Biologia, Bratislava, 62/6: 650—656, 2007 Section Cellular and Molecular Biology DOI: 10.2478/s11756-007-0144-y Screening of different contaminated environments for polyhydroxyalkanoates-producing bacterial strains Shafiq ur Rehman,NaziaJamil* & Shahida Husnain Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore 54590, Pakistan; e-mail: jamil [email protected] Abstract: Total sixteen bacterial strains were isolated and purified from the samples collected from sugarcane molasses soil, sewage water and long-chain-hydrocarbon-contaminated area of the Punjab University, Lahore, Pakistan. Tolerance to different antibiotics was studied and strains showed multiple antibiotic resistance. All strains were characterized for Gram stain, biochemical reactions and polyhydroxyalkanoate (PHA) production. Total fourteen strains were Gram negative and two were Gram positive, while biochemically nine PHA producers showed affiliation to Pseudomonas, Enterobacter, Citrobacter, Bacillus and Escherichia. Screening for PHA production was done by Sudan black staining and nine out of sixteen strains exhibited PHA producing ability. PHA production was optimized for different growth parameters, like nitrogen concentration, pH and temperature. PHA extraction was done by solvent extraction method. Bacterial strains US1 and M1 accumulated up to 30% PHA of their cell dry weight on PHA extraction by solvent extraction method. Bacterial strain US1 was identified by 16S rRNA gene analysis as P. aeruginosa (DQ455691). PHA production was confirmed by PCR amplification of 500 bp fragment from PHA polymerase (Pha C) gene; five strains from nine PHA producers gave positive results on PCR. Pha C gene fragment of US1 was sequenced and submitted to Gene Bank under the accession number DQ455690. The amino acid sequence showed homology using the protein BLAST at 129–132 sites with different PHA synthases of the Pseudomonas sp. Key words: sewage water; polyhydroxyalkanoates; PhaC gene; Pseudomonas sp.; conserved gene. Abbreviations: mcl, medium-chain-length; MIC, minimal inhibitory concentration; PDA, PHA-detection agar; PHAs, polyhydroxyalkanoates; scl, short-chain-length. Introduction ply of carbon source (Du et al. 2001; Du & Yu 2002). Approximately 150 different hydroxyalkanoic acids are Synthetic polymers (known as plastics) have become known at present to occur as constituents of PHAs. The significant since the 1940s, and since then they are re- use of PHAs as raw material is generally believed to re- placing glass, wood and other constructional materials, duce the environmental impact of plastic wastes mainly and even metals in many industrial, domestic and en- due to their biodegradability (Poirier et al. 1995). vironmental applications (Poirier et al. 1995). Due to PHAs produced by bacteria consist of three main their persistence in the environment, globally peoples types: (i) polymers composed of short-chain-length (scl) are now more sensitive to the impact of discarded plas- monomers; (ii) polymers composed of medium-chain- tic on the environment, including deleterious effects on length (mcl) monomers; and (iii) polymers composed wildlife and on the aesthetic qualities of cities and for- of scl-mcl monomers. scl PHA consists of monomeric est. The increased cost of solid waste disposal as well as subunits 3 to 5 carbons in length, while mcl PHA con- the potential hazards from waste incineration such as sists of monomers 6 to 14 carbons in length and scl-mcl dioxin emission from polyvinyl chloride makes synthetic PHA copolymer consists of monomeric subunits 4 to plastic a waste management problem (Ojumu et al. 12 carbons in length (Ojumu et al. 2004). The major 2004). Polyhydroxyalkanoates (PHAs) are polyesters of cost of PHA production is the substrate and the sepa- 3–hydroxyalkanoic acids produced as intracellular gran- ration process (Bruce et al. 1990). De Lima et al. (1999) ules by many different bacteria (Steinb¨uchel & Hein studied different PHA-producing bacterial strains iso- 2001). PHAs are synthesized and accumulated intra- lated from a sugarcane agro-ecosystem for their resis- cellularly in most bacteria under unfavorable growth tance to certain antibiotics. Choosing an antimicrobial condition, such as limitation of nitrogen, phosphorus, with the desirable selective toxicity is difficult and the oxygen or magnesium in the presence of excess sup- exact amount to be applied must previously be deter- * Corresponding author c 2007 Institute of Molecular Biology, Slovak Academy of Sciences Characterization of polyhydroxyalkanoates-producing bacterial strains 651 mined since an excessive concentration may modify the · 7H2O 2.25, CuSO4 · 5H2O 1, MnSO4 · 5H2O0.5,CaCl2 · global performance of the microorganism of interest or 2H2O2.0,Na2B4O7 · 10H2O 