Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 222-224

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 222-224

Biosynthesis Pathway Related Production of Medium Chain Length Polyhydroxyalkanoates

LIN Huilan , SHI Wenhui , WU Qiong ,  CHEN Jinchun , XU Jun †, CHEN Guoqiang

Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;
†Institute of Polymer Science and Engineering, Department of Chemical  Engineering, Tsinghua University, Beijing 100084, China

Received: 2000-08-31; revised: 2001-02-17

Code Number: ts01070

Abstract:   

Pseudomonas flava HBE06 isolated from oil-contaminated soil was found to produce polyesters consisting of medium chain length polyhydroxyalkanoates (mcl PHA). The strain mainly synthesized PHA containing 3-hydroxyoctanoate (C₈ or HO), 3-hydroxynonanoate (C₉ or HN), 3-hydroxydecanoate (C₁₀  or HD), and 3-hydroxyunidecanoate (C₁₁  or HUD) as monomers when grown on various substrates. It was found that the monomer ratio (especially C₁₀ /C₈ or C₁₁ /C₉) was related to the PHA biosynthesis pathway. When PHA was synthesized via the de novo fatty acid pathway, the molar ratio of C₁₀ /C₈was greater than 2. If PHA was synthesized from b-oxidation of fatty acids, C₁₀ /C₈was less than 1. Pseudomonas stutzeri 1317 is another mcl PHA synthesizing bacteria whose C₁₀ /C₈ ratio is also related to the synthesis pathway. When the two synthesis pathways were used together, the C₁₀ /C₈ ratio could be adjusted according to the ratio of the mixed substrates.

Key  words: polyhydroxyalkanoates (PHA); Pseudomonas flava; Pseudomonas stutzeri

Introduction   

Polyhydroxyalkanoates, abbreviated as PHA, is a family of intracellular biopolymers synthesized by many bacteria as carbon and energy storage compounds[1,2] . Attempts have been made to produce PHA for biodegradable packaging[3-5].    

Various bacterial strains have different PHA biosynthesis pathways. Gern et al, studying the biosynthesis pathway of Pseudomonas putidausing   ¹³ C nuclear magnetic resonance, concluded that the strain synthesized PHA via the de novo fatty acid synthesis pathway when acetate was the sole carbon source, while b-oxidation of fatty acids occurred as the PHA synthesis pathway when decanoate or octanoate was used[6].

This paper reports on the production of medium chain length PHA by Pseudomonas flava HBE06 and Pseudomonas stutzeri 1317 isolated from oil-contaminated soil collected from an oilfield in Northern China. The relationship between PHA monomer composition and PHA synthesis pathways is discussed. This is the first time the PHA synthesis pathway of P. flava has been studied in China.

1 Materials and Methods   

1.1 Strains and cultivation conditions

Strains HBE06 and 1317 isolated from oil-contaminated soil near the Huabei Oilfield were identified by the Institute of Microbiology, Academica Sinica in Beijing, as a strain of Pseudomonas flava and a strain of Pseudomonas stutzeri, respectively.

The strains were grown in a mineral medium (pH 7) containing (gDK·L ): (NH ₄) 2SO₄ 0.5, MgSO₄ 0.4, Na₂HPO₄ 3.8, and KH₂PO₄ 2.65, supplemented with 1 mL ·L^-1  of trace element solution containing (g) CoCl₂ 0.218, FeCl₃ 9.7, CaCl₂ 7.8, NiCl₃ 0.118, CrCl₆ ·H₂O 0.105, and CuSO₄ ·5H₂O 0.156 in 1 L of 0.1 mol ·L^-1  HCl. Glucose, lauric acid, tridecanoate were used as carbon sources in the studies.

The strains were grown aerobically at 30 °C for 24 h. 150 mL of the culture was incubated in a 500-mL conical flask shaken at 200 r/min for 48 hours on a rotary shaker (NBS, Series 25 D, New Brunswick, USA). The cells were harvested by centrifugation (5000 g) for 10 min.

