International Conference on the Role of Universities in Hands-On Education Rajamangala University of Technology Lanna, Chiang-Mai, Thailand 23-29 August

2009

BENEFICIAL INDIGENOUS MICROORGANISM PRODUCTS IN MICROBIOLOGY

Charunee Meechoui* Arissara Wanna Kingkan Wongjee Kunnika Khuntong Sumalee Promrukhachate and Sawit Meechoui

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ABSTRACT Indigenous microorganisms is a term of small beneficial microbes from that local area (Tancho, 2004) which used in natural or organic agriculture system for soil remediation, composting, water treatment and livestock etc. Beneficial indigenous microorganism products consisted of the mixed cultures of beneficial and naturally living microorganisms that can be useful to increase microbial diversity of soils and plants indirectly. To realize how to develop and produce the beneficial indigenous microorganism products being suitable eastern wisdom technology, its necessary to integrate the knowledge of microbiology biochemistry and fermentation technology. This was studied the typical predominant microorganisms growth as microorganism ecology. For the appropriated local wisdom fermentation was based on local submerged fermentation in fedbatch process following as the local wisdom procedure. By this appropriated fermentation technology, the living-fertile soil use natural inoculum and molasses as the carbon source. This methodology were refer to Charunee and Sumalee (2550) that have been studied from batch culture fermentation of IMO by viable count and direct microscopic count of motile spirillum, lactobacilli, and budding yeast.

The results showed a growth pattern of predominant indigenous microorganism according a time by naturally submerge fed-batch process fermentation. The predominant microorganisms in the first 2 day was nitrogen fixing living motile spirilla group that showed curve rod shape by gram stain which shortly exponential phase stationary phase and decline phase. The second group of microorganism was lactobacilli which grown 2-3 day after starting fermentation, and longer period growth phase than the first, the lowest pH level was occurred in this period.The last predominant group was budding yeast grown about 5-9 day the final fermentation which was film forming in appearance. The addition of soil inocula and molasses every 10 day take a next cycle growth of microbial series that make microbial population concentrated especially lactobacilli group which effective high quality of the indigenous microorganisms inocula. In addition , t he ecological effects on the sequence of a microorganism growth pattern were discussed. Keywords : beneficial indigenous microorganisms products, appropriated technology

Lampang Agricultural Research and Training Center, P.O. Box 89 Lampang 52000, Thailand Tel. 6654-342553 *Corresponding author. e-mail address : charunee.rmutl@gmail.com

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International Conference on the Role of Universities in Hands-On Education Rajamangala University of Technology Lanna, Chiang-Mai, Thailand 23-29 August 2009

INTRODUCTION The inoculum IMO preparation was done by using fertility soil in the forest, namely living soil. It was fermented with molasses as carbon source. Fertile forest soil was as several microorganisms groups. It may be done directly by using submerge culture fermentation method with molasses solution for inoculums IMO culture, or by firstly adding different organic materials such as rice hull, rice bran or cooked rice to ferment with the soil as followed solid state fermentation method, namely Baiyodofermented soil. Then finally to do as submerge culture fermentation. During fermentation period, each microorganism groups will growth as following inoculums soil microorganism ecology. However, in Thailand, inoculums IMO culture product development and the appropriated inoculums IMO technology , introduced from the east wisdom of Japan and Korea, should be modified by integrating some knowledge bases, such as microbiology, fermentation technology and biochemistry, etc. This research was studied the natural IMO microbiological fermentation corresponded the period of time by following batch and fed-batch process. In addition, the ecological results of the sequent ordering microorganism growth were predicted.

MATERIALS AND METHODS The local IMO inoculums preparation for this research was modified by both of Japanese local wisdom method and the method from Tancho (2004). It was done as submerge culture by fed-batch fermentation. Fertile forest soil, 200 g, obtained fulfilled microorganisms source was mixed with 5% molasses solution, as carbon source for microorganisms growth in 30 liter plastic tank. Then the newer mixed solution was added every 7 days until 1 month. And finally, the amount of each predominant three microorganism groups; such as curve rod, lactobacilli and budding yeast were sampling everyday to study their growth change under microscope under the gram strain counting method. It was replicated 5 fields /slide. Their change was plotted to graph against the time.

