J. Gregory Zeikus

Microbial composition and characterization of prevalent methanogens and acetogens isolated from syntrophic methanogenic granules

The microbial species composition of methanogenic granules developed on an acetate-propionate-butyrate mixture was characterized. The granules contained high numbers of adhesive methanogens (1012/g dry weight) and butyrate-, isobutyrate-, and propionate-degrading syntrophic acetogens (1011/g dry weight), but low numbers of hydrolytic-fermentative bacteria (109/g dry weight). Prevalent methanogens in the granules included: Methanobacterium formicicum strain T1N and RF, Methanosarcina mazei strain T18, Methanospirillum hungatei strain BD, and a non-filamentous, bamboo-shaped rod species, Methanothrix/Methanosaeta-like strain M7. Prevalent syntrophic acetogens included: a butyrate-degrading Syntrophospora bryantii-like strain BH, a butyrate-isobutyrate degrading non-spore-forming rod, strain IB, a propionate-degrading sporeforming oval-shaped species, strain PT, and a propionate-degrading none-spore-forming sulfate-reducing rod species, strain PW, which was able to grow syntrophically with an H2-utilizing methanogen. Sulfate-reducing bacteria did not play a significant role in the metabolism of H2, formate, acetate and butyrate but they were involved in propionate degradation.

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H2−CO2. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H2 released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H2 plus HCOinf3sup-or produced H2 from formate to a steady-state level at which point the Gibbs free energy (ΔG′) available for formate synthesis or H2 production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H2 pressure. The relative rates of conversion of formate and H2 were apparently controlled by the ΔG′ available for formate synthesis, hydrogen production, methane production from formate and methane production from H2. Results from 14C-tracer tests indicated that a rapid isotopic exchange between HCOO- and HCOinf3sup-occurred during the growth of M. formicicum on H2−CO2. Data from metabolism of 14C-labelled formate to methane suggested that formate was initially split to H2 and HCOinf3sup-and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H2−CO2 was inhibited although production of methane was not Formate synthesis from H2 was also inhibited.

Metabolic properties and kinetics of methanogenic granules

Two types of mesophilic methanogenic granules (R- and F-granules) were developed on different synthetic feeds containing acetate, propionate and butyrate as major carbon sources and their metabolic properties were characterized. The metabolic activities of granules on acetate, formate and H2-CO2 were related to the feed composition used for their development. These granules performed a reversible reaction between H2 production from formate and formate synthesis from H2 plus bicarbonate. Both types of granules exhibited high activity on normal and branched volatile fatty acids with three to five carbons and low activity on ethanol and glucose. The granules performed a reversible isomerization between isobutyrate and butyrate during butyrate or isobutyrate degradation. Valerate and 2-methylbutyrate were produced and consumed during propionate-butyrate degradation. The respective apparent Km (mm) for various substrates in disrupted R- and F-granules was: acetate, 0.43 and 0.41; propionate, 0.056 and 0.038; butyrate, 0.15 and 0.19; isobutyrate, 0.12 and 0.19; valerate, 0.15 and 0.098. Both granules had an optimum temperature range from 40 to 50° C for H2-CO2 and formate utilization and 40° C for acetate, propionate and butyrate utilization and a similar optimum pH.

Effect of storage on the performance of methanogenic granules

Abstract Anaerobic methanogenic granules were stored under anaerobic condition at 4° and 22°C for 1- to 18-month period to evaluate the effect of storage on degradation of volatile fatty acids (VFAs), including acetate, propionate and butyrate, and on methane production. The length of storage period affected the activities of different microbial trophic groups. During storage at 22°C, the degradation rates for all the three acids decreased gradually. At low temperature (4°C), reduction in degradation rates of acetate and propionate was relatively slower than that at 22°C. Reduction in butyrate degradation rate was faster (by 45%) during the first month of storage at 4°C, but the rate declined afterwards. Nevertheless, the granules maintained, although at reduced level, their metabolic activities for all three VFAs even after storage for 18 months. Higher decay coefficients were obtained at 22°C than those at 4°C. For a relatively short period (1–5 months), granules can be stored at ambient temperature (approx. 20–22°C) with limited loss in their VFA degradation rates. However, granules can maintain higher levels of VFA degradation rates when they are stored at low (4°C) rather than at ambient temperature. Reactor studies indicated that the granules can completely recover their original VFA degradation rates in three days when stored for 31 d at 22°C. The granules stored at 22°C for 9 months were used successfully as inoculum to start a laboratory-scale reactor. The original VFA degradation rates of the granules were achieved after 15–20 d of reactor operation at 35°C.

Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183.

