Amaret Bhumiratana's Homepage

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Research Works :

Biological control of insect pests and vectors using Bacillus thuringiensis based biopesticides.

Recent efforts have been made to reduce the population of insect pests and vectors by using biological means. These are thought to hold considerable advantages over chemical controls, which often have long residual action and may be toxic to non-target organisms. Also, insect populations have developed resistance to a number of these chemicals. Many investigators including Prof. Amaret’s group have reported larvicidal effects on various insects by various strains of Bacillus thuringiensis. Certain subspecies of B. thuringiensis are highly effective against important Lepidopteran insect pests such as Plutella xylostella, Trichoplusia ni, and Spodoptera itura. Whereas, other subspecies of B. thuringiensis are highly effective against Dipteran or Coleopteran larvae. In order to promote the utilization of B. thuringiensis as biological controlling agents of insect pests and vectors in Thailand, it is important to develop effective methods of genetic improvement for strains of B. thuringiensis as well as appropriate fermentation methods for achieving "local production" of the biopesticides.

The conjugation-like gene transfer process used successfully in this study allows one to freely move genes from one strain of B. thuringiensis to others. The development of appropriate plasmid vectors as well as the ability to insert various genes into the vector systems and to transfer the hybrid plasmids into B. thuringiensis, both in vitro and in vivo, will lead to means for obtaining genetically manipulated strains of B. thuringiensis. Also, the results obtained from indept study on the molecular cloning and sequencing of S-layer protein gene from B. thuringiensis will, hopefully, lead to better understanding of the conjugation-like gene transfer process in B. thuringiensis.

Additionally, indepth study of the carbohydrate metabolism, physiology and genetics of B. thuringiensis have been carry on. Prof. Amaret Bhumiratana has been initiating and co-ordinating research network on “Physiology and genetics of Bacillus thuringiensis” which supports by the Thailand Research Fund. This research project could support at least six faculty staffs and 10 graduate students to carry on research work on this research area.

Prof. Amaret’s research work has also been involved in the development of an innovative fermentation technique for production of B. thuringiensis. The technique is based on the use of cell recycle system using ceramic membrane as the method to recycle the cell mass. It has been found that the productivity of the B. thuringiensis can be increased by ten folds through the use of cell recycle system as compared to the conventional batch type fermentation system. Furthermore, after achieving the development of appropriate formulations, the economic feasibility for the industrial production of B. thuringiensis based biopesti-cides at 100 tons per year capacity has been determined.

More over, supplement of some enzymes such as chitinase has been used for development the appropriate formulations. The chitinase genes have been cloned and combined with the delta-endotoxin gene of B. thuringiensis for engineering the B. thuringiensis strains.

Thus, Prof. Amaret research program has not only elucidated new finding and more indept understandings on the physiology and genetics of B. thuringiensis but will also lead to possible realization of industrial level “local production” of B. thuringiensis based biopesticides in Thailand.

Publications of research works concerning Bacillus thuringiensis based biopesticides. :

Panbangred, W., Pantuwatana, S. and Bhumiratana, A. (1979). Toxicity of Bacillus thuringiensis toward Aedes aegypti larvae. J. Invertebr. Pathol. 33: 340-347.

Somsanti, W., Pantuwatana, A., and Bhumiratana, A. (1982). Role of the Bacillus thuringiensis toward Aedes aegypti larvae. J. Invertebr. Pathol. 39: 41-48.

Silapanuntakulo, S., Pantuwatana, S., Bhumiratana, A., and Charoensiri, K. Bacillus sphaericus strain 1593 and Bacillus thuringiensis serotype 11-14 against mosquito larvae in different kinds of environment. J. Invertebr. Pathol. 42: 387-392.

Dharmsthiti, S.C., Pantuwatana, S., and Bhumiratana, A. (1985). Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus strain 1593 in media using by product from monosodium glutamate factory. J. Invertebr. Pathol. 46: 231-238.

Dharmsthiti, S.C., and Bhumiratana, A. (1985). Production of Bacillus thuringien- sis var. israelensis and Bacillus sphaericus strain 1593 on media using by product from a monosodium glutamate factory. Microbial Utilizat. Renew. Resour. 4: 357-365.

