Screening of Different Contaminated Environments For

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 (Steinbüchel & 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 &Steinbüchel 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 Antibiotics resistance analyzed by BLAST at the NCBI server (http://www.ncbi. Plate dilution technique was used for determining the min- nlm.nih.gov/BLAST/) and submitted to Gene Bank under imal inhibitory concentration (MIC) to the selected antibi- the accession number DQ455691 and DQ455690, respec- otics, amoxicillin (Amx), tetracycline (Tet) and ciprofloxa- tively. Amino acid sequence of PhaC amplified gene frag- cin (Cip), on nutrient agar plates (Sambrook et al. 2001) by ment (DQ455690) was found through online ExPASy Trans- incubating at 37 ◦C for 24 h. late tool (http://www.expasy.ch/tools/dna.html). The cor- rect amino acid sequence was found from 3–5 reading frame Screening and optimization for PHA production 3. The protein sequences matched by BLAST were further All purified strains were grown on PHA-detection agar analyzed by CLUSTALW program (http://www.ebi.ac.uk/ (PDA) consisted of (g/L): (NH4)2SO4 (2), KH2PO4 (13.3), clustalw/) for phylogenetic tree formation and Genedoc MgSO4 (1.3), citric acid (1.7), carbon source (2), trace el- software (http://www.nrbsc.org/gfx/genedoc/) was used ement solution 10 mL/L (g/L, FeSO4 · 7H2O 10, ZnSO4 for conserved domain finding. 652 S. ur Rehman et al.

Table 1. Screening for potential PHA producers and antibiotic resistance to PHA-producing strains.a

Turbidity Antibiotic resistance Strain # Sudan black staining PHA % Glu Suc Amox Tet Cip

UW1 ++ – +ve S S S – UOS1 +++ – +ve 650 50 50 14 UOS2 – + –ve S S S – UOS3 +++ +++ +ve 1000 S 50 9 US1 ++++ +++ +ve 650 300 S 27 US2 ++++ + +ve 1000 300 50 25 EO1 – – –ve S S 50 – M1 ++++ ++++ +ve 650 300 50 30 M2 – – –ve 50 S S – M3 + + +ve 1000 300 S 6 M4 ++++ + +ve 400 S S 16 M5 + + +ve 1000 300 50 – M6 + + +ve 1000 200 S 17 M7 + +++ +ve 400 S S 16 M8 +++ – –ve 900 50 S – M9 +++ + +ve S 50 50 20 a Legend: – = no growth, + = slightly turbid, +++ = turbid, ++++ = more turbid, Glu = glucose, Suc = sucrose, S = sensitive, Amx = amoxicilline, Tet = tetracycline, Cip = ciproﬂoxacine.

Table 2. Biochemical properties of PHA producers.a

Sugar fermentation Strain No. Growthb TSI Oxidase Catalase Citrate Indole MR/VP Urease Probable genera Glu Suc Lac Mal

UOS1 LF A/A/–/+ –ve +ve –ve –ve –ve/+ve –ve +ve –ve +ve –ve Enterobacter UOS3 LF A/A/–/– –ve +ve –ve –ve +ve/–ve –ve +ve –ve +ve +ve Escherichia US1 NLF K/K/–/– +ve +ve +ve –ve –ve/–ve –ve –ve –ve –ve –ve Pseudomonas US2 NLF K/K/–/– –ve +ve +ve –ve –ve/–ve –ve –ve –ve –ve –ve Pseudomonas M1 NLF K/K/–/+ +ve +ve +ve –ve –ve/+ve –ve –ve –ve –ve –ve Pseudomonas M3 NLF K/K/–/– +ve +ve +ve –ve –ve/–ve –ve –ve –ve –ve –ve Pseudomonas M4 LF A/A/–/+ –ve +ve +ve –ve –ve/–ve –ve +ve –ve +ve –ve Bacillus M6 NLF K/K/–/– +ve +ve +ve –ve –ve/–ve –ve –ve –ve –ve –ve Pseudomonas M9 LF A/A/–/– –ve +ve +ve –ve +ve/–ve –ve +ve –ve +ve –ve Citrobacter a NLF = Non-lactose fermentor, LF = lactose fermentor, K = alkaline, A = acidic, NM = non-motile, Glu = glucose, Suc = sucrose, Lac = lactose, Mal = maltose. b Growth on Mac Conkey agar.

