Progress 10/01/07 to 09/30/12
Outputs
Target Audience: Target audiences included microbiologists at the Society for Industrial Microbiology meeting who visited the poster describing protease and chitinase activity of several isolates we have studied in our laboratory. Another target audience was readers of the publication in Internet Journal of Microbiology. Efforts included the poster presentation and peer reviewed publication described above. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The project attempted to isolate novel, lignocellulose-degrading microorganisms, including thermotolerant and thermophilic isolates, that could find uses in industrial processes. Change in knowledge included isolation and characterization of several prokaryotic and eukaryotic microorganisms. In the work of Ph.D. student Kathleen Gibson, a prokaryotic isolate that grows at 50 C and which produces both carboxymethylcellulase (CMCase) and cellulase was found to be a Bacillus strain, based on small subunit ribosomal RNA sequencing. It is thermotolerant, growing in a range of 27 to 55 C, with an optimum pH of 6.5, and is microaerophilic. Under microaerophilic, acidic incubation at 50 C, its cellulase activity was two orders of magnitude higher than under aerobic, neutral pH incubation at 25 C. The importance of cellulose decomposition by such microaerophiles has not received much attention previously. This strain also produces amylase, xylanase, and protease. The other prokaryotic strain of interest produces both cellulase and CMCase, grows between 50 and 70 C, produces protease, and appears to be motile. Undergraduate research resulted in isolation of seven new fungal isolates that are positive for one or more of the extracellular enzymes laccase, catechol oxidase, and lignin peroxidase. Several new chitinase-producing microbial isolates were also obtained from different South Dakota environments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Murthy ,N.K.S., and B.H. Bleakley. 2012. Simplified Method of Preparing Colloidal Chitin Used For Screening of Chitinase-Producing Microorganisms. The Internet Journal of Microbiology. Vol. 10 Number 2.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2012
Citation:
NKS Murthy, N.K.S., B.H. Bleakley, A. Dangel, and N. Haag. 2012. Examination of Bacillus Biological Control Agents for Extracellular Chitinase and Protease Activities. Abstract (163). In: Proceedings of Society for Industrial Microbiology Annual Meeting and Exhibition, Washington, D.C.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Activities included mentoring four undergraduate students on the project, and one Ph. D. graduate student. The products included physical collection of materials from South Dakota sites including the Homestake Mine, and other locations in the state, to be used as inoculum to isolate lignocellulose-degrading microorganisms. PARTICIPANTS: Individuals who worked on the project included the principal investigator (Bruce Bleakley), who oversaw the project, developed protocols, and trained student laboratory workers; four undergraduate students (Renee Speidel, Colleen Friel, Jessica Luke, and Ben Curnow), who carried out lab analyses of microorganisms; and one Ph.D. graduate student (Kathleen Gibson) who helped supervise the undergraduates while also being involved in microbial pure culture work. Collaborators and contacts included William Gibbons of the SDSU Biology/Microbiology Department, whose laboratory is working in collaboration with ours to obtain new lignocellulose degrading microbial strains. TARGET AUDIENCES: Target audiences included microbiologists at the Society for Industrial Microbiology meeting who visited the poster describing CMCase activity of some isolates from a Homestake Mine biofilm. Efforts included the poster presentation described above. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge included isolation and characterization of several prokaryotic and eukaryotic microorganisms from Homestake Mine inoculum. Microbial isolates were obtained by culturing on several types of media and at temperatures of 27, 50, and 70 Celsius. During the past year, partial characterization of 111 additional isolates was completed, bringing the total number of isolates characterized by Ph. D. student Kathleen Gibson in this study to 286. Microorganisms were screened for ability to utilize carboxymethylcellulase (CMCase) activity, and cellulase activity (ability to use fibrous cellulose). Of the 54 fungal strains screened, all grown at 27 C, seven were positive for both CMCase and cellulase. For the prokaryotes, of 51 isolates able to grow at 50 C, only one had both CMCase and cellulase; and of 24 isolates able to grow at 70 C, only one had both CMCase and cellulase. Undergraduate research suggested three fungal isolates from the Homestake Mine are able to produce laccase, catechol oxidase, and lignin peroxidase.
