Biologia, Bratislava, 62/6: 650—656, 2007Section Cellular and Molecular BiologyDOI: 10.2478/s11756-007-0144-y

Screening of different contaminated environmentsfor polyhydroxyalkanoates-producing bacterial strains

Shafiq ur Rehman, Nazia Jamil* & Shahida Husnain

Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore 54590, Pakistan; e-mail:jamil nazi@yahoo.com

Abstract: Total sixteen bacterial strains were isolated and purified from the samples collected from sugarcane molassessoil, sewage water and long-chain-hydrocarbon-contaminated area of the Punjab University, Lahore, Pakistan. Toleranceto different antibiotics was studied and strains showed multiple antibiotic resistance. All strains were characterized forGram stain, biochemical reactions and polyhydroxyalkanoate (PHA) production. Total fourteen strains were Gram negativeand 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 outof sixteen strains exhibited PHA producing ability. PHA production was optimized for different growth parameters, likenitrogen concentration, pH and temperature. PHA extraction was done by solvent extraction method. Bacterial strains US1and M1 accumulated up to 30% PHA of their cell dry weight on PHA extraction by solvent extraction method. Bacterialstrain US1 was identified by 16S rRNA gene analysis as P. aeruginosa (DQ455691). PHA production was confirmed byPCR amplification of 500 bp fragment from PHA polymerase (Pha C) gene; five strains from nine PHA producers gavepositive results on PCR. Pha C gene fragment of US1 was sequenced and submitted to Gene Bank under the accessionnumber DQ455690. The amino acid sequence showed homology using the protein BLAST at 129–132 sites with differentPHA 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

Synthetic polymers (known as plastics) have becomesignificant since the 1940s, and since then they are re-placing glass, wood and other constructional materials,and even metals in many industrial, domestic and en-vironmental applications (Poirier et al. 1995). Due totheir persistence in the environment, globally peoplesare now more sensitive to the impact of discarded plas-tic on the environment, including deleterious effects onwildlife and on the aesthetic qualities of cities and for-est. The increased cost of solid waste disposal as well asthe potential hazards from waste incineration such asdioxin emission from polyvinyl chloride makes syntheticplastic a waste management problem (Ojumu et al.2004). Polyhydroxyalkanoates (PHAs) are polyesters of3–hydroxyalkanoic acids produced as intracellular gran-ules by many different bacteria (Steinbuchel & Hein2001). PHAs are synthesized and accumulated intra-cellularly in most bacteria under unfavorable growthcondition, such as limitation of nitrogen, phosphorus,oxygen or magnesium in the presence of excess sup-

ply of carbon source (Du et al. 2001; Du & Yu 2002).Approximately 150 different hydroxyalkanoic acids areknown at present to occur as constituents of PHAs. Theuse of PHAs as raw material is generally believed to re-duce the environmental impact of plastic wastes mainlydue to their biodegradability (Poirier et al. 1995).PHAs produced by bacteria consist of three main

types: (i) polymers composed of short-chain-length (scl)monomers; (ii) polymers composed of medium-chain-length (mcl) monomers; and (iii) polymers composedof scl-mcl monomers. scl PHA consists of monomericsubunits 3 to 5 carbons in length, while mcl PHA con-sists of monomers 6 to 14 carbons in length and scl-mclPHA copolymer consists of monomeric subunits 4 to12 carbons in length (Ojumu et al. 2004). The majorcost of PHA production is the substrate and the sepa-ration process (Bruce et al. 1990). De Lima et al. (1999)studied different PHA-producing bacterial strains iso-lated from a sugarcane agro-ecosystem for their resis-tance to certain antibiotics. Choosing an antimicrobialwith the desirable selective toxicity is difficult and theexact 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 theglobal performance of the microorganism of interest orremain in the product altering its properties (Oliveiraet al. 2000).PHA synthase is the crucial enzyme, with hydrox-

yacyl coenzyme A as the substrate, in synthesizingPHAs. More than 59 PHA synthase genes have beencloned from 45 bacterial species and broadly catego-rized into four different classes, based on their in vivosubstrate specificities, amino acid sequences, and sub-unit composition (Rehm 2003). Class I and class IIPHA synthases comprise enzymes consisting of only onetype of subunit PhaC (Qi & Rehm 2001). The class IIPHA synthase genes PhaC1 and PhaC2 that favour mcl3HA have been identified and characterized in Pseu-domonas sp. including Pseudomonas aeruginosa (Timm& Steinbuchel 1992), Pseudomonas oleovorans (Huis-man et al. 1991), Pseudomonas sp. 61–3 (Matsusakiet al. 1998), and Pseudomonas mendocina (Hein et al.2002). The different studies showed that both of themexhibited very similar properties; only in rare cases theyshowed some differences on the contents and monomercomposition of the accumulated PHA (Qi et al. 1997;Hein et al. 2002).In this study, different contaminated environments

