Four  thermotolerant  acetic  acid  bacteria designated  as  CMU1, CMU2, CMU3 and CMU4 were isolated from six honey samples produced by three native bee species in northern Thailand, namely the dwarf honey bee (Apis  florea), Asian honey bee (A. cerena) and giant honey bee (A. dorsata). All isolates were tested for their tolerance to acetic acid and ethanol at 30C and 37C. It was found that they grew only in a medium containing 1% (v/v) acetic acid at 30C. However, isolate CMU4 showed the highest toleration to  ethanol, viz. 10% (v/v) and 9% (v/v) at 30C and 37C respectively. Morphological and biochemical examination indicated that all isolates were members of the genus Gluconobacter

Wasu Pathom-aree

Four  thermotolerant  acetic  acid  bacteria designated  as  CMU1, CMU2, CMU3 and CMU4 were isolated from six honey samples produced by three native bee species in northern Thailand, namely the dwarf honey bee (Apis  florea), Asian honey bee (A. cerena) and giant honey bee (A. dorsata). All isolates were tested for their tolerance to acetic acid and ethanol at 30C and 37C. It was found that they grew only in a medium containing 1% (v/v) acetic acid at 30C. However, isolate CMU4 showed the highest toleration to  ethanol, viz. 10% (v/v) and 9% (v/v) at 30C and 37C respectively. Morphological and biochemical examination indicated that all isolates were members of the genus Gluconobacter