Panyim, S., Kertbundit, S., Hiranyavasit, W., Wilairat, P., Ponglikitmongkol, M., Bhumiratana, A., and Pantuwatana, S. (1985). Molecular cloning and expression in Escherichia coli of delta-endotoxin gene of Bacillus thuringiensis H-14. Microbial Utilizat. Renew. Resour. 4: 214-217.

Loprasert, S., Pantuwatana S., and Bhumiratana, A. (1986). Transfer of plasmids pBC 16 and pC194 into Bacillus thuringiensis subsp. israelensis. J. Invertebr. Pathol. 48: 325-334.

Bhumiratana, A. (1987). Production and Utilization of Bacterial Agents to Control Agriculture Insect Pests in Thailand. Extension Bulletin no. 257. Food and Fertilizer Technology Center. Taiwan. Republic of China.

Pantuwatana, S., Bhumiratana, A., Panyim, S., and Wilairat, P. (1987). Improvement of bacterial agents for control of mosquito vectors. In "Proceedings of His Majesty's Fifth Cycle Commemorative Conference of USAID Science Research Grantees" held in Nakorn Pathom, Thailand. 24-26 July 1987, p. 65-72.

Wiwat, C., Panbangred, W., Bhumiratana, A., and Okada, H. (1989). Inter-subspecies transfer of plasmids and chromosomal genes in Bacillus thuringiensis. Microbial Utilizat. Renew. Resour. 6: 395-403.

Wiwat. C., Panbangred, W., and Bhumiratana, A. (1990). Transfer of plasmids and chromosomal genes amongst subspecies of Bacillus thuringiensis J. Industrial Microbiol. 6: 19-27.

Trisrisook, M., Pantuwatana, S., Bhumiratana, A., and Panbangred, W. (1990). Molecular cloning of the 130 kDa mosquitocidal gene in Escherichia coli and Bacillus sphaericus. Appl. Environ. Microbiol. 56: 1710-1716.

Bhumiratana, A. (1991). Local production of Bacillus sphaericus. In "Bacterial Control of Mosquitos and Black Flies." D. Sutherland and H. de Barjac (eds) Rutgers University press. USA.

Tantipaiboonwut, S., Panbangred, W., Bhumiratana, A., and Pantuwatana, S. (1991). Selection of insecticidal activity of Bacillus thuringiensis subsp. kurstaki. Microbial Utilizat. Renew. Resour. 7: 283-286.

Wiwat, C., Panbangred, W., Pantuwatana, S., Mongkolsuk, S., and Bhumiratana, A. (1992).

Bhumiratana A., Panbangred, W., Thepsingha, W., and Wiwat, C. (1994). Research and development for microbial based biopesticides in Thailand. International Workshop on Application and Control of Microorganism in Asia. Tokyo. pp. 23-32.

Wiwat, C., Panbangred, W., Mongkolsuk, S., Pantuwatana, S., and Bhumiratana, A. (1995). Inhibition of a conjugation-like gene transfer process in Bacillus thuringiensis subsp. israelensis by the anti-S-layer protein antibody. Current Microbiol. 30 : 69-75.

Sirichotpakorn, N., Panbangred, W., Pantuwatana, S., and Bhumiratana, A. (1996). The effect of cryIVB terminator fragments on expression of chloramphenicol acetyltransferase gene in Bacillus thuringiensis subsp. israelensis. J.Sci.Soc. Thailand. 22 : 121-130.

Bhumiratana, A. (1996). Research and Development on microbial controls of insect pests in Thailand. Outstanding Scientists. 1982-1996. NSTDA publication. pp. 198-213.

Wiwat, C., Lertcanawanichkul, M., Siwayapran, P., Pantuwatana, S., and Bhumiratan, A. (1996). Expression of chitinase encoding genes from Aeromonas hydrophila and Pseudomonas maltophilia in Bacillus thuringiensis subsp. israelensis. Gene. 179 : 119-126.

Panjaisee, S., Charoenpornwatana, S., Pantuwatana, S., Bhumiratana, A., and Panbangred, W. (1997). High production of Cry IVB protein does not lead to formulation of crystalline inclusions in Bacillus thuringiensis subsp. israelensis. World J. of Microbiol. and Biotechnol. 13:319-327.