were screened for potential PHA producers and theirability to show resistance against selected antibioticswere monitored. Also the growth conditions of maxi-mum PHA producers were optimized as well as a frag-ment from PHA synthase gene was sequenced and an-alyzed.

Material and methods

Environments selectedThe environments selected included Punjab Universityworkshop, oil shop, engine oil, sewage water and sugar-cane molasses soil. All samples and the strains isolated werecoded as UW, UOS, EO, US and M respectively.

Isolation of bacteriaAll samples were properly diluted and spread on the nutrientagar (peptone 5 g/L, beef extract 3 g/L, agar 15 g/L). ThepH was adjusted to 7.0, incubated for 24 h at 37◦C. Gramstaining was performed for all purified strains (Cheesbrough2001). Biochemical analyses were done by performing thecatalase test, oxidase test, triple sugar iron reactions, citrateutilization test, urease test, indole test, motility test, Voges-Proskaur test, methyl red test and sugars fermentation test(Cheesbrough 2001).

Antibiotics resistancePlate dilution technique was used for determining the min-imal inhibitory concentration (MIC) to the selected antibi-otics, amoxicillin (Amx), tetracycline (Tet) and ciprofloxa-cin (Cip), on nutrient agar plates (Sambrook et al. 2001) byincubating at 37◦C for 24 h.

Screening and optimization for PHA productionAll purified strains were grown on PHA-detection agar(PDA) consisted of (g/L): (NH4)2SO4 (2), KH2PO4 (13.3),MgSO4 (1.3), citric acid (1.7), carbon source (2), trace el-ement solution 10 mL/L (g/L, FeSO4 · 7H2O 10, ZnSO4

· 7H2O 2.25, CuSO4 · 5H2O 1, MnSO4 · 5H2O 0.5, CaCl2 ·2H2O 2.0, Na2B4O7 · 10H2O 0.23, (NH4)6MO7O24 0.1, HCl10 mL); pH of the medium was adjusted to 7.0. Glucose andsucrose were used as carbon sources for screening and opti-mization experiments. PHA producers were detected macro-scopically by observing the turbidity on PDA. Producerswere further screened by Sudan black B staining (Arnold etal. 1999). Optimization of different growth parameters, i.e.the incubation at different temperature, pH, harvest timeand carbon source, were done by growing on PDA mediumin shake flask. Biomass and PHA % was calculated afterinterval of 24 h. Firstly, the time was optimized for PHAextraction. Different nitrogen concentrations, i.e. 0%, 0.1%,0.2% and 0.4%, were used, while temperature and pH wereoptimized by incubating different sets of shake flask culturesat 30◦C, 37◦C and 42◦C temperature and pH 6 and 7.

Extraction of PHAPHA extraction was done by solvent extraction methods(Arnold et al. 1999). In solvent extraction, bacterial cellswere collected by centrifugation and dry cell weight wascalculated. The chloroform was added (50 times the drycell weight) and incubated at 95◦C for 10 min. Cooled toroom temperature, and mixed solution overnight with stir-ring. The solution was filtered to remove the cell debris, fil-trate was precipitated by adding a mixture of methanol andwater (7:3 v/v) (five volumes of chloroform) to the filtrate.Filtered the precipitated PHA and weighed.

Genomic DNA isolation, amplification and sequencingAll selected strains were grown in nutrient broth for 24h and genomic DNA was isolated by Fermentas genomicDNA isolation kit. 16S rRNA gene was amplified usingPrimus96 (PeQLab) with PCR master mix, forward primer16S-3 (5’-CCCGGGAACGTATTCACCG-3’) and the re-verse primer 16S-5 (5’-GCYTAAYACATGCAAGTCGA-3’)(Cameron 2002). PCR was carried out by denaturing DNAat 110◦C for 10 min followed by 30 cycles of amplification(95◦C for 2 min, 52◦C for 1 min and 72◦C for 2 min).

