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Title: Light and electron microscopic examinations of methane-producing biofilms from anaerobic fixed-bed reactors. [Methanothrix spp. ; Methanosarcina spp]

Abstract

Ultrastructural examinations were performed on biofilms from eight anaerobic fixed-bed reactors filled with various packing materials and operated on fresh swine waste. By using light, UV, scanning, and transmission electron microscopy, the distribution of a diverse microbial population composed of bacteria and a few yeasts was determined. This is the first time that the ultrastructure of in situ anaerobic digestor biofilms has been reported. A large number of methanogenic bacteria were identified by their fluorescence under 420 nm of radiation. Of these, two morphologically distinct types were most prevalent in the films. Methanothrix spp. was present in high numbers at the film surface, whereas Methanosarcina spp. were commonly embedded in the lower regions of the of the film. Inhabitants of the film were surrounded by an exopolysaccharide matrix that was very dense toward the base. An extensive network of channels was observed throughout the matrix that may facilitate gas and nutrient exchange to the lower regions of the film.

Authors:
; ; ; ;
Publication Date:
Research Org.:
Univ. of Florida, Gainesville
OSTI Identifier:
6147589
Resource Type:
Journal Article
Resource Relation:
Journal Name: Appl. Environ. Microbiol.; (United States); Journal Volume: 48:1
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; BIOREACTORS; SAMPLING; METHANOGENIC BACTERIA; MORPHOLOGY; FLUORESCENCE; OPTICAL MICROSCOPY; PACKED BED; SCANNING ELECTRON MICROSCOPY; TRANSMISSION ELECTRON MICROSCOPY; BACTERIA; ELECTRON MICROSCOPY; LUMINESCENCE; MICROORGANISMS; MICROSCOPY 140504* -- Solar Energy Conversion-- Biomass Production & Conversion-- (-1989); 550700 -- Microbiology; 090122 -- Hydrocarbon Fuels-- Preparation from Wastes or Biomass-- (1976-1989)