Molecular cloning of S-layer protein gene from Bacillus thuringiensis. In "Biotechnology and Environmental Science : Molecular Approaches" S. Mongkolsuk, P.S. Lovett, and J.E. Trempy (eds). Plenum Press, U.S.A.

Tantimavanich, S., Pantuwatana, S., Bhumiratana, A., and Panbangred, W. (1997). Cloning of a chitinase gene into Bacillus thuringiensis subsp. aizawai for enhanced insecticidal activity. J.Gen.Appl.Microbiol. 43 : 341-347.

Tantimavanich, S., Pantuwatana, S., Bhumiratana, A., and Panbangred, W. (1998). Multiple chitinase enzymes from a single gene of Bacillus lichenifirmis TP 1. J.Ferment.Bioengin. 85 : 259-265.

Wiwat, C., Siwayaprahm, P., and Bhumiratana, A. (1999). Purification and characterization of chitinase from Bacillus circulaus No. 4.1 Curr. Microbiol. 39: 131-140.

Wiwat, C., Thaithanun, S. Pantuwatana, S., and Bhumiratana, A. (2000). Toxicity of chitinase-producing Bacillus thuringiensis spp. kurstaki HD-1(G) toward plutella xylostella. J.Invertbr. Pathol. 76 : (in press).

Improvement of manufacturing techniques for small and medium fermentation industry in Thailand.

Prof. Amaret’s research work concerning soy sauce involved both basic research as well as applications to small and medium factories summarized in a article “Technology transfer for small and medium soy sauce fermentation factories in Thailand: a consortium approach” which was results published in the Journal of Food Research International.

There are a number of soybean fermentation products in Thailand. These include soy sauce, soybean paste, fermented soybean curd and fermented black soybeans. Although there are two or three large factories, the soybean fermentation industry mainly comprises of small and medium size factories. The manufacturing techniques employed by these small factories are usually those passed from generation to generation with little or no improvement. Due to the lack of qualified personnel and low financial investment, there are minimal research and development activities by either large or small factories. The lack of research and development activity greatly inhibits the capability of these factories to improve either their production technology or the quality of their products. A few years ago, a small research and development unit was set up through the support of the Thailand Research Fund (TRF) and a consortium of small and medium size soy sauce manufactures. This unit was called the Quality Control and Training Center for Soybean Fermentation (QCTC-Soybean Fermentation). By working closely with the consortium, this unit has initiated research and development activities that have benefited members of the consortium. The objectives of the QCTC-Soybean Fermentation program have been to improve the quality of soybean fermentation products and to improve manufacturing techniques. Some examples of QCTC- achievements include the introduction of improved microbial inocula (Aspergillus oryzae, Zygosaccharomyces rouxii and Tetratogenococcus halophilus) for soy sauce fermentation, the introduction of new technique for preparing koji, the use of large fiberglass tanks instead of small earthenware containers for second stage soy sauce fermentation, and the introduction of cost effective waste treatment systems. These inputs have helped the small industry consortium members to improve the quality of their products and/or to reduce production costs. Prof. Amaret’s research activities in this area not only lead to publications in scientific journals but also lead to improvements of manufacturing techniques in a number of small and medium soy sauce factory in Thailand as follows.

Creating of a factory producing Aspergillus oryzae spore inoculum.
More than 10 factories are presently using pure culture techniques for production of Koji.
Applications of new soy bean cooking technique, new technique for koji production, and the use of large fiber glass tank for momori fermentation are now practiced in a number of small and medium soy sauce factories.
Formation of QCTC unit, a consortium of small factories (over 10 factories) and Thai soy sauce association (over 30 factories), has established sustained linkages between university’s research unit and small and medium soy sauce factories.

Publications of research works concerning soy sauce production :

Bhumiratana, A., Flegel, T.W., Glinsukon, T., and Somporn, W. (1980). Isolation and analysis of molds from soy sauce koji in Thailand. Appl.Environ.Microbiol. 39: 430- 435.

Bhumiratana, A. (1980). Traditional fermented foods in Thailand. Proceedings of the Oriental Fermented Foods. Taiwan, Republic of China.

Takada, N., Iwashiro, T., Yagyu, J., Bhumiratana, A., Flegel, T.W., and Oshima, Y. (1980). Microflora in various Thai fermented foods. Microbial Utilizat. Renew. Resour. 1: 85-91.