Pha C polymerase gene was amplified using Primus96(PeQLab) with PCR master mix, forward primer 179–L (5’-ACAGATCAAGTTCTACATCTTCGAC-3’) and the re-verse primer 179–R (5’-GGTGTTGTCGTTCCAGTAGA-GGATGTC-3’) (Solaiman et al. 2000). PCR was carriedout by denaturing DNA at 110◦C for 10 min followed by 30cycles of amplification (95◦C for 2 min, 56◦C for 1 min and72◦C for 2 min). DNA sequencing was done in the Centre forExcellence in Molecular Biology, Lahore (Pakistan) on DNAsequencing system (Applied Biosystems, 3100/ga3100-1696-013) by Dideoxy DNA sequencing method.

Bioinformatics toolsThe sequences of 16S rRNA and PhaC gene fragment wereanalyzed by BLAST at the NCBI server (http://www.ncbi.nlm.nih.gov/BLAST/) and submitted to Gene Bank underthe accession number DQ455691 and DQ455690, respec-tively. Amino acid sequence of PhaC amplified gene frag-ment (DQ455690) was found through online ExPASy Trans-late tool (http://www.expasy.ch/tools/dna.html). The cor-rect amino acid sequence was found from 3–5 reading frame3. The protein sequences matched by BLAST were furtheranalyzed by CLUSTALW program (http://www.ebi.ac.uk/clustalw/) for phylogenetic tree formation and Genedocsoftware (http://www.nrbsc.org/gfx/genedoc/) was usedfor 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 resistanceStrain # Sudan black staining PHA %

Glu Suc Amox Tet Cip

UW1 ++ – +ve S S S –UOS1 +++ – +ve 650 50 50 14UOS2 – + –ve S S S –UOS3 +++ +++ +ve 1000 S 50 9US1 ++++ +++ +ve 650 300 S 27US2 ++++ + +ve 1000 300 50 25EO1 – – –ve S S 50 –M1 ++++ ++++ +ve 650 300 50 30M2 – – –ve 50 S S –M3 + + +ve 1000 300 S 6M4 ++++ + +ve 400 S S 16M5 + + +ve 1000 300 50 –M6 + + +ve 1000 200 S 17M7 + +++ +ve 400 S S 16M8 +++ – –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 fermentationStrain 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 EnterobacterUOS3 LF A/A/–/– –ve +ve –ve –ve +ve/–ve –ve +ve –ve +ve +ve EscherichiaUS1 NLF K/K/–/– +ve +ve +ve –ve –ve/–ve –ve –ve –ve –ve –ve PseudomonasUS2 NLF K/K/–/– –ve +ve +ve –ve –ve/–ve –ve –ve –ve –ve –ve PseudomonasM1 NLF K/K/–/+ +ve +ve +ve –ve –ve/+ve –ve –ve –ve –ve –ve PseudomonasM3 NLF K/K/–/– +ve +ve +ve –ve –ve/–ve –ve –ve –ve –ve –ve PseudomonasM4 LF A/A/–/+ –ve +ve +ve –ve –ve/–ve –ve +ve –ve +ve –ve BacillusM6 NLF K/K/–/– +ve +ve +ve –ve –ve/–ve –ve –ve –ve –ve –ve PseudomonasM9 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

Isolation, purification and characterizationPHAs are intracellular carbon and energy reserve mate-rials that are accumulated by a variety of microorgan-isms under certain unbalanced growth conditions, suchas high carbon to nitrogen ratio (Lee 1996). Samplesanalysed in the present study were collected from theUniversity of Punjab (Lahore, Pakistan) oil shop, auto-mobile workshop, sewage, engine oil and sugarcane mo-lasses soil, as these sites help in PHA accumulation in acell due to nutrition depletion conditions. Sixteen bac-terial strains were isolated form all samples. Two of theisolated strains were Gram-positive (M4 and M7) and14 were Gram-negative. On PDA media, in presence ofglucose as carbon source, nine strains, (UW1, UOS1,UOS3, US1, US2, M1, M4, M8 and M9) showed turbidcolonies which indicates PHA production (Arnold etal. 1999), four bacterial strains (M3, M5, M6 and M7)showed slightly turbid colonies, two strains (UOS2 andEO1) showed no turbidity and M2 showed no growth(Table 1). PHA granules were observed in nine strains(UOS1, UOS3, US1, US2, M1, M3, M4, M6 and M9)

in all cells, when stained with Sudan black staining.Sudan black is a lipophilic dye attached to PHA andgives purple colour to PHA granules with pink blackground (Schegel et al. 1970). In three strains (UW1,M5 and M7), granules were observed in some cells,whereas in four bacterial strains (USO2, EO1, M2 andM8) no granule was observed. On the basis of these re-sults nine bacterial strains (UOS1, UOS3, US1, US2,M1, M3, M4, M6 and M9) were selected as PHA pro-ducers.In presence of sucrose as carbon source, four strains

