fermentation were ethanol, isobutyrate, propionate, lactate, formate and CO2. Strain DVT was resistant to penicillin, D-cycloserine, streptomycin and tetracycline (200 micrograms ml-1). The G + C content was 50 mol%. 16S rRNA gene sequence analysis indicated that it was closely related to Salinivibrio costicola (97.7%) and this was confirmed by DNA-DNA hybridization (93% relatedness). However, phenotypic characteristics such as halotolerance, gas production, growth at 50 degrees C, antibiotic resistance, sugar-utilization spectrum and phylogenetic signatures are sufficiently different from Salinivibrio costicola to warrant designating strain DVT as a new subspecies of Salinivibrio costicola, Salinivibrio costicola subsp. vallismortis subsp. nov. (= DSM 8285T).

An intracellular delivery vehicle for protein transduction of micro-dystrophin

The Fv fragment of an antibody that selectively targets and penetrates skeletal muscle in vivo was produced as a fusion protein with a micro-dystrophin for use as a delivery vehicle to transport micro-dystrophin into muscle cells. Fv-micro-dystrophin was produced as a secreted protein by transient transfection of Fv-micro-dystrophin cDNA in COS-7 cells and as a non-secreted protein by permanent transfection in Pichia pastoris. Isolated Fv-micro-dystrophin was shown to be full-length by Western blot analysis. Fv-micro-dystrophin penetrated multiple cell lines in vitro, and it localized to the plasma membrane of a cell line with membrane beta-dystroglycan. In the absence of membrane beta-dystroglycan, it localized to the cytoplasm. Antibody-mediated transduction of micro-dystrophin into muscle cells is a potential therapy for dystrophin-deficient muscular dystrophies.

Methane Estimation for Methanogenic and Methanotrophic Bacteria

Methanogenic and methanotrophic bacteria frequently occur in the same habitats, the methanogens producing methane and the methanotrophs consuming the methane produced by the methanogens. The activities of both bacterial groups can be monitored by measuring methane in the atmospheres of cultures and ecosystem samples. However, there is a greater reliance on methane measurement for methanogenic than for methanotrophic bacteria. In this chapter, we cover methods for estimating methane and associated gases. A few specialized methods for each group will also be considered.

The Conversion of Lignocellulosics to Fermentable Sugars: A Survey of Current Research and Applications to CELSS

An overview of the options for converting lignocellulosics into fermentable sugars as applied to the Closed Ecological Life Support System (CELSS) is given. A requirement for pretreatment is shown as well as the many available options. At present, physical/chemical methods are the simplest and best characterized options, but enzymatic processes will likely be the method of choice in the future. The use of pentose sugars by microorganisms to produce edibles at levels comparable to conventional plants is shown. The possible use of mycelial food production on pretreated but not hydrolyzed lignocelluloscis is also presented. Simple tradeoff analysis among some of the many possible biological pathways to regeneration of waste lignocellulosics was undertaken. Comparisons with complete oxidation processes were made. It is suggested that the NASA Life Sciences CELSS program maintain relationships with other government agencies involved in lignocellulosic conversions and use their expertise when the actual need for such conversion technology arises rather than develop this expertise within NASA.