Short Communication: Isolation of polyhydroxyalkanoate-producing bacteria from an integrated-farming pond and palm-oil mill effluent ponds. G. Redzwan ...
World Journal of Microbiology & Biotechnology 13, 707±709
Short Communication: Isolation of polyhydroxyalkanoate-producing bacteria from an integrated-farming pond and palm-oil mill ef¯uent ponds
G. Redzwan, S.-N. Gan and I.K.P. Tan* Bacterial isolates from two environments, an integrated-farming pond in the university and palm-oil mill ef¯uent (POME) ponds at a local palm-oil-processing factory, were screened for polyhydroxyalkanoates (PHAs). Initially Sudan Black B staining was performed to detect lipid cellular inclusions. Lipid-positive isolates were then grown in a nitrogen-limiting medium containing 2% (w/v) glucose to promote accumulation of PHA before the subsequent Nile Blue A staining. The PHA extracted from positive isolates was con®rmed by nuclear magnetic resonance (NMR) spectroscopy. The proportion of PHA-positive bacterial isolates was higher in the POME ponds compared to the integrated-farming pond. Key words: Bacteria, integrated-farming pond, isolation, palm-oil mill ef¯uent, PHA, screening.
PHAs have thermoplastic properties, they are biodegradable, and can be synthesized from renewable resources such as carbohydrates and lipids as the fermentation feedstocks. All these properties make this class of microbial polyester very attractive as a source of alternative materials to conventional petrochemicalbased plastics. The biosynthesis and accumulation of PHA is a common phenomenon in many species of bacteria as a form of carbon and energy reserve (Dawes & Senior 1973) or as an electron sink mechanism (Page & Knosp 1989). As with most microbial products, the search for new producer organisms is a continuous process, necessitated by the desire for higher product yield, more ef®cient utilization and conversion of speci®c raw materials, tolerance to environmental conditions, and novel end-products. This study focused on the isolation of bacterial strains from two different types of environments in Malaysia, and screening of the isolates for PHA-producing abilities. The two environments were: an integrated-farming pond which incorporated ®sh culture, and a series of POME ponds. POME ponds are standard treatment areas in every palm-oil mill: raw waste and washings ¯ow into the ponds and remain there until the chemical oxygen demand (COD) and biological
G. Redzwan and I.K.P. Tan are with the Institute of Advanced Studies, S.-N. Gan is with the Department of Chemistry, University of Malaya, 50603 Kuala Lumpur, Malaysia; Fax: 60-3-756 8940. *Corresponding author.
oxygen demand (BOD) are suf®ciently reduced before discharge into the common environment.
Materials and Methods Isolation of Bacterial Strains Bacterial isolates were obtained by dilution-streaking of nutrient agar (NA) plates of sediment taken from the sides of the integrated-farming pond, and liquid from the surface of three consecutive POME ponds which held progressively-treated ef¯uent. Screening for PHA-producing Bacteria All the bacterial isolates were subjected to an initial Sudan Black B staining (Burdon 1946) to detect the presence of lipid granules in the bacteria. A positively-stained isolate was considered a potential PHA producer. However, before the second staining with PHA-speci®c Nile Blue A (Ostle & Holt 1982), the isolate was ®rst induced to accumulate PHA by culturing in E2 medium, a nitrogen-limiting medium (Lageveen et al. 1988) containing 2% (w/v) glucose. Con®rmation of PHA PHA-positive isolates were grown in much larger quantities in the E2 + 2% glucose medium. After 48 h, the bacterial cells were harvested and lyophilized. Polymers were extracted from the lyophilized cells according to Lageveen et al. (1988), dissolved in deuterated chloroform (CDCl3) at a concentration of 20 mg/ml, and analyzed on a JEOL JNM-GSX 270 Fourier Transformation (FT) NMR system. Identi®cation of PHA-producing Bacteria The PHA-positive bacterial isolates were identi®ed according to routine biochemical tests and the use of the API kit (Biomerieux Co.).