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 Maejo Int. J. Sci. Technol.  2009, 3(01), 71-76 Maejo International  Journal of Science and Technology ISSN 1905-7873 Available online at www.mijst.mju.ac.th Full Paper Isolation of acetic acid bacteria from honey Kanlaya Kappeng and Wasu Pathom-aree*  Microbiology Section, Department of Biology, Faculty of Science, Chiang Mai University,  Chiang Mai 50200, Thailand  *Corresponding author, e-mail: wasu@chiangmai.ac.th  Received: 19 August 2008  / Accepted: 5 February 2009 / Published: 10 February 2009   Abstract:  Four  thermotolerant  acetic  acid  bacteria designated  as  CMU1, CMU2, CMU3 and CMU4 were isolated from six honey samples produced by three native bee species in northern Thailand, namely  the  dwarf  honey  bee  (Apis    florea), Asian honey bee (A. cerena) and giant honey bee (A. dorsata). All isolates were tested for their tolerance to acetic acid and ethanol at 30C and 37C. It was found that they grew only in a medium containing 1% (v/v) acetic acid at 30C. However, isolate CMU4  showed  the  highest  toleration  to   ethanol, viz. 10% (v/v) and 9% (v/v) at 30C and 37C respectively. Morphological and biochemical examination indicated that all isolates were members of the genus Gluconobacter.   Keywords: acetic acid bacteria, honey, enrichment culture technique, Gluconobacter  Introduction  Honey is a nectar and sweet deposit from plants which is gathered, modified and stored in the honeycomb by honey bees [1]. There are five major bee species used in honey production industry in the northern part of Thailand. Four native species are the dwarf honey bee (Apis florea), small dwarf honey bee (Apis adreniformis), Asian honey bee (A. cerena) and giant honey bee (A. dorsata). The remaining European honey bee (A. mellifera) is the only introduced species [2].   Maejo Int. J. Sci. Technol.  2009, 3(01), 71-76     72   Acetic acid bacteria are a large group of obligate aerobic Gram-negative bacteria which are commonly found in association with various kinds of sugary material. They are divided into four groups, namely Acetobacter, Acidomonas, Frateuria and Gluconobacter, these being slightly different in some physiological  characteristics  [3].  The  two  most  studied  acetic  acid  bacteria  are  Acetobacter  and Gluconobacter due to their economical importance. Acetic acid bacteria are found in association with honey  and  honey  bees  [4-6].  However,  to  date,  apparently  no  studies  attempting  to  isolate  these bacteria from Thai honey have been reported. In this study, the selective enrichment procedure for the isolation of acetic acid bacteria of particular thermotolerant strains from honey was carried out.   Materials and Methods   Selective isolation of acetic acid bacteria by enrichment culture technique    A total of six honey samples from dwarf honey bees (Apis florea), Asian honey bees (A. cerena) and giant honey bees (A. dorsata) were collected from local honey farms in San Kamphang district, Chiang Mai, Thailand. Approximately 1 ml of each sample was added to the enrichment broth (20 ml) containing 0.5% glucose, 2% glycerol, 1% yeast extract, 1% peptone, 1.5% potato extract, 4% ethanol and 1% Acti-dione (an antifungal). The mixture was incubated at 37 oC for up to one week. After 1, 3 and 5 days of incubation, one loop-full of enrichment broth was streaked onto a potato agar plate (containing 0.5% glucose, 2% glycerol, 1% yeast extract, 1% peptone, 1.5% potato extract, 4% ethanol  (v/v)  and  0.003%  bromcresol  purple).  All  colonies  showing  a  yellow halo zone were collected for further study. The change of pH in the enrichment broth was also observed after 1, 3, 5 and 7 days of incubation.   Phenotypic characterisation of isolates  Morphological, physiological and biochemical characteristics of all pure isolates were examined according to Bergey’s Manual of Determinative Bacteriology [3]. The ability of the isolate to oxidise acetate  to  CO2  and  H2O was used to distinguish between members of the genera Acetobacter and Gluconobacter.   Acid and alcohol tolerance of isolates    All isolates were tested for their ability to grow in seed culture agar supplemented with 1-4 % (v/v) acetic acid and that containing 5-15% (v/v) ethanol at 30 and 37 oC. Inoculum was prepared by growing each isolate on seed culture medium for 48 h and then transferred to the test plates. All plates were  incubated  for  up  to  5  days.  The  growth  of  each  isolate  was  compared  between  the  two temperatures.  Results and Discussion   Four isolates of acetic acid bacteria (Table 1) were successfully recovered from honey samples using enrichment technique. Two isolates, CMU1 and CMU2 were isolated from honey of A. florae whereas isolate CMU3 and CMU4 were from honey of A. cerena and A. dorsata respectively. The  Maejo Int. J. Sci. Technol.  2009, 3(01), 71-76     73 successful isolation was due to the efficacy of enrichment culture to promote the growth of acetic acid bacteria present in the samples. It is evident from Figure 1 that the numbers of acetic acid bacteria in the culture  broth  increased  with  time  during  the  enrichment  period.  As  a  result,  the  pH  values  were decreased accordingly (Figure 1). Many reports have also addressed the usefulness of the enrichment culture technique in selective isolation of targeted microorganisms [7-10].               Table 1.   Morphological characterisation of acetic acid bacterial isolates  Isolate  Source of honey  Days of enrichment  Gram stain and morphology CMU1  Apis  florae  3-7  Gram-negative, short rod CMU2  Apis  florae  3-7  Gram-variable, short rod CMU3  Apis cerena  7  Gram-variable, short rod CMU4  Apis dorsata  7  Gram-variable, short rod  As shown in Table 1, Gram staining revealed that the isolates were either Gram-negative or Gram-variable, short rod bacteria, which is a typical character of acetic acid bacteria. All of them were identified as Gluconobacter sp. due to their inability to oxidise acetate (Table 2). All isolates recorded the same phenotypic profile among themselves, thus suggesting that they belonged to the same taxa. Most acetic acid bacteria are known to be mesophilic with an optimum temperature for growth around 30oC. A slight increase in temperature results in a dramatic decrease in growth of these organisms [11,12]. However, all isolates obtained in this study grew well at 37oC, the character which suggested that these isolates may be thermotolerant strains. No members of the genus Acetobacter were obtained from any of the samples tested.   