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Biosynthesis of medium-chain-length poly(hydroxyalkanoates) from soy


Biotechnology Letters (2006) 28: 157–162 DOI 10.1007/s10529-005-5329-2

? Springer 2006

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) from soy molasses
Daniel K.Y. Solaiman1,*, Richard D. Ashby1, Arland T. Hotchkiss Jr2 & Thomas A. Foglia1
Fats, Oils and Animal Coproducts Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania, 19038, USA 2 Crop Conversion Science and Engineering Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania, 19038, USA *Author for correspondence (Fax: +1-215-233-6795; E-mail: dsolaiman@errc.ars.usda.gov)
Received 8 November 2005; Accepted 10 November 2005

Key words: biopolymer, PHA, Pseudomonas, Pseudomonas corrugata, ra?nose, short-chain galactooligosaccharides, stachyose

Abstract Pseudomonas corrugata was selected from a screening process for the bioconversion of inexpensive soy molasses into medium-chain-length poly(hydroxyalkanoates) (mcl-PHA). We obtained yields of 1.5 g cell dry weight (CDW)/l culture with growth medium supplemented with 2% (w/v) soy molasses, and of an average of 3.4 g CDW/l with 5% (w/v) soy molasses. Crude PHAs were obtained at 5–17% of CDW. The most prominent repeat-unit monomers in the PHAs were 3-hydroxydodecanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, and 3-hydroxytetradecenoate. This work represents the ?rst description of fermentative mcl-PHA production from the soy molasses. Introduction Poly(hydroxyalkanoates) (PHAs) are bacterial polyesters produced and sequestered as intracellular granules by many microorganisms. PHAs are commonly grouped into two major categories: short-chain-length (scl-) PHA in which the repeat units of the polymer are the hydroxy fatty acids (HFAs) of 4–6 carbon chain length; and medium-chain-length (mcl-) PHA where the repeat units are HFAs are >6 carbon chain length. The scl-PHAs are thermoplastic-like because of their relatively high crystallinity. On the other hand, mcl-PHAs have minimal crystallinity
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and hence are amorphous and exhibit elastomeric and adhesive properties. Because PHAs are biodegradable and biocompatible, and can be produced by fermentation of renewable feedstocks, they are thus considered as attractive ‘‘green’’ substitutes for petroleum-derived polymers in such applications as in medicine, drug-delivery agents, agriculture and horticulture, ?bers, and other consumer products (Zinn et al. 2001, Lenz & Marchessault 2005). To help lower the production cost, an active area of PHA research is the use of inexpensive renewable agricultural and industrial coproducts as feedstocks for PHA production. Various molasses streams with high carbohydrate content are generated in many industrial processes and several studies have shown that these streams can support PHA biosynthesis (Gouda et al. 2001,

158 Wu et al. 2001). Soy molasses is a coproduct stream of soybean processing. It is high in potentially fermentable carbohydrates (up to 30% w/v) and is less expensive ($ one-?fth) than the commonly used glucose substrate. Since the cost of the carbon substrate has been suggested to contribute to >50% of the production cost of bioproducts (Braunegg et al. 2004), the inexpensive soy molasses is thus an attractive feedstock to use to reduce fermentative production costs. Examples of the use of soy molasses to produce industrial chemicals or bioproducts include lactic acid synthesis with Lactobacillus salivarius (Montelongo et al. 1993), butanol production with Clostridium beijerinckii (Qureshi et al. 2001), and sophorolipid synthesis with Candida bombicola (Solaiman et al. 2004). In this paper, we describe the use of soy molasses as a potential low-cost substrate to produce mcl-PHA from Pseudomonas corrugata. bottom (3 prongs) Erlenmeyer ?asks, respectively. Soy molasses was added to the medium to a ?nal concentration of 2 or 5% (w/v) before sterilization. Inoculation was done by the addition of a 1/100-volume aliquot of an overnight (16–20 h) culture of P. corrugata 388 to the medium. Unless otherwise speci?ed, incubation was performed at 30 °C and 250 rpm shaking for 3 days. Cells were collected by centrifugation and washed once with cold distilled water. Cells were then lyophilized and weighed to obtain cell dry weight (CDW). To obtain the crude PHA, the lyophilized cells were extracted with chloroform; cell debris was removed by ?ltration through Whatman No. 1 paper, and the solvent evaporated on a rotary-evaporator. The solvent-free residue was weighed to obtain the crude PHA yields. Repeat-unit analysis of PHA Crude PHA samples were subjected to acid-catalyzed methanolysis, the liberated hydroxy fatty acid (HFA) monomers were silylated, and the compositions of these HFA monomers were analyzed as detailed in Ashby et al. (2004). HPLC analysis of sugars in soy molasses Sucrose, raf?nose, and stachyose were determined using a Dionex DX-500 HPLC system consisting of a GP50 gradient pump, a CarboPac PA1 column and guard column, an ED40 electrochemical detector (gold working electrode, pH reference electrode and the quadruple potential waveform), an LC25 chromatography oven (30 °C), a PC10 pneumatic controller (post-column addition of 500 mM NaOH), and an AS3500 autosampler. The isocratic mobile phase was 100 mM NaOH for 30 min. Individual sugars were identi?ed by comparison to standards. As shown in Figure 1, the HPLC protocol used in this study satisfactorily resolved the three major sugars in soy molasses.