Results and discussion in all cells, when stained with Sudan black staining. Sudan black is a lipophilic dye attached to PHA and Isolation, puriﬁcation and characterization gives purple colour to PHA granules with pink black PHAs are intracellular carbon and energy reserve mate- ground (Schegel et al. 1970). In three strains (UW1, rials that are accumulated by a variety of microorgan- M5 and M7), granules were observed in some cells, isms under certain unbalanced growth conditions, such whereas in four bacterial strains (USO2, EO1, M2 and as high carbon to nitrogen ratio (Lee 1996). Samples M8) no granule was observed. On the basis of these re- analysed in the present study were collected from the sults nine bacterial strains (UOS1, UOS3, US1, US2, University of Punjab (Lahore, Pakistan) oil shop, auto- M1, M3, M4, M6 and M9) were selected as PHA pro- mobile workshop, sewage, engine oil and sugarcane mo- ducers. lasses soil, as these sites help in PHA accumulation in a In presence of sucrose as carbon source, four strains cell due to nutrition depletion conditions. Sixteen bac- (UOS3, US1, M1 and M7) showed turbid colonies and terial strains were isolated form all samples. Two of the granules were observed in all cells. Seven strains (UOS2, isolated strains were Gram-positive (M4 and M7) and US2, M3, M4, M5, M6 and M9) showed slightly turbid 14 were Gram-negative. On PDA media, in presence of colonies and granules in some cells were observed, three glucose as carbon source, nine strains, (UW1, UOS1, strains (UW1, UOS1 and M2) showed no growth and UOS3, US1, US2, M1, M4, M8 and M9) showed turbid two strains (EO1 and M8) showed non-turbid colonies colonies which indicates PHA production (Arnold et (Table 1). With regard to biochemical characterization al. 1999), four bacterial strains (M3, M5, M6 and M7) PHA-producing strains showed resemblance to Pseu- showed slightly turbid colonies, two strains (UOS2 and domonas, Enterobacter, Citrobacter, Bacillus and Es- EO1) showed no turbidity and M2 showed no growth cherichia (Table 2). On the basis of the 16S rRNA gene (Table 1). PHA granules were observed in nine strains sequence, the strain US1 was identiﬁed as Pseudomonas (UOS1, UOS3, US1, US2, M1, M3, M4, M6 and M9) aeruginosa (Accession No. DQ455691). Characterization of polyhydroxyalkanoates-producing bacterial strains 653

Fig. 1. Comparison of PHA percentage ( ) and biomass () production at diﬀerent nitrogen concentrations by strain M1 and US1.

Antibiotic resistance and PHA production UOS1, UOS3, EO1, M2, M4 and M7) were sensitive for Most strains showed resistance to amoxicillin, a com- tetracycline, and five strains (US1, US2, M1, M3 and monly used antibiotic, whereas most were sensitive to M5) showed maximum MIC (300 µg/mL) for tetracy- ciprofloxacin, a less frequent antibiotic. This may be cline. The bacterial strains showed a wide range of re- due to large use of these antibiotics in the community sistance against amoxicilline. Only four strains (UW1, (hospital waste and sewage contamination to the envi- UOS2, EO1, and M9) were sensitive for amoxicilline. ronment). Exposure of an antibiotic in the environment Five strains (UOS1, US2, M3, M5 and M6) showed re- leads the bacterium to develop the resistance against sistance at 1000 µg/mL amoxicilline. multiple antibiotics. Resistance to different antibiotics is spread among bacterial population of different envi- Effect of nitrogen, pH and temperature on PHA pro- ronments. Atlas (1984) pointed out diversity changes duction in response to environmental stress, showing both ten- Bacterial strains US1 and M1 were selected for opti- dencies: (a) increase in diversity by selective toxicity, mization experiments due to the maximum production eliminating dominant organism; and (b) diversity de- of PHA among all strains. At 0.0% nitrogen concentra- creases by elimination of many species due to toxic- tion, no growth of US1 and M1 was observed. Maximum ity or increase in particular populations. The minimal PHA production was observed at 0.2% concentration. inhibitory concentrations (MICs) for three antibiotics The optimum time for PHA production was found to (amoxicilline, tetracycline and ciprofloxacine) were esti- be 72 h (Fig. 1). While at 0.4% concentration biomass mated for all the isolated strains (Table 1). Except M9, and PHA production was less as compared to 0.1% and all strains showed resistance to amoxicillin, a commonly 0.2% concentrations. At 30 ◦C M1 showed maximum used antibiotic. Most strains were resistant to the three growth at pH 7 but minimum at pH 6. M1 showed max- tested antibiotics. All strains showed very low resistance imum growth at pH 6 and 42 ◦C. When comparing the to ciprofloxacine (< 50 µg/mL). Seven strains (UW1, US1 and M1, growth at different pH and temperatures 654 S. ur Rehman et al.