(UOS3, US1, M1 and M7) showed turbid colonies andgranules were observed in all cells. Seven strains (UOS2,US2, M3, M4, M5, M6 and M9) showed slightly turbidcolonies and granules in some cells were observed, threestrains (UW1, UOS1 and M2) showed no growth andtwo strains (EO1 and M8) showed non-turbid colonies(Table 1). With regard to biochemical characterizationPHA-producing strains showed resemblance to Pseu-domonas, Enterobacter, Citrobacter, Bacillus and Es-cherichia (Table 2). On the basis of the 16S rRNA genesequence, the strain US1 was identified as Pseudomonasaeruginosa (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 productionMost strains showed resistance to amoxicillin, a com-monly used antibiotic, whereas most were sensitive tociprofloxacin, a less frequent antibiotic. This may bedue to large use of these antibiotics in the community(hospital waste and sewage contamination to the envi-ronment). Exposure of an antibiotic in the environmentleads the bacterium to develop the resistance againstmultiple antibiotics. Resistance to different antibioticsis spread among bacterial population of different envi-ronments. Atlas (1984) pointed out diversity changesin response to environmental stress, showing both ten-dencies: (a) increase in diversity by selective toxicity,eliminating dominant organism; and (b) diversity de-creases by elimination of many species due to toxic-ity or increase in particular populations. The minimalinhibitory concentrations (MICs) for three antibiotics(amoxicilline, tetracycline and ciprofloxacine) were esti-mated for all the isolated strains (Table 1). Except M9,all strains showed resistance to amoxicillin, a commonlyused antibiotic. Most strains were resistant to the threetested antibiotics. All strains showed very low resistanceto ciprofloxacine (< 50 µg/mL). Seven strains (UW1,

UOS1, UOS3, EO1, M2, M4 and M7) were sensitive fortetracycline, and five strains (US1, US2, M1, M3 andM5) showed maximum MIC (300 µg/mL) for tetracy-cline. The bacterial strains showed a wide range of re-sistance against amoxicilline. Only four strains (UW1,UOS2, EO1, and M9) were sensitive for amoxicilline.Five strains (UOS1, US2, M3, M5 and M6) showed re-sistance at 1000 µg/mL amoxicilline.

Effect of nitrogen, pH and temperature on PHA pro-ductionBacterial strains US1 and M1 were selected for opti-mization experiments due to the maximum productionof PHA among all strains. At 0.0% nitrogen concentra-tion, no growth of US1 and M1 was observed. MaximumPHA production was observed at 0.2% concentration.The optimum time for PHA production was found tobe 72 h (Fig. 1). While at 0.4% concentration biomassand PHA production was less as compared to 0.1% and0.2% concentrations. At 30◦C M1 showed maximumgrowth at pH 7 but minimum at pH 6. M1 showed max-imum growth at pH 6 and 42◦C. When comparing theUS1 and M1, growth at different pH and temperatures

654 S. ur Rehman et al.

Fig. 2. Production of PHA by the strains US1 (�) and M1 ( ) at different temperatures (30◦C, 37◦C and 42◦C) and pH 6 (A) and 7(B), grown on PDA (glucose, 72 h).

Fig. 3. Amplification of PHA polymerase gene fragment of allstrains. Lanes 1 to 10: marker DNA, UOS1, UOS3, US1, US2,M1, M3, M4, M6 and M9.

