Abstract: The effectiveness of three enrichment broths, namely potato medium, seed culture medium and sterile distilled water for the isolation of thermotolerant acetic acid bacteria from flowers and fruits was investigated. The numbers of successful isolation obtained from each medium were nearly the same. Seed culture medium was found to give the highest numbers of successful isolation from flower samples whereas potato medium and sterile distilled water gave higher numbers for the isolation from fruits. However, there were no statistically significant differences between the media tested (α>0.05).
INTRODUCTION
Acetic acid bacteria are obligate aerobic Gram negative bacteria which are capable of oxidize ethanol to acetic acid (Matsushita et al., 1994). They are classified in the family Acetobacteraceae and widely distributed in natural habitats (Holt et al., 1994). Members of this family are useful in industrial applications including production of vinegar and L-ascorbic acid (Deppenmeier et al., 2002). The global warming crisis in the past few years is a serious challenge to fermentation industries since a large cooling system is required for maintaining the optimum temperature. Recently, there has been interest in thermotolerant bacteria due to their economic profits (Saeki et al., 1997a). Isolation, identification and characterization of acetic acid bacteria were set in progress to develop new microbial resources for oxidative fermentation (Moonmangmee et al., 2000; Adachi et al., 2003).
The standard procedure for isolation of acetic acid bacteria from samples of natural origin usually involve the use of enrichment culture technique since acetic acid bacteria are not present as dominant specie (Yamada et al., 2000; Lisdiyanti et al., 2002; Jojima et al., 2004). However, to date, no studies attempting to evaluate the efficiency of enrichment media for the isolation of acetic acid bacteria have been reported. Hence, it is the purpose of this study to compare the effect of three different enrichment media for the isolation of acetic acid bacteria especially thermotolerant strains from flowers and fruits.
MATERIALS AND METHODS
This research was carried out at Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, Thailand during June 2002-March 2003.
Samples
Fifteen species of flower (Table 1) commonly found in
northern part of Thailand were collected from area within Chiang Mai University
main campus. Fifteen fruit samples (Table 2) were purchased
from local market.
Selective Isolation of Thermotolerant Acetic Acid Bacteria
Acetic acid bacteria were isolated from each sample by enrichment culture
technique using three enrichment broths: potato medium (0.5% glucose, 1% yeast
extract, 1% peptone, 2% glycerol and 1.5% potato extract), seed culture medium
(0.5% glucose, 0.5% yeast extract, 0.5% peptone and 0.5% glycerol) and sterile
distilled water. Each enrichment broth was supplemented with 4% ethanol (v/v)
and the initial pH was adjusted to 7.0. Approximately 5 g of flowers or fruits
were added to the medium and incubated at 37°C for 3-5 days to select thermotolerant
strains. Acetic acid bacteria were isolated by streaking the content of each
enrichment broth onto potato agar plate to which 0.003% bromocresol purple,
4% ethanol (v/v) and 1.5% agar were added. The colonies producing yellow halo
were selected and presumptively identified as acetic acid bacteria. All selected
colonies were restreaked onto fresh new plate to obtain pure culture and subjected
to detailed taxonomic characterization.
Taxonomic Characterization
Morphological, physiological and biochemical characteristics of pure isolates
were examined according to the ninth edition of Bergey’s Manual of Determinative
Bacteriology (Holt et al., 1994). The ability of the isolate to oxidize
acetate to CO2 and H2O was used to distinguish between
members of the genus Acetobacter and Gluconobacter.
Statistical Analysis
The significance of differences in numbers of successful isolation was evaluated
with the non-parametric Cochran Q test (Rall et al., 2005) using SPSS
software package.
RESULTS AND DISCUSSION
The present communication compared the effect of three different enrichment media for the isolation of thermotolerant acetic acid bacteria from flowers and fruits. Acetic acid bacteria usually present in small numbers so it is necessary to increase their numbers to detectable level using enrichment culture technique. An enrichment culture usually provides nutrients and environmental conditions that favor the growth of microorganisms of interest. In this study, ethanol was used as a selective nutrient to select acetic acid bacteria. All media were success in promoting the growth of acetic acid bacteria. This result supported the use of enrichment culture as standard procedure for the isolation of acetic acid bacteria from environmental samples (Yamada et al., 2000; Lisdiyanti et al., 2002; Jojima et al., 2004).
Of the media tested, seed culture medium gave the highest numbers of successful isolation from flowers (Table 1) whereas potato medium and sterile distilled water gave the best result for fruit samples (Table 2).
| Table 1: | Isolation of thermotolerant acetic acid bacteria from 15 flowers using three different enrichment media |
| + : Successful isolation; - : Unsuccessful isolation | |
| Table 2: | Isolation of thermotolerant acetic acid bacteria from 15 fruits using three different enrichment media |
| +: Successful isolation; - : Unsuccessful isolation | |
However, statistical analysis revealed that there were no significant differences between the media tested. This result suggested that these media could be used interchangeably. Comparison of these results with those earlier studies was difficult as so little relevant research has been done and many studies on the isolation of acetic acid bacteria were limited to only the mesophilic group (Yamada et al., 2000; Lisdiyanti et al., 2002; Hanmoungjai et al., 2007).
All isolates were identified as Gluconobacter sp. due to their inability to oxidize acetate. Most acetic acid bacteria are known to be mesophilic with an optimum temperature for growth around 30°C. A slight increase in temperature resulted in a dramatic decrease in growth of these organisms (Saeki et al., 1997b). All isolates grew well at 37°C, the character which suggested that these isolates may be thermotolerant strains (Saeki et al., 1997a; Moonmangmee et al., 2000; Adachi et al., 2003). The observation that all isolates were member of the genus Gluconobacter indicated that flowers may be good sources for isolation of acetic acid bacteria. This is in agreement with previous report that natural habitats of Gluconobacter strains are flowers and fruits (Gupta et al., 2001). Several thermotolerant Gluconobacter strains had also been isolated from flowers, fruits and vegetables collected in Chiang Mai (Jaiinphon et al., 2002). Most of these isolates showed ability to utilize sorbitol, an indication of sorbitol dehydrogenase activity. This enzyme is responsible for the oxidation of D-sorbitol to L-sorbose, a precursor in fructose and ascorbic acid production (Matsushita et al., 1994).
CONCLUSIONS
The obtained results indicated that the three media tested are equally effective in enrichment purpose for the isolation of thermotolerant acetic acid bacteria present in flowers and fruits. However, the use of sterile distilled water supplemented with 4% ethanol (v/v) was recommended as the enrichment medium which led to the reduction in both time and cost in media preparation. These results also added to a list of references support the usefulness of enrichment culture technique for isolation of acetic acid bacteria from environmental samples.
ACKNOWLEDGMENTS
The authors are grateful to Department of Biology, Faculty of Science, Chiang Mai University for all facilities and Laboratory provided. Thanks also go to Associate Professor Dr. Araya Jatisatien for statistical analysis and CMU Herbarium for identification of all flower samples.