Fig. 2. Production of PHA by the strains US1 ()andM1( ) at diﬀerent temperatures (30 ◦C, 37 ◦Cand42◦C) and pH 6 (A)and7 (B), grown on PDA (glucose, 72 h).

Fig. 4. Phylogenetic tree of DQ455690 (US1) with YP 793525 (PaeruginosaUCBPP-PA14), NP 253743 (PaeruginosaPAO1), EAZ61501 (Paeruginosa2192), EAZ55677 (PaeruginosaC3719) and YP 793527 (PaeruginosaUCBPP-PA14) prepared using the program CLUSTALW.

analysis as Pseudomonas sp. (Accession No. DQ455691) showing 94% homology to Pseudomonas aeruginosa. Fig. 3. Amplification of PHA polymerase gene fragment of all Strain US1 was isolated from a sample collected from strains. Lanes 1 to 10: marker DNA, UOS1, UOS3, US1, US2, the University sewage. Sewage water consists of efflu- M1, M3, M4, M6 and M9. ent from different departments, hospital and domestic wastes, which is the probable cause for 94% homology to Pseudomonas aeruginosa (AF417876) 16S rRNA showed that US1 was a faster grower than M1 at all gene. This strain was able to utilize glucose and sucrose different pH and temperatures studied so far (Fig. 2). as carbon source for PHA production. The PCR proto- Extraction of PHA from all granule-producing col was developed using genomic DNA purified from 5 strains (UW1, UOS1, UOS3, US1, US2, M1, M3, M4, strains (UOS1, US1, US2, M4 and M6). Results showed M5, M6, M7 and M9) was done by solvent extraction that a 540 bp PCR product was obtained (Fig. 3). The method. Chloroform and other chlorinated hydrocar- size of the PCR product agrees with the length of the bons solubilised all PHAs. PHA extraction was done phaC gene flanked by the I-179LI-179R primer-pair. after 72 h and all bacterial strains produced PHA ex- Amplified gene fragment from US1 was sequenced. On cept for M7, which showed no growth in PHA detection sequencing the PCR product of PhaC from US1, it gave broth with glucose as carbon source. Bacterial strains length of about 445 bp. The DNA sequence has 59% M1 and US1 produce maximum PHA% of their cell dry GC and 41% AT content. DNA sequence was analyzed weight (30% and 27%, respectively).The decrease in the by finding proper ORF and homology to other PhaC biomass and PHA content between 72 to 96 h of growth proteins in the databases. 3’-5’ ORF 3 have no stop was observed that is due to the depletion of nutrient and codon and codes for 147 amino acids. Using the BLAST production of toxic materials in the medium; at this search it showed homology of maximum of 132 amino time bacteria also start using PHA as carbon source. acid residues (90%). The homologues sequences belong Du et al. (2001) reported that PHAs were synthesized to PhaC1 (90%) and PhaC2 (87%) showing maximum and intracellularly accumulated in most bacteria under homology to PhaC1. Five out of nine PHA producers unfavourable growth condition, such as limitation of ni- showed positive results on amplification of gene frag- trogen, phosphorus, oxygen or magnesium in the pres- ment of PHA polymerase. The other four strains were ence of excess supply of carbon source. Yuksekdag et al. also PHA producers but not amplified on PCR that (2004) studied the effect of carbon and nitrogen sources may be due to the non-complementation of their PHA and incubation times on poly-β-hydroxybutyrate syn- synthase to primers, or these strains may not belong thesis. to the PHA synthase class II (Timm & Steinbüchel 1992; Shamala et al. 2003). Solaiman et al. (2000) used Ribotyping and PCR amplification of PhaC polymerase the PCR-based strategy to confirm the PHA produc- gene ers from different Pseudomonas sp. after screening by Bacterial strain US1 was identified by 16S rRNA gene the Sudan black staining. PCR results showed that Characterization of polyhydroxyalkanoates-producing bacterial strains 655

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