Publications
- Murthy, N. K. S., B. H. Bleakley, and R. Speidel. 2011. Partial characterization of chemoheterotrophic, mesophilic bacterial isolates from a pigmented biofilm in the Homestake Gold Mine. Abstract P99; in SIM Annual Meeting and Exhibition Abstracts; online at http://sim.confex.com/sim/2011/webprogram/meeting.html
- Murthy, N. K. S., and B. H. Bleakley. 2011. Lipase activity of Bacillus strains used for biological control of Fusarium Head Blight. Abstract, p. 151; In: S. Canty, A. Clark, A. Anderson-Scully, D. Ellis and D. Van Sanford (Eds.), Proceedings of the 2011 National Fusarium Head Blight Forum ( pp. 154-158). East Lansing, MI/Lexington, KY: U.S. Wheat & Barley Scab Inititiative
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: OUTPUTS: Activities included mentoring three undergraduate students on the project, and one Ph.D. graduate student. The products included physical collection of materials from South Dakota sites including the Homestake Mine, and other sites for use as inoculum to isolate lignocellulose-degrading microorganisms. PARTICIPANTS: Individuals who worked on the project included the principal investigator (Bruce Bleakley), who oversaw the project, developed protocols, and trained student laboratory workers; three undergraduate students (Ashley Boyd, Seth Harris, and Renee Speidel), who carried out lab analyses of microorganisms; and one Ph.D. graduate student (Kathleen Gibson), who helped supervise the undergraduates while also being involved in microbial pure culture work. Collaborators and contacts included William Gibbons of the SDSU Biology/Microbiology Department, whose laboratory is working in collaboration with ours to obtain new lignocellulose degrading microbial strains. TARGET AUDIENCES: Target audiences included microbiologists at the Society for Industrial Microbiology meeting who attended the poster describing the CMCase activity of some sulfur oxidizing bacteria isolated from Homestake Mine inoculum. Efforts included the poster presentation described above. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge included isolation and characterization of several prokaryotic and eukaryotic microorganisms from Homestake Mine inocula. A plate screening for culturable unicellular bacteria, fungi, and actinomycetes resulted in isolation of 175 separate mesophilic isolates. Because of limitations in time and personnel, only selected fungal and actinomycete isolates were screened for carboxymethylcellulase (CMCase) activity using plate assays, as well as for ability to produce clearing zones on plates containing powdered cellulose. Nine actinomycete isolates, and six fungal isolates, were positive for CMCase activity. Seven of the nine actinomycete isolates produced clearing of powdered cellulose; while only one of the fungal isolates did. Anaerobic enrichment cultures produced visible microbial growth that formed a biofilm on filter paper. Further work will attempt to isolate pure cultures from these anaerobic enrichments. Work with nine mesophilic, culturable unicellular bacteria and one actinomycete obtained from Homestake Mine biofilm material showed that the actinomycete had the greatest CMCase activity of the biofilm isolates. Work continued with fibrous cellulose dyed with Remazol Brilliant Blue R (RBBR) for use as an indicator of cellulolytic activity. Two isolates used in Kathleen Gibson's work tested positive for dye release, and will be studied in more detail.
Publications
- Harris, S.T., K.A. Gibson, and B.H. Bleakley. 2010. Isolation and partial characterization of three sulfur-reducing bacteria from South Dakotas Homestake Mine. Paper number 16671. In Abstracts of 60th Annual Meeting of the Society for Industrial Microbiology. August 1-5, 2010. San Francisco, CA.
- Bleakley, B.H., and D.L. Crawford. 2010. Exchange of a conjugative plasmid at different soil moisture levels between Streptomyces species colonizing artificial soil aggregates. The Internet Journal of Microbiology. 2010 Volume 8 Number 2; online.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: Activities included mentoring two undergraduate students on the project, and one Ph.D. graduate student. The products included physical collection of materials from South Dakota sites, obtained during two new sampling trips to the Homestake Mine, and from other hot temperature environments to use as inocula to isolate thermophilic lignocellulose degrading microorganisms. PARTICIPANTS: Individuals who worked on the project included the principal investigator (Bruce Bleakley), who oversaw the project, developed protocols, and trained student laboratory workers; two undergraduate students (Ashley Boyd and Seth Harris), who carried out lab analyses of microorganisms; and one Ph.D. graduate student (Kathleen Gibson) who helped supervise the undergraduates while also doing her own research and protocol development. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge included isolating and screening several prokaryotic and eukaryotic microorganisms for carboxymethylcellulase (CMCase) activity. About 100 new microbial isolates were obtained, to add to the isolates we had previously. All isolates were screened for cellulolytic activity by plating onto carboxymethylcellulose agar (CMC agar), and checking for hydrolysis zones after Congo Red staining. Isolation of thermophilic isolates that could grow at 70 C was done using gellan gum plates (with calcium chloride as polymerizing agent),with or without CMC. Work with cultures on CMC plates at 50 C and 70 C presented two problems. First, the polymerizing cations in gellan plates caused a precipitate to form that appeared to prevent growth. Secondly, the high temperature of the incubator caused the plates to dry out quickly, preventing effective staining and destaining of colonies on plates. To eliminate the need for the CMC agar, a different screening method for cellulase producers is being developed. Fibrous cellulose dyed with Remazol Brilliant Blue R (RBBR) will be used as an indicator of cellulolytic activity. If isolates produce cellulase, the dye will be released into solution, causing a change in absorbance. Initial attempts to dye paper disks with the RBBR and use them for cellulase screening indicated that the disks did not have sufficient surface area to provide sensitive assays. Use of the dyed cellulose powder should be more useful for assays.