0.23, (NH4)6MO7O24 0.1, HCl remain in the product altering its properties (Oliveira 10 mL); pH of the medium was adjusted to 7.0. Glucose and et al. 2000). sucrose were used as carbon sources for screening and opti- PHA synthase is the crucial enzyme, with hydrox- mization experiments. PHA producers were detected macro- scopically by observing the turbidity on PDA. Producers yacyl coenzyme A as the substrate, in synthesizing were further screened by Sudan black B staining (Arnold et PHAs. More than 59 PHA synthase genes have been al. 1999). Optimization of different growth parameters, i.e. cloned from 45 bacterial species and broadly catego- the incubation at different temperature, pH, harvest time rized into four different classes, based on their in vivo and carbon source, were done by growing on PDA medium substrate specificities, amino acid sequences, and sub- in shake flask. Biomass and PHA % was calculated after unit composition (Rehm 2003). Class I and class II interval of 24 h. Firstly, the time was optimized for PHA PHA synthases comprise enzymes consisting of only one extraction. Different nitrogen concentrations, i.e. 0%, 0.1%, type of subunit PhaC (Qi & Rehm 2001). The class II 0.2% and 0.4%, were used, while temperature and pH were optimized by incubating different sets of shake flask cultures PHA synthase genes PhaC1 and PhaC2 that favour mcl ◦ ◦ ◦ 3HA have been identified and characterized in Pseu- at 30 C, 37 Cand42C temperature and pH 6 and 7. domonas sp. including Pseudomonas aeruginosa (Timm &Steinb¨uchel 1992), Pseudomonas oleovorans (Huis- Extraction of PHA man et al. 1991), Pseudomonas sp. 61–3 (Matsusaki PHA extraction was done by solvent extraction methods (Arnold et al. 1999). In solvent extraction, bacterial cells et al. 1998), and Pseudomonas mendocina (Hein et al. were collected by centrifugation and dry cell weight was 2002). The different studies showed that both of them calculated. The chloroform was added (50 times the dry exhibited very similar properties; only in rare cases they cell weight) and incubated at 95 ◦C for 10 min. Cooled to showed some differences on the contents and monomer room temperature, and mixed solution overnight with stir- composition of the accumulated PHA (Qi et al. 1997; ring. The solution was filtered to remove the cell debris, fil- Hein et al. 2002). trate was precipitated by adding a mixture of methanol and In this study, different contaminated environments water (7:3 v/v) (five volumes of chloroform) to the filtrate. were screened for potential PHA producers and their Filtered the precipitated PHA and weighed. ability to show resistance against selected antibiotics were monitored. Also the growth conditions of maxi- Genomic DNA isolation, amplification and sequencing mum PHA producers were optimized as well as a frag- All selected strains were grown in nutrient broth for 24 ment from PHA synthase gene was sequenced and an- h and genomic DNA was isolated by Fermentas genomic alyzed. DNA isolation kit. 16S rRNA gene was amplified using Primus96 (PeQLab) with PCR master mix, forward primer 16S-3 (5’-CCCGGGAACGTATTCACCG-3’) and the re- Material and methods verse primer 16S-5 (5’-GCYTAAYACATGCAAGTCGA-3’) (Cameron 2002). PCR was carried out by denaturing DNA Environments selected ◦ at 110 C for 10 min followed by 30 cycles of amplification The environments selected included Punjab University ◦ ◦ ◦ (95 Cfor2min,52C for 1 min and 72 Cfor2min). workshop, oil shop, engine oil, sewage water and sugar- Pha C polymerase gene was amplified using Primus96 cane molasses soil. All samples and the strains isolated were (PeQLab) with PCR master mix, forward primer 179–L (5’- coded as UW, UOS, EO, US and M respectively. ACAGATCAAGTTCTACATCTTCGAC-3’) and the re- verse primer 179–R (5’-GGTGTTGTCGTTCCAGTAGA- Isolation of bacteria GGATGTC-3’) (Solaiman et al. 2000). PCR was carried All samples were properly diluted and spread on the nutrient ◦ out by denaturing DNA at 110 C for 10 min followed by 30 agar (peptone 5 g/L, beef extract 3 g/L, agar 15 g/L). The ◦ ◦ ◦ cycles of amplification (95 Cfor2min,56C for 1 min and pH was adjusted to 7.0, incubated for 24 h at 37 C. Gram ◦ 72 C for 2 min). DNA sequencing was done in the Centre for staining was performed for all purified strains (Cheesbrough Excellence in Molecular Biology, Lahore (Pakistan) on DNA 2001). Biochemical analyses were done by performing the sequencing system (Applied Biosystems, 3100/ga3100-1696- catalase test, oxidase test, triple sugar iron reactions, citrate 013) by Dideoxy DNA sequencing method. utilization test, urease test, indole test, motility test, Voges- Proskaur test, methyl red test and sugars fermentation test (Cheesbrough 2001). Bioinformatics tools The sequences of 16S rRNA and PhaC gene fragment were