Bhumiratana, A., Flegel, T.W., Impoolsup, A., Okada, H., and Oshima, Y. (1980). Isolation and analysis of molds from soy sauce koji in Thailand. Microbial Utilizat. Renew. Resour. 1: 92-100.

Flegel, T.W., Bhumiratana, A., and Srisutipruti, A. (1981). The problematic occurrence of tyrosine crystals in the Thai soy bean paste, Tao Chieo. Appl. Environ. Microbiol. 41: 746-752.

Impoolsup, A., Bhumiratana, A., and Flegel, T.W. (1981). Characterization of proteases from Aspergillus flavus var. columnaris. Appl. Environ. Microbiol. 42: 619-628.

Flegel, T.W., Bhumiratana, A., Impoolsup, A., Takada, N., Oshima, Y., and Okada, H. (1981). Studies on Aspergillus flavus var. columnaris in soy sauce koji. Microbial Utilizat. Renew. Resour. 2: 59-63.

Impoolsup, A., Flegel, T.W., Bhumiratana, A., Oshima, Y., and Okada, H. (1981). Characterization of proteases in soy sauce koji produced in Thailand. Microbial. Utilizat. Renew. Resour. 2: 217-231.

Chancharoensin, S. and Bhumiratana, A. (1983). Production of L-lysine by homoserine auxotrophic mutant of Corynebacterium glutamicum (Hom-). Thai. J. Agric. Sci. 16: 315-337.

Yoshida, W., Chancharoensin, S., and Bhumiratana, A. (1983). Glucoamylase production by solid cultivation of Aspergillus niger 1112. Annual report of International Center of Cooperative Research and Development in Microbial Engineering, Japan. 6: 297-278.

Bhumiratana, A., Flegel, T.W., Impoolsup, A., Kaenjak, A., Chancharoensin, S., Shinmyo, A., and Okada, H. (1983). Research and development in soy sauce manufacturing technique in Thailand. Microbial Utilizat. Renew. Resour. 3: 99-107.

Chancharoensin, S., and Bhumiratana, A. (1984). Isolation and characterization of high L-lysine producing mutant of Corynebacterium glutamicum (Hom-). Thai J. Agric. Sci. 16: 263-278.

Dharmsthiti, S.C., Yoshida, T., and Bhumiratana, A., (1985). Production of glucoamylase from Aspergillus niger. Microbial. Utilizat. Renew. Resour. 4: 107-114.

Dharmsthiti, S.C., Flegel, T.W., and Bhumiratana, A. (1986). Isolation and characterization of amylases from Aspergillus flavus var. columnaris a soy sauce koji mold. ASEAN Food. J. 2: 59-63.

Bhumiratana, A., Dharmsthiti, S., Jiratatananon, R., Lothong, N., Pichyangkura, S., Bhumiphamon, O., and Sakumavase, J. (1987). Status of enzyme technology in Thailand. Microbial Utilizat. Renew. Resour. 5: 92-96.

Kalayanamitr, A., Bhumiratana, A., Flegel, T.W., Glinsukon, T., and Shinmyo, A. (1987). Occurrence of toxicity among protease, amylase, and color mutants of a nontoxic soy sauce koji mold. Appl. Environ. Microbiol. 53: 1980-1982.

Bhumiratna, A., Flegel, T.W., Lothong, N., and Suwanarit, P. (1988). A manual of soy sauce production for small scale manufacturers in the ASEAN countries. Text and Journal Corporation Co., Ltd. Thailand.

Mongkolwai., T., Assawanig, A., Amnajsongsiri, C., Flegel, T.W., and Bhumiratana, A. (1997). Technology transfer for small soy sauce production factories… a consortium approach. J. of Food Research International. 30: 555-563.

Juthasmith, P., Udomsopagit, S., Suphantharika, M., and Bhumiratana, A. (1997). Isolation and characterization of a glutamate oxidase producing microorganism. (in press).

Udomsopagit, S., Suphantharika, M., Kunnecke, W., Bilitewski, U., and Bhumiratana, A. (1998). Determination of L-glutamate in various commercial soy sauce products using flow injection analysis with a modified electrode. World J. of Microbiol. And Biotechnol. 14: 543-549.