SummaryA new thermophilic Bacillus strain 3183 (ATCC 49341) was isolated from hot-spring sediments. The organism grew on pullulan as a carbon source and showed optimum pH and temperature at pH 5.5 and 62° C, respectively, for growth. The strain reduced nitrate to nitrite both aerobically and anaerobically. It produced extracellular thermostable pullulanase and saccharidase activities which degraded pullulan and starch into maltotriose, maltose, and glucose. Medium growth conditions for pullulanase production were optimized. The optimum pH and temperature for pullulanase activity were at pH 6.0 and 75° C, respectively. The enzyme was stable at pH 5.5-7.0 and temperature up to 70° C in the absence of substrate. The Km for pullulan at pH 6.0 and 75° C was 0.4 mg/ml. The pullulanase activity was stimulated and stabilized by Ca2+. It was inhibited by ethylenediaminetetraacetate (EDTA), beta and gamma-cyclodextrins but not by alpha-cyclodextrin and reagents that inhibit essential enzyme SH-groups.

Comparison of rod- versus filament-type methanogenic granules: microbial population and reactor performance

Two types of methanogenic granules capable of high chemical oxygen demand removal rates were developed in laboratory-scale upflow reactors at 35° C. One granule type (R-granules) had a rod-type Methanothrix-like species as the predominant species whereas the other (F-granules) had a filament-type M. soehngenii-like acetate-utilizer as the predominant species. These two types of granules were compared in terms of operational performance, physical-chemical characteristics and microbial population. The R-granules had a higher density [65–70 vs 39–43 g suspended solids (SS)/l], specific gravity (1.03 vs 1.01) and specific volumetric methane production rate (180 vs 120 l CH4/l granules per day) than the F-granules. Acetate, propionate and butyrate degraders in both types of granules had similar specific growth rates. The most probable number enumeration indicated that both types of granule had the same population levels (cells/g SS) in terms of methanogens (H2-CO2-, formate- and acetate-utilizing) and syntrophic acetogens. Hydrolytic-fermentative bacteria were present in greater number in the F-granules than in the R-granules. The R-granules had a higher cell density than the F-granules. The differences in operational performance were due mainly to their different microbial composition, especially the predominant acetate-utilizing methanogens in the granules. The long-filamentous M. soehngenii-like rods in the F-granules appeared to be responsible for their lower density and large-sized granules.

Characterization of thermostable α-glucosidase from Clostridium thermohydrosulfuricum 39E

SummaryClostridium thermohydrosulfuricum 39E produced a cell-bound α-glucosidase. It was partially purified 140-fold by solubilizing with Triton X-100, ammonium sulfate treatment, DEAE-Sepharose CL-6B, octyl-Sepharose and acarbose-Sepharose affinity chromatography. The optimum temperature for the action of the enzyme was at 75°C. It had a half-life of 35 min at 75°C, 110 min at 70°C and 46 h at 60°C. The enzyme was stable at pH 5.0–6.0 and had an optimum pH at 5.0–5.5. It hydrolyzed the α-1,4-linkages in maltose, maltotriose, maltotetraose and maltohexaose, the rate decreasing in order of higher-sized oligosaccharides. The enzyme preparation also hydrolyzed the α-1,6 linkages in isomaltose and isomaltotriose. It rapidly hydrolyzed p-nitrophenyl α-d-glucoside (pNPG). The Km values for maltose, isomaltose, panose, maltotriose, and pNPG were 1.85, 2.95, 1.72, 0.58, and 0.31 mm, respectively, at pH 5.5 and 60°C. The enzyme produced glucose from all these substrates. The enzyme preparation did not require any metal ion for activity. The α-glucosidase activity was inhibited by acarbose.

Construction of inducible secretion vectors and their application for the secretion of foreign extracellular and intracellular proteins in Bacillus subtilis

Abstract We have constructed inducible secretion vector (pISA412) which has (i) a unique BamHI site as a cloning site between the signal sequence and transcriptional terminator of Bacillus subtilis α-amylase gene (amyE), and (ii) penI-penJ operon and promoter-operator regions of penP (penicillinase gene) from Bacillus licheniformis as a regulation system upstream to the amyE signal sequence. Using pISA412, we examined the expression in B. subtilis of several cloned secretory protein genes such as the penicillinase gene from B. licheniformis (penP), α-amylase genes from Bacillus stearothermophilus (amyT) and human salivary (hsa), and the thermostable endoglucanase gene from Clostridium thermocellum (engD). In each case, gene expression was induced by the addition of 2-(2′-carboxyphenyl) benzoyl-6-aminopenicillanic acid (CBAP). Significant levels of extracellular activity of these gene products could be detected. Furthermore, when an intracellular enzyme gene (β-galactosidase gene of Escherichia coli; lacZ) was subcloned in the secretion vector, β-galactosidase activity could be detected in the culture supernatant. We also constructed a secretion vector, pISA t8412, with the temperature-sensitive repressor (P70L) in order to induce gene expression simply by shifting temperature of the culture from 30°C to 48°C. Expression of the cloned gene into the pISAts412 could be controlled by the temperature shift. These results indicate that pISA412 and pISA ts412 vectors are useful inducible secretion vectors in B. subtilis.