012345670 3 5 7Time (days)pH value0200040006000800010000120001400016000Total acetic acid bacteira (cfu/ml)Apis florea Apis cerena Apis dorsata Figure  1.    Change in numbers of acetic acid bacteria in culture broth and pH value during enrichment period  Maejo Int. J. Sci. Technol.  2009, 3(01), 71-76     74 Table 2.  Biochemical characterisation of acetic acid bacterial isolates Biochemical test  CMU1  CMU2  CMU3  CMU4 Overoxidation  -  -  -  - Catalase  +  +  +  + Growth in ethanol  +  +  +  + Production of gluconate +  +  +  + Ketogenesis from glycerol -  -  -  - Cellulose production  -  -  -  - Soluble pigment  -  -  -  - Note: + = positive; - = negative    The four isolates were tested for their ability to grow in broths supplemented with acetic acid and alcohol at 30 and 37oC. It was found that all isolates could grow in 1% (v/v) acetic acid only at 30oC.  However,  these  isolates  showed  remarkable  tolerance  to  ethanol,  particularly  isolate  CMU4, which was able to grow well in a medium containing as high as 10% ethanol (v/v) at 30oC (Table 3). In general, the alcohol tolerance decreased with higher temperature (Tables 3, 4). It seems that there may be  some  relationship  between  thermotolerance,  acetic  acid  resistance  and  ethanol  resistance characteristic  in  this  group  of  bacteria.  This  suggestion  is  supported  by  the  observation  that thermotolerant growth in the presence of acetic acid and high ethanol concentration is an outstanding characteristic of acetic acid bacteria from Thailand [11]. However, little has so far been reported about the mechanism of either acetic acid or ethanol resistance. Further investigation has to be done to test this hypothesis.  Table 3.  Alcohol tolerance of acetic acid bacterial isolates at 30oC  Percentage of alcohol (v/v)  Isolate 6  7  8  9  10 CMU1  ++++  ++++  ++++  +++  - CMU2  ++++  ++++  +++  -  - CMU3  ++++  ++++  ++++  +++  - CMU4  ++++  ++++  ++++  ++++  +++ Note : ++++ = very good growth; +++ = good growth; - = no growth  Surprisingly, in this study, the use of enrichment medium with ethanol which usually favours the development of Acetobacter yielded only Gluconobacter. This is probably due to the nature of honey with very high concentration of sugar, which allows only the growth of osmotolerant or osmophilic microorganisms including member of the genus Gluconobacter. This result is in agreement     Maejo Int. J. Sci. Technol.  2009, 3(01), 71-76     75 Table 4.  Alcohol tolerance of acetic acid bacterial isolates at 37oC  Percentage of alcohol (v/v)  Isolate 6  7  8  9  10 CMU1  ++++  ++++  +++  -  - CMU2  ++++  +++  -  -  - CMU3  ++++  ++++  +++  -  - CMU4  ++++  ++++  ++++  +++  - Note : ++++ = very good growth; +++ = good growth; - = no growth   with earlier reports by Ruiz-Argueso and Rodriguez-Navarro [4,5] who examined the microorganisms of  ripening  honey  from an apiary in Madrid, Spain. They found that Lactobacillus viridescens and Gluconobacter were two main groups of bacteria present in honey. These authors concluded that the microbial flora of honey appears to be the result of chance as honey bees come into contact with several microorganisms during their visit to various niches. Though Gluconobacter was found in association with  the  honey  bees,  it  is  not  known  whether  the  presence  of  the  acetic  acid  bacteria  has  any significance for the bees or any hive products including honey [6].  Conclusions   The first isolation of acetic acid bacteria from honey in Thailand was reported in the present study, the results of which have also provided evidence that the enrichment culture technique is useful in promoting the growth of acetic acid bacteria. These bacteria present in honey also show  high tolerance to ethanol, which suggests their usefulness in fermentation industry.  Acknowledgement The  authors  are  indebted  to  the  Department  of  Biology,  Faculty  of  Science,  Chiang  Mai University for all facilities and laboratories provided.   References  1.    J. A. Snowdon and D. O. Cliver,  “Microorganisms in honey”, Int. J. Food Microbiol., 1996, 31, 1-         26. 2.   O. Songram, S. Sittipraneed, and S. Klinbunga, “Mitochondrial DNA diversity and genetic          differentiation of the honeybee (Apis cerena) in Thailand”, Biochem. Gen., 2006, 44, 256-269. 3.   J. G. Holt, N. R. Krieg, P. H. A. Sneath, J. T. Stanley, and S. T. Williams, “Bergey’s Manual of        Determinative Bacteriology”, 9th Edn., Williams and Wilkins, Baltimore, 1994. 4.   T. Ruiz-Argueso and A. Rodriguez-Navarro, “Gluconic acid producing bacteria from honey bees        and ripening honey”, J. Gen. Microbiol., 1973, 76, 211-216.  Maejo Int. J. Sci. Technol.  2009, 3(01), 71-76     76 5.   T. Ruiz-Argueso and A. Rodriguez-Navarro, “Microbiology of ripening honey”, Appl. Microbiol.,        1975, 30, 893-896. 6.   B. Lambert, K. Kersters, F. Gossele, J. Swings, and J. De Ley, “Gluconobacters from honey bees”,        Antonie van Leeuwenhoek, 1981, 47, 147-157. 7.   Jaiinphon, K. Thonglem, and W. Pathom-aree, “Selective isolation of sorbitol utilizing acetic acid        bacteria.” Chiang Mai J.  Sci., 2002, 29, 189-192. 8.   S. Sudsakda, W. Srichareon, and W. Pathom-aree, “Comparison of three enrichment broths for the       isolation of thermotolerant acetic acid bacteria from flowers and fruits.”, Res. J. Microbiol., 2007,        2, 792-795. 9.   F. Duthatai and W. Pathom-aree, “Application of chemical dyes as colour indicator for selective        isolation of acetic acid bacteria.” Res. J. Microbiol., 2007, 2, 885-888. 10. A. Moryadee and W. Pathom-aree, “Isolation of thermotolerant acetic acid bacteria from fruits for        vinegar production.”, Res. J. Microbiol., 2008, 3, 209-212. 11. A. Saeki, G. Theeragool, K. Matsushita, H. Toyama, N. Lotong, and O. Adachi, “Development of        thermotolerant acetic acid bacteria useful for vinegar fermentation at higher temperatures”, Biosci.        Biotechnol. Biochem., 1997, 61, 138-145. 12.  A. Saeki, M. Taniguchi, K. Matsushita, H. Toyama, G. Theeragool, N. Lotong, and O. Adachi,        “Microbiological aspects of acetate oxidation by acetic acid bacteria, unfavorable phenomena in        vinegar fermentation”, Biosci. Biotechnol.Biochem., 1997, 61, 317-323.    © 2009 by Maejo University, San Sai, Chiang Mai, 50290 Thailand. Reproduction is permitted for noncommercial purposes. 

Isolation of acetic acid bacteria from honey

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