1.2 Analytical methods

The polyesters in the dried cells were methyl esterified in the mixture (1 : 1, volume ratio) consisting of chloroform and methanol with 3% concentrated sulfuric acid[7]. The lower chloroform solution was collected for gas chromatographic analysis (Beifen, SQ-204, Beijing, China). Various 3-HA methyl esters purchased from Sigma Chemical Company were used as standards to determine the polymer composition both quantitatively and qualitatively.

2 Results and Discussion   

Glucose can be utilized by many bacteria for PHA synthesis via the de novo fatty acid synthesis pathway[6, 8, 9]. A similar phenomenon was observed in Pseudomonas flava HBE06. The strain showed significant growth and PHA accumulation in media with various glucose concentrations. The PHA contents ranged from 2.1%-52.1% of the cell dry weight. The PHA monomers included b-hydroxyhexanoate (C₆ or HHx), C₈, C₁₀ and C₁₂ . C₈and C₁₀  were the most abundant, accounting for 90% (molar percentage) of the PHA composition (data not shown).  When the glucose concentration was more than 20 g ·L^-1 , the PHA content remained around 40%-50% of the cell dry weight (Fig.1). The C₁₀/C₈ratio remained almost constant at around 2.8.

Similar  results were obtained when gluconate, acetate or succinate was used as the sole carbon source (data not shown). These substrates were utilized to generate acetyl-CoA for the de novo PHA synthesis.

Lauric acid can be b-oxidized to synthesize PHA via a fatty acid b-oxidation pathway. When lauric acid was used as a sole carbon source, the PHA content and the C₁₀/C₈ratio remained almost constant despite variation of the lauric acid concentration (Fig.2). The C₁₀/C₈ratio was constant at around  0.6 . Similar results were obtained when tetradecanoate or palmitoate was used as the sole carbon source (data not shown).

The results indicated that the PHA content and C₁₀/C₈ratio were not affected by the glucose or lauric acid concentration. However, the PHA metabolic pathway affected the C₁₀/C₈ratio. Although the two C₁₀/C₈ ratio for PHA synthesized from glucose and lauric acid remained constant, the PHA compositions from glucose and lauric acid were significantly different. For PHA from glucose, C₁₀/C₈ was about 2.8, while for PHA from lauric acid, C₁₀/C₈was approximately 0.67. This difference was attributed to their different synthesis pathways. Consequently, the C₁₀ /C₈ratio might be used as an indicator of the PHA synthesis pathway when various carbon sources are used.

This hypothesis was also verified when an odd-carbon-number fatty acid tridecanoate was supplied as the substrate for cell growth. The odd-carbon-number PHA monomers, C₇, C₉ and C₁₁  would be synthesized from b-oxidation of tridecanoate, while the even-carbon-number PHA monomers of C₈and C₁₀ could come only from the de novo fatty acid synthesis pathway. When Pseudomonas flava HBE06 was cultivated on tridecanoate, the PHA monomers included C₆, C₇, C₈, C₉, C₁₀ and C ₁₁ , with C₉ and C₁₁  as the most abundant monomers in the PHA. The C₁₀  and C₈contents in the PHA were similar to those synthesized from glucose, demonstrating the simultaneous existence of the de novo fatty acid synthesis and b-oxidation pathways. 

Pseudomonas stutzeri 1317 was also reported to posses these two synthesis pathways[8, 9] , synthesizing mcl PHA both via the de novo fatty acid synthesis pathway and the b-oxidation pathway when both glucose and lauric acid were simultaneously supplied as substrates in the medium. The two pathways in P. stutzeri 1317 produced a variation of the C₁₀/C₈ratio over a short range (Table 1).

In  summary, Pseudomonas flava HBE06 preferred to synthesize PHA with C₈, C₉, C₁₀, and C₁₁  as the major monomers. When the synthesis is based on the de novo fatty acid synthesis pathway, the C₁₀/C₈ratio is greater than 2. When the synthesis is based on b-oxidation, the C₁₀/C₈ ratio is less than 1. Therefore, the monomer composition can be adjusted using both pathways.

When lauric acid and glucose were supplied together to cultivate Pseudomonas stutzeri 1317, various C₁₀/C₈ratios ranging from  0.36  to  3.49  were observed. This result further confirmed that ratios of the major monomers of mcl PHA are related to the biosynthesis pathway and can be adjusted by using two pathways.

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