ã 1997 Rapid Science Publishers World Journal of Microbiology & Biotechnology, Vol 13, 1997
707
G. Redzwan, S.-N. Gan and I.K.P. Tan
Results and Discussion Isolation and Screening of PHA-producing Bacteria The use of Sudan Black B as the ®rst-line screening for PHA-producing bacteria was based on a report by Williamson & Wilkinson (1958) who observed that 89% of the lipid granules in Bacillus megaterium contained PHA when the bacteria was cultured in an unbalanced growth medium. In this study, it was assumed that those isolates which were not stained positively with Sudan Black B did not form lipid granules and hence also did not form PHA because the polyester is lipidic. All the isolates obtained from the three POME ponds stained positively with Sudan Black B, and close to 50% of these isolates stained positively with Nile Blue A, meaning that half of all the isolates from the POME ponds contain PHA (Table 1). On the other hand, only 15 of the 18 isolates obtained from the integrated-farming pond contain lipid inclusions, and of these, only ®ve (or 28% of the total number of isolates) contained PHA. Compared to the integrated-farming pond, the POME ponds have a higher content of oil (triacylglycerols) and its breakdown products such as diacylglycerols, monoacylglycerols and fatty acids. It is reasonable that the micro¯ora in that environment would be able to utilize oils and their degradative products for metabolism, and convert them to related compounds such as storage materials within the cell. It is interesting to note that a higher proportion (48%) of the lipid-positive isolates from the POME ponds contain PHA, compared to only 33% of the lipid-positive isolates from the integratedfarming pond. This indicates the potential of obtaining good PHA-producing bacteria which could utilize POME, and hence convert an industrial pollutant into a useful product. All of the Nile Blue A positively-stained isolates were con®rmed to accumulate poly-3-hydroxybutyrate (PHB) (Figure 1A,B). Seventeen of the 18 PHBpositive isolates were identi®ed as Bacillus sp. while one was identi®ed as an Acinetobacter sp.
Table 1. Number of isolates obtained and which reacted positively with Sudan Black B and Nile Blue A. Source
Number of isolates
Integrated-farming pond Untreated POME pond Treated POME pond I Treated POME pond II
708
18 8 7 12
Isolates staining positively with Sudan Black B
Isolates staining positively with Nile Blue A
15 8 7 12
5 4 3 6
World Journal of Microbiology & Biotechnology, Vol 13, 1997
Figure 1. (A) 67.8 MHz 13C NMR spectrum, and (B) 270 MHz 1H NMR spectrum of the extracted polymer from an isolate which stained positively with Nile Blue A.
Selection for a Wider Range of Bacteria Which Produce Different Types of PHA Different bacteria may utilize different carbon substrates (e.g. sugars, fatty acids) for PHA production; some have speci®c requirements for substrate and limiting nutrients in order to accumulate PHA (Dawes & Senior 1973). Therefore, the second-step screening process should be expanded to include carbon substrates other than glucose, and limiting medium other than nitrogen, to induce PHA accumulation in isolates showing lipid inclusions. This way, a wider range of bacteria which produce different types of PHA may be selected for.
Acknowledgement The studies were funded by the University of Malaya Vote F 62/95.
References Burdon, K.L. 1946 Fatty acid material in bacteria and fungi revealed by staining dried, ®xed, slide preparations. Journal of Bacteriology 52, 665±678.
Polyhydroxyalkanoate from bacteria Dawes, E.A. & Senior. P.J. 1973 The role and regulation of energy reserve polymer in microorganisms. Advances in Microbial Physiology 10, 135±265. Lageveen, R.G., Huisman, G.W., Preusting, H., Ketelaar, P., Eggink, G. & Witholt, B. 1988 Formation of polyesters by Pseudomonas oleovorans: Effects of substrates on formation and composition of poly-(R)-3-hydroxyalkanoates and poly-(R)-3hydroxyalkenoates. Applied and Environmental Microbiology 54, 2924±2932. Ostle, A.G. & Holt, J.G. 1982 Nile Blue A as a ¯uorescent stain for poly-b-hydroxybutyrate. Applied and Environmental Microbiology 44, 238±241.
Page, W.J. & Knosp, O. 1989 Hyperproduction of poly-bhydroxybutyrate during exponential growth of Azotobacter vinelandii UWD. Applied and Environmental Microbiology 55, 1334±1339. Williamson, D.H. & Wilkinson, J.F. 1958 The isolation and estimation of poly-b-hydroxybutyrate inclusions of Bacillus sp. Journal of General Microbiology 19, 198±209.
(Received in revised form 2 January 1997; accepted 3 January 1997)
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