showed that US1 was a faster grower than M1 at alldifferent pH and temperatures studied so far (Fig. 2).Extraction of PHA from all granule-producing

strains (UW1, UOS1, UOS3, US1, US2, M1, M3, M4,M5, M6, M7 and M9) was done by solvent extractionmethod. Chloroform and other chlorinated hydrocar-bons solubilised all PHAs. PHA extraction was doneafter 72 h and all bacterial strains produced PHA ex-cept for M7, which showed no growth in PHA detectionbroth with glucose as carbon source. Bacterial strainsM1 and US1 produce maximum PHA% of their cell dryweight (30% and 27%, respectively).The decrease in thebiomass and PHA content between 72 to 96 h of growthwas observed that is due to the depletion of nutrient andproduction of toxic materials in the medium; at thistime bacteria also start using PHA as carbon source.Du et al. (2001) reported that PHAs were synthesizedand intracellularly accumulated in most bacteria underunfavourable growth condition, such as limitation of ni-trogen, phosphorus, oxygen or magnesium in the pres-ence of excess supply of carbon source. Yuksekdag et al.(2004) studied the effect of carbon and nitrogen sourcesand incubation times on poly-β-hydroxybutyrate syn-thesis.

Ribotyping and PCR amplification of PhaC polymerasegeneBacterial strain US1 was identified by 16S rRNA gene

Fig. 4. Phylogenetic tree of DQ455690 (US1) with YP 793525(P aeruginosa UCBPP-PA14), NP 253743 (P aeruginosa PAO1),EAZ61501 (P aeruginosa 2192), EAZ55677 (P aeruginosa C3719)and YP 793527 (P aeruginosa UCBPP-PA14) prepared using theprogram CLUSTALW.

analysis as Pseudomonas sp. (Accession No. DQ455691)showing 94% homology to Pseudomonas aeruginosa.Strain US1 was isolated from a sample collected fromthe University sewage. Sewage water consists of efflu-ent from different departments, hospital and domesticwastes, which is the probable cause for 94% homol-ogy to Pseudomonas aeruginosa (AF417876) 16S rRNAgene. This strain was able to utilize glucose and sucroseas carbon source for PHA production. The PCR proto-col was developed using genomic DNA purified from 5strains (UOS1, US1, US2, M4 and M6). Results showedthat a 540 bp PCR product was obtained (Fig. 3). Thesize of the PCR product agrees with the length of thephaC gene flanked by the I-179LI-179R primer-pair.Amplified gene fragment from US1 was sequenced. Onsequencing the PCR product of PhaC from US1, it gavelength of about 445 bp. The DNA sequence has 59%GC and 41% AT content. DNA sequence was analyzedby finding proper ORF and homology to other PhaCproteins in the databases. 3’-5’ ORF 3 have no stopcodon and codes for 147 amino acids. Using the BLASTsearch it showed homology of maximum of 132 aminoacid residues (90%). The homologues sequences belongto PhaC1 (90%) and PhaC2 (87%) showing maximumhomology to PhaC1. Five out of nine PHA producersshowed positive results on amplification of gene frag-ment of PHA polymerase. The other four strains werealso PHA producers but not amplified on PCR thatmay be due to the non-complementation of their PHAsynthase to primers, or these strains may not belongto the PHA synthase class II (Timm & Steinbuchel1992; Shamala et al. 2003). Solaiman et al. (2000) usedthe PCR-based strategy to confirm the PHA produc-ers from different Pseudomonas sp. after screening bythe Sudan black staining. PCR results showed that

Characterization of polyhydroxyalkanoates-producing bacterial strains 655

Fig. 5. Conserved domain finding and homology of DQ455690 (US1, P aeruginosa) with YP 793525 (P aeruginosa UCBPP-PA14),NP 253743 (P aeruginosa PAO1), EAZ61501 (P aeruginosa 2192), EAZ55677 (P aeruginosa C3719) and YP 793527 (P aeruginosaUCBPP-PA14) prepared using the program Genedoc.

the primers used in this study, based on PseudomonasPhaC polymerase conserved sequence, also amplifiedEnterobacter and Bacillus, which indicated the con-served sequences between different genera.The top five hit sequences were then subject on

CLUSTALW alignment and phylogenetic tree forma-tion. The sequences belong to P. aeruginosa UCBPP-PA14, P. aeruginosa PAO1, P. aeruginosa 2192, P.aeruginosa C3719, and P. aeruginosa UCBPP-PA14(Fig. 4). Our putative protein showed much affiliationwith the sequences of PhaC1 as compared to PhaC2(Fig. 5). In the phylogenetic tree the putative aminoacids sequence showed a common ancestry to bothPhaC1 and PhaC2 (Fig 4). It is suggested that in bothenzymes, there are conserved regions due to similarityin their function.

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Received January 19, 2007Accepted September 10, 2007