RESULTS DISCUSSION AND CONCLUSTION For fed-batch fermentation of inoculums IMO (in Figure 1.3), the different characteristic growth of microorganisms were found. However, it was summarized only three predominant microorganism groups were determined as 1.) curve rod bacteria, motile spirilla living former bacteria, 2.) lactobacilli group, and 3.) budding yeast group. This methodology were refer to Charunee and Sumalee (2550) that have been studied from batch culture fermentation of IMO by viable count and direct microscopic count of motile spirillum, lactobacilli, fission yeast and budding yeast. (From those experiment, Charunee and Sumalee (2550) The figure 1.1 show typical time course fermentation of indigenous microorganisms by direct microscopic count. And figure 1.2 show predominant major groups of microorganisms isolated during typical time course fermentation of

indigenousmicroorganism products) From this experiment, firstly the response growth curve was - 407 -

International Conference on the Role of Universities in Hands-On Education Rajamangala University of Technology Lanna, Chiang-Mai, Thailand 23-29 August 2009

mostly the motile spirilla population increased and then decreased sharply in medium pH nutrient. This bacteria growth changed quickly in exponential phase, stationary phase and decline phase of the growth pattern. It was hypothesized that this bacteria growth firstly predominant and short growth period because of nitrogen shortage condition at the beginning of fermentation. But it was able to fix nitrogen in the air and then grew so fast and much more than the others.

160000000 140000000 120000000 100000000 lactobacilli 80000000 60000000 40000000 20000000 0 0 1 2 3 4 5 6 7 8 9

Schizosaccharomyces

cell/ml
motile spirilla

budding yeast

Time (day)

Figure 1.1 Typical time course fermentation of indigenous microorganisms by direct microscopic count.

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International Conference on the Role of Universities in Hands-On Education Rajamangala University of Technology Lanna, Chiang-Mai, Thailand 23-29 August 2009

Figure 1.2 Predominant major groups of microorganisms isolate during typical time course fermentation of indigenous microorganism products Left above : motile spirilla (1000x) 0.5x2.3-2.5 m Right above : lactobacilli (1000x) 0.4x10-20 m Left below : fission yeast , Schizosaccharomyces (1000x) 4x12 m Right below : budding yeast (400x) 4.5x12 m

Bright field observation microscopy of motile spirilla living cell, the first major group during typical time course fermentation. (left above) Grams stain microscopy of lactobacilli, the second group of microorganisms which plays an important role in this system. (right below). The fission yeast Schizosaccharomyces, unicellular eukaryote which is the third group in this ecosystem grows together with lactobacilli. (left below) And the last group is budding yeast which grows after another group is at final stage Then this bacteria population had declined by death and composed to be organic nitrogen

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International Conference on the Role of Universities in Hands-On Education Rajamangala University of Technology Lanna, Chiang-Mai, Thailand 23-29 August 2009

source for the other microorganism living. In this research, it was isolated pure culture to classify. This was spiral bacteria, movable and well growth in lack nitrogen source in media. For the secondary phase of fermentation growth curve, it found lactobacilli bacteria population mainly increase in low pH media condition. On the other, its growth made lower pH media. Finally, the budding yeast group, the third generation microorganism growth was shown as the lactobacilli declining. Its growth as a thin film on the surface of media could protect oxidation reaction by air to prevent acetic acid bacteria growth. It may be the way to preserve the usage IMO for plant. On the other had, the observed predominantly IMO growth pattern in the batch had change as a system and finally had low pH 3.9 for IMO inoculums culture product. On the fed-batch fermentation, it affected to inoculums culture quality, especially for lactic bacteria

REFFERENCES Charunee Meechoui and Sumalee Promrukhachate. 2550. Application Azospirillum and Rhodopseudomonas of as Biofertilizers for Rice. Complete Research Report. Rajamangala University of Technology. Tancho, A. 2004. Natural Agriculture. National Science and Technology Development Agency. Bangkok. Thailand. 146 p. ISBN: 974-226-108-3 (in Thai)

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