AssociateProfessor
Department of Microbiology
University of Manitoba
Winnipeg, Manitoba
Canada R3T 2N2
Telephone: (204) 474-8320
FAX: (204) 474-7603
E-mail: Richard_Sparling@umanitoba.ca
I) Methane producing bacteria
This research work wishes to better understand various aspects of biosynthetic processes in methane producing bacteria using Methanosphaera stadtmanae as the major model organism. The relevance of the study of methanogenic organisms stems from the fact that i) they act as terminal electron acceptor in a variety of anthropogenic and natural anaerobic communities (swamps and sediments, digestive tracts of ruminants and also of humans, domestic and agricultural liquid and solid wastes, land fills and secondary digesters of municipal waste treatment plants), ii) they produce methane, a potential fuel, as end product, and iii) they are members of the Archaean Domain, a group of primitive organisms with very unusual metabolic traits and enzymatic co-factors. This work is in 2 parts.
(a)-The use of C1-carriers for biosynthesis
A tetrahydrofolate (H4F) dependent serine hydroxymethyltransferase has been demonstrated in several representatives of the Methanomicrobiales. The enzyme from Methanospirillum hungatei has been purified to homogeneity and is being characterised for comparison with the tetrahydromethanopterin (H4MPT) dependent enzyme described in other methanogens. This finding was rather unexpected and novel since H4F has not been detected in M. hungatei, however, the major pterin of this organism, H4MPT does not react with this enzyme. The discovery of a non-H4MPT dependent serine hydroxymethyltransferase may explain the 13C-labelling work performed in other laboratories.
I am currently looking for a graduate student to continue this investigation. This would involve 1) determining whether other H4F dependent enzyme activities can be found in the methanogens for which we have detected a H4F dependent serine hydroxymethyltransferase and/or 2) determining the genetic sequence corresponding to this protein for comparison with sequences from other methanogens to aid in elucidating the differences between the tetrahydrofolate vs tetrahydromethanopterin dependent serine hydroxymethyl transferases in methanogens.
(b)-Electron transfer from H2 for biosynthesis in Methanosphaera stadtmanae.
All electrons for reductive biosynthetic reactions in this organism must come from H2, which is the only source of electrons supplied to the cell. However, the means of electron transfer from H2 to the general biosynthetic electron carrier NADP+ is unclear. In extracts we have found all activities necessary to transfer electrons from H2 to NADP+ except 1, and have proposed a model for electron transfer. Currently 2 of the enzymes involved are being purified and characterised (Hydrogenase and NADP+-F420 oxidoreductase). F420 is the direct donor of electrons to NADP+. A third and the most novel of the enzymes uncovered is a methyl-viologen dependent F420 dehydrogenase which may be involved in the transfer of electrons from H2 to F420. We were the first to report the activity of such an enzyme in a H2 utilising methanogen. Once this enzyme has been characterised in M. stadtmanae, its presence will be looked for in other H2 oxidizing methanogens.
II) Mechanism of production of methyl mercury by anaerobic bacteria.
The production of methyl mercury by anaerobic bacteria is a significant problem behind man-made reservoirs (e.g. hydroelectric reservoirs) because methyl mercury, which in large concentrations is a neurotoxin, accumulates in the flesh of fish. Fish caught in such reservoirs often exceed the maximal permissible limit of methyl mercury for human consumption. Various lab strains of environmentally significant anaerobic bacteria are been tested for their capacity to methylate mercury. Once the breath of methylating organism types has been determined (at this time we have only found that certain strains of sulphate reducing bacteria are capable of mercury methylation), these will be tested under different growth conditions to see under which environment conditions mercury methylation would be maximized/minimized. From such experiments suggestions may be made with respect to the control of methylation in reservoirs and will provide new data for computer simulation models currently been devised.
Thanks to the work in this lab apparent contradictions in some of the environmental field work has been explained (i.e. the fact that the sulphate reducing bacteria inhibitor molybdate was effective at halting mercury methylation in the absence of detectible sulphate in the sediment water). We are also the first lab to observe mercury methylation in pure bacterial cultures at environmentally relevant concentrations of mercury using defined media.
III) A collaborative project with Drs J. Oleszkiewicz and S. Cenkowski in the faculty of Engineering is in the planning stage to look at the feasibility of dry anaerobic digestion of poultry manure for biogas and nutrient recovery.
RECENT PUBLICATIONS
Sparling, R., L. T. Holt, and Z. Lin. 1993. Active transport of leucine in Methanosphaera stadtmanae. Can. J. Microbiol. 39: 749-753.
Sparling, R., M. Blaut, and G. Gottschalk. 1993. Bioenergetic studies of Methanosphaera stadtmanae, an obligate H2/methanol utilising methanogen. Can. J. Microbiol. 39: 742-748.
Wong, D., D. Juck, K. Terrick, Z. Lin and R. Sparling. 1994. Electron transfer reactions for the reduction of NADP+ in Methanosphaera stadtmanae. FEMS Lett. 120: 285-290.
Lin, Z., and R. Sparling. 1995. Oxidation/reduction of methanol, formaldehyde, serine and formate in Methanosphaera stadtmanae. Can. J. Microbiol. 41: 1048-1053.
Basu, S.K., J.A. Oleszkiewicz, and R. Sparling. 1996 Dehalogenation of 2-chlorophenol (2-CP) in anaerobic batch cultures. Water Research 30:315-322.
Themel, K., R. Sparling and J. Oleszkiewicz. 1996 Anaerobic dehalogenation of 2-chlorophenol by mixed bacterial cultures in absence of methanogenesis. Environ. Technol. (in the press).