Citation Formats

Robinson, R.W., Akin, D.E., Nordstedt, R.A., Thomas, M.V., and Aldrich, H.C.. Light and electron microscopic examinations of methane-producing biofilms from anaerobic fixed-bed reactors. [Methanothrix spp. ; Methanosarcina spp]. United States: N. p., 1984. Web.
Robinson, R.W., Akin, D.E., Nordstedt, R.A., Thomas, M.V., & Aldrich, H.C.. Light and electron microscopic examinations of methane-producing biofilms from anaerobic fixed-bed reactors. [Methanothrix spp. ; Methanosarcina spp]. United States.
Robinson, R.W., Akin, D.E., Nordstedt, R.A., Thomas, M.V., and Aldrich, H.C.. 1984. "Light and electron microscopic examinations of methane-producing biofilms from anaerobic fixed-bed reactors. [Methanothrix spp. ; Methanosarcina spp]". United States. doi:.
@article{osti_6147589,
title = {Light and electron microscopic examinations of methane-producing biofilms from anaerobic fixed-bed reactors. [Methanothrix spp. ; Methanosarcina spp]},
author = {Robinson, R.W. and Akin, D.E. and Nordstedt, R.A. and Thomas, M.V. and Aldrich, H.C.},
abstractNote = {Ultrastructural examinations were performed on biofilms from eight anaerobic fixed-bed reactors filled with various packing materials and operated on fresh swine waste. By using light, UV, scanning, and transmission electron microscopy, the distribution of a diverse microbial population composed of bacteria and a few yeasts was determined. This is the first time that the ultrastructure of in situ anaerobic digestor biofilms has been reported. A large number of methanogenic bacteria were identified by their fluorescence under 420 nm of radiation. Of these, two morphologically distinct types were most prevalent in the films. Methanothrix spp. was present in high numbers at the film surface, whereas Methanosarcina spp. were commonly embedded in the lower regions of the of the film. Inhabitants of the film were surrounded by an exopolysaccharide matrix that was very dense toward the base. An extensive network of channels was observed throughout the matrix that may facilitate gas and nutrient exchange to the lower regions of the film.},
doi = {},
journal = {Appl. Environ. Microbiol.; (United States)},
number = ,
volume = 48:1,
place = {United States},
year = 1984,
month = 7
}
  • The short-term effects of temperature on methanogenesis from acetate or CO/sub 2/ in a thermophilic (58/sup 0/C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with /sup 14/C-labeled methane precursors (/sup 14/CH/sub 3/COO/sup -/ or /sup 14/CO/sub 2/). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60/sup 0/C and was completely inhibited at 65/sup 0/C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58/sup 0/C and did not grow or produce methane at 65/sup 0/C. An accidental shift of digestormore » temperature from 58 to 64/sup 0/C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from /sup 14/CH/sub 3/COO/sup -/ was optimal at 65/sup 0/C and completely inhibited at 75/sup 0/C. Methanogenesis from /sup 14/CO/sub 2/ in the sludge was optimal at 65/sup 0/C and still proceeded at 75/sup 0/C. A CO/sub 2/-reducing methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75/sup 0/C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65/sup 0/C produced more methane than sludge incubated at 60/sup 0/C, and no acetate accumulated at 65/sup 0/C. Methanogenesis was severely inhibited in sludge incubated at 70/sup 0/C, but since neither acetate nor H/sub 2/ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. 19 references.« less
  • CO and H{sub 2} have been implicated in methanogenesis from acetate, but it is unclear whether they are directly involved in methanogenesis or electron transfer in acetotrophic methanogens. The authors compared metabolism of H{sub 2}, CO, and formate by cultures of the thermophilic acetotrophic methanogens Methanosarcina thermophila TM-1 and Methanothrix sp. strain CALS-1. M. thermophila accumulated H{sub 2} to partial pressures of 40 to 70 Pa, as has been previously reported for this and other Methanosarcina cultures. In contrast, Methanothrix sp. strain CALS-1 accumulated H{sub 2} to maximum partial pressures near 1 Pa. Growing cultures of Methanothrix sp. strain CALS-1more » initially accumulated CO, which reached partial pressures near 0.6 Pa during the middle of methanogenesis; this was followed by a decrease in CO partial pressure to less than 0.01 Pa by the end of methanogenesis. Late-exponential-phase cultures of Methanothrix sp. strain CALS-1, in which the CO partial pressure was decreased , accumulated CO to 0.16 Pa, whereas cultures to which ca. 0.5 Pa of CO was added consumed CO until it reached this partial pressure. Results suggest that CO plays a role in Methanothrix sp. strain CALS-1 similar to that of H{sub 2} in M. thermophila and are consistent with the conclusion that CO is an intermediate in a catabolic or anabolic pathway in Methanothrix sp. strain CALS-1; however, they could also be explained by passive equilibrium of CO with a metabolic intermediate.« less
  • A new acetotrophic marine methane-producing bacterium that was isolated from the methane-evolving sediments of a marine canyon is described. Exponential phase cultures grown with sodium acetate contained irregularly shaped cocci that aggregated in the early stationary phase and finally differentiated into communal cysts that released individual cocci when ruptured or transferred to fresh medium. The irregularly shaped cocci (1.9 +/- 0.2 mm in diameter) were gram negative and occurred singly or in pairs. Cells were nonmotile, but possessed a single fimbria-like structure. Micrographs of thin sections showed a monolayered cell wall approximately 10 nm thick that consisted of protein subunits.more » The cells in aggregates were separated by visible septation. The communal cysts contained several single cocci encased in a common envelope. An amorphous form of the communal cyst that had incomplete separation and internal membrane-like vesicles was also present in late exponential phase cultures. Sodium acetate, methanol, methylamine, dimethylamine, and trimethylamine were substrates for growth and methanogenesis; H/sub 2/-CO/sub 2/ (80:20) and sodium formate were not. The optimal growth temperature was 35 to 40/sup 0/C. The optimal pH range was 6.5 to 7.0. Both NaCl and Mg/sub 2//sup +/ were required for growth, with maximum growth rates at 0.2 M NaCl and 0.05 M MgSO/sub 4/. The DNA base composition was 41 +/- 1% guanine plus cytosine. Methanosarcina acetivorans is the proposed species. C2A is the type strain (DSM 2834, ATCC 35395). 43 references.« less
  • Anaerobic digestion processes with rapid production of methane are economical methods of waste stabilization. At the present time, these processes require highly trained technical staff and analytical equipment. The rudiments of a simple computer process control system are described to alleviate the need for analytical equipment and trained staff. Gas production was chosen as the controlled variable since it is a very sensitive indicator of digestor performance and can be measured rapidly. Feed rate was chosen as the manipulated variable. The aim was to develop a program which would maintain the performance of the process under normal operating conditions, and,more » in the event of a severe upset, would signal that a problem existed so remedial action could be taken. The digestor used was a down-flow stationary fixed film reactor. The waste used was a synthetic sugar waste containing 1% (w/v) sugar (10g chemical oxygen demand/L). The desired rate of biogas production was chosen as 250L/day at an approximate methane content of 50%.« less
  • Aceticlastic methanogens and other microbial groups were enumerated in a 58 degrees C laboratory-scale (3 liter) anaerobic digestor which was fed air-classified municipal refuse, a lignocellulosic waste (loading rate = 1.8 to 2.7 g of volatile solids per liter per day: retention time = 10 days). Two weeks after start-up, Methanosarcina sp. was present in high numbers (10/sup 5/ to 10/sup 6/ CFU/ml) and autofluorescent Methanosarcina clumps were abundant in sludge. After 4 months of operation, numbers of Methanosarcina sp. dropped 2 to 3 orders of magnitude and large numbers (most probable number = 10/sup 6/ to 10/sup 7/ /ml)more » Methanothrix sp. were found. Methanothrix sp. had apparently displaced Methanosarcina sp. as the dominant aceticlastic methanogen. During the period when Methanothrix sp. was dominant, acetate concentrations varied between 0.3 and 1.5 mumol/ml during the daily feeding cycle, and acetate was the precursor of 63 to 66% of the methane produced during peak digestor methanogenesis. The apparent Km value obtained for methanogenesis from acetate, 0.3 mumol/ml, indicated that the aceticlastic methanogens were saturated for substrate during most of the digestor cycle. CO2 reducing methanogens were capable of methanogenesis at rates more than 12 times greater than those usually found in the digestor. Added propionate (4.5 mumol/ml) was metabolized slowly and slightly inhibited methanogenesis. Added n-butyrate, isobutyrate, or n-valerate (4.5 mumol/ml each) were broken down within 24 h. Isobutyrate was oxidized to acetate, a novel reaction possibly involving isomerization to n-butyrate. The rapid growth rate and versatile metabolism of Methanosarcina sp. make it a likely organism to be involved in start-up whereas the low Km value of Methanothrix sp. for acetate may cause it to be favored in stable digestors operated with long retention times.« less