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Activities included mentoring two undergraduate students on the project, one Ph.D. graduate student, and one M.S. student. The products included physical collection of materials from South Dakota sites including the Homestake Mine, Hot Springs, compost piles, and other sites for use as inoculum to isolate thermophilic lignocellulose-degrading microorganisms. PARTICIPANTS: Individuals who worked on the project included the principal investigator (Bruce Bleakley), who oversaw the project, developed protocols, and trained student laboratory workers; two undergraduate students (Ashley Boyd and Tami Messenger), who carried out lab analyses of microorganisms; one Ph.D. graduate student (Kathleen Gibson), who helped supervise the undergraduates while also being involved in microbial pure culture work; and one M.S. student (Jennifer Morgan) who worked with the biological control strains. Collaborators and contacts included William Gibbons of the SDSU Biology/Microbiology Department, whose laboratory is working in collaboration with ours to obtain new lignocellulose degrading microbial strains; and Gary Yuen of the University of Nebraska who we collaborate with on biological control work. TARGET AUDIENCES: Target audiences included microbiologists at the Society for Industrial Microbiology meeting who attended the poster describing the CMCase and xylanase activities of our Bacillus strains that are used as biological control agents of plant pathogenic fungi. Efforts included the poster presentation described above. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Change in knowledge included isolation and characterization of several prokaryotic and eukaryotic microorganisms from the inocula. Using Hot Springs inocula, 28 filamentous fungal cultures were isolated, and 125 unicellular bacteria. All the fungi grow at 27 C but not 50 C; and 17 of the 28 fungi used carboxymethylcellulose (CMC) in plate assays. Eight of the 125 unicellular bacteria hydrolyzed CMC; of these eight, two grew at 50 C. Selected bacterial cultures identified using partial small subunit rDNA sequencing were: Bacillus thuringiensis, growing at 27 C but not 50 C and having weak CMCase activity; and Bacillus amyloliquefaciens, growing at both temperatures and having strong CMCase activity. Using Homestake Mine inoculum, 13 filamentous fungal cultures wereisolated, and 32 unicellular bacteria. All the fungi grow at 27 C but not 50 C; and one fungal isolate used CMC in plate assays. Eight of the 32 unicellular bacteria hydrolyzed CMC; and all grew at both temperatures. Selected bacterial strains identified using partial small subunit rDNA sequencing were: three Bacillus pumilus strains; two Bacillus licheniformis strains; and three Bacillus subtilis-subtilis strains. We also verfied that four Bacillus amyloliquefaciens strains we use as biological control agents of plant pathogenic fungi produce CMCase and xylanase.
Publications
- Morgan, J.L., and B.H. Bleakley. 2008. Physiologic profiling and carbon source utilization of four Bacillus strains used as biological control agents of FHB. p. 44. In: Canty, S.M., E. Walton, A. Clark., D. Ellis. J. Mundell, and D.A. Van Sanford (Eds.), Proceedings of the National Fusarium Head Blight Forum; 2008 Dec 2-4; Indianapolis, IN. Lexington, KY: University of Kentucky
- Morgan, J.L., and B.H. Bleakley. 2008. Use of most probable number and PCR methods to estimate populations of Bacillus strain 1BA applied to wheat and barley for biological control of FHB. p. 45. In: Canty, S.M., E. Walton, A. Clark., D. Ellis. J. Mundell, and D.A. Van Sanford (Eds.), Proceedings of the National Fusarium Head Blight Forum; 2008 Dec 2-4; Indianapolis, IN. Lexington, KY: University of Kentucky
- Ruden, K.R., L.E. Osborne, B.H. Bleakley, J. Morgan, and B.E. Ruden. 2008. 2008 Uniform trials for the performance of biological control agents in the suppression of Fusarium Head Blight in South Dakota. p. 58. In: Canty, S.M., E. Walton, A. Clark., D. Ellis. J. Mundell, and D.A. Van Sanford (Eds.), Proceedings of the National Fusarium Head Blight Forum; 2008 Dec 2-4; Indianapolis, IN. Lexington, KY: University of Kentucky
- Bleakley, B.H., and J. Morgan. 2008. Field Plot Studies of Survival and Growth of the Biocontrol Agent Bacillus Strain 1BA Applied To Wheat Heads. Abstract; In Abstracts of the 108 th General Meeting of the American Society for Microbiology; Boston, MA. ASM Press, Washington, D.C.
- Bleakley, B.H., and J. Morgan. 2008. Cellulase and xylanase activities of four Bacillus amyloliquefaciens strains used as biocontrol agents to antagonize plant pathogenic fungi. Abstract; Poster abstract P35; p. 112. In: The Society for Industrial Microbiology's (SIM) Annual Meeting and Exhibition program; August 10 - 14, 2008; San Diego, CA.
- Jochum, C.C., G. Y. Yuen, K.R. Ruden, B.H. Bleakley, J. Morgan, L. Osborne, L.E. Sweets, S. Halley and K. Kinzer. 2008. 2008 Results from the uniform evaluation of biological agents for the control of Fusarium Head Blight on wheat and barley. pp. 32-35. In: Canty, S.M., E. Walton, A. Clark., D. Ellis. J. Mundell, and D.A. Van Sanford (Eds.), Proceedings of the National Fusarium Head Blight Forum; 2008 Dec 2-4; Indianapolis, IN. Lexington, KY: University of Kentucky.
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