Materials and methods Bacteria and culture media Pseudomonas corrugata 388, originally isolated from alfalfa roots by F.L. Lukezic (Pennsylvania State University, University Park, PA), and P. chlororaphis NRRL B-2075 were obtained from Dr. W.F. Fett (Eastern Regional Research Center/ARS/USDA, Wyndmoor, PA). P. oleovorans strains NRRL B-778, NRRL B-14682, and NRRL B-14683; and P. resinovorans NRRL B-2649 were obtained from the ARS Culture Collection (NCAUR, Peoria, IL). P. putida KT2442 was a gift of Prof. R.A. Gross (Polytechnic University, Brooklyn, NY). Bacteria were grown in E* medium (for medium composition see Brandl et al. 1988) with soy molasses as the sole carbon source. Incubation of shake-?ask cultures was carried out at 30 °C at 200–250 rpm rotary shaking. Soy molasses in the form of soy solubles (50% moisture and 30% carbohydrates) was supplied by Central Soya (Gibson City, IL).

PHA production from P. corrugata E* medium (0.5 and 1.0 L) was prepared and sterilized (by autoclaving) in 1- and 2-L beveled-

Results and discussion Several strains of PHA-producing Pseudomonas were surveyed for their ability to metabolize the

45.0 40.0 35.0

Table 1. Consumption of sucrose, ra?nose, and stachyose in soy molasses by Pseudomonas strains.

Bacterial straina

Sugar Consumptionb Sucrose Ra?nose ) ) ) ) ) ) ) Stachyose ) ) ) ) ) ) )

Response [nC]

30.0 25.0 20.0 15.0 10.0 5.0 0.0 -5.0 0.0



P. chlororaphis NRRL B-2075 P. corrugata 388 P. oleovorans NRRL B-778 P. oleovorans NRRL B-14682 P. oleovorans NRRL B-14683c P. putida KT2442 P. resinovorans NRRL B-2649

± + ) ) ) ) )







a Cells were grown in culture tubes containing 3 ml E* medium supplemented with 1 and 2% soy molasses. Incubation was performed at 30 °C and 250 rpm rotary shaking for 3 days.

Time [min]
Fig. 1. High performance anion exchange chromatography – pulsed amperometric detection analysis of soy molasses. Sucrose (A), ra?nose (B) and stachyose (C) were identi?ed by comparing retention times with standards injected separately.

Sugar consumption was monitored by HPLC peak areas. For P. chlororaphis, an $50% decrease of HPLC peak areas attributed to sucrose was observed regardless of the initial soymolasses concentration. c Variously known as strain ATCC 29347, GPo1, or TF4-1L.


sucrose and major short-chain galactooligosaccharides (i.e. ra?nose and stachyose) of soy molasses. The strains selected for the survey were P. resinovorans, P. corrugata, P. oleovorans (3 strains), P. chlororaphis, and P. putida. We previously had identi?ed these organisms as having certain unique characteristic in regard to PHA production such as metabolizing intact triacylglycerols (Ashby & Foglia 1998), growing and synthesizing mcl-PHA at moderately elevated temperatures (Solaiman et al. 2002), synthesizing scl-PHA only or a scl/mcl-PHA blend (Solaiman & Ashby 2005), or producing rhamnolipid biosurfactants (Gunther et al. 2005). In this study, their ability to metabolize sucrose (Su), ra?nose (Ra) and stachyose (St), which are the major sugars in soy molasses, was investigated in a chemically de?ned medium E* originally formulated for an improved mcl-PHA synthesis in Pseudomonas. Accordingly, cells were incubated in E* medium supplemented with 1 and 2% (w/v) soy molasses for 2 days at 30 °C and 250 rpm. At the end of incubation, cells were pelleted by centrifugation and the resultant cell-free spent

medium was collected and analyzed for sugar content by HPLC. The results of the survey showed that none of the strains metabolized Ra and St (Table 1). This is not surprising in view of the absence of a-galactosidase enzyme in Pseudomonas. To date, only one instance of the identi?cation of an a-galactosidase 27A in P. ?uorescens subsp. cellulosa has been described (Halstead et al. 2000). The fate of Su in the culture medium, however, varied with each organism (Table 1). Our results showed that P. corrugata completely consumed the Su in the medium, and P. chlororaphis reduced the Su content of the medium ($50%) with a concomitant production of an unidenti?ed metabolite (data not shown). The other Pseudomonas strains, however, did not metabolize sucrose under the experimental conditions used in this work. Although the ability of P. corrugata to metabolize sucrose is well documented (Catara et al. 1997), our results established for the ?rst time that the organism is still capable of utilizing this sugar in a complex medium environment. Sucrose metabolism by P. chlororaphis also has been described (Bergey’s

Table 2. Cell-mass productivity and PHA yield of P. corrugata grown on soy molassesa. Culture volume 500 ml 1l

Soy-soluble content (w/v) 2% 5% 2% 5%

Cell mass productivity (g CDW/l)b 1.5 3.2 1.5 3.6

Crude PHA yield (% CDW)c 17 7 6 5

The shake-?ask cultures were grown in 1- (for 500 ml culture) or 2-l (for 1 l culture) capacity Erlenmeyer ?asks having 3-pronged beveled bottom. Incubation was performed at 30 °C and 250 rpm for 3 days. b Lyophilized cell mass was expressed in g cell dry weight (CDW). c Crude PHA was extracted from lyophilized cell with chloroform and subsequently dried to an adhesive-like substance by removal of the solvent in a rotory evaporator.

Manual of Systematic Bacteriology, 1984). Our results show that as a component of the E* + soy molasses medium, the sucrose is only partially metabolized by P. chlororaphis NRRL B-2075 under the present experimental conditions. The literature lacks information on sucrose utilization by P. oleovorans. The results of this study showed that three strains from this species did not metabolize sucrose in soy molasses. We did not observe consumption of sucrose in soy molasses by P. resinovorans and P. putida as these organisms are not known to metabolize this dissaccharide (Bergey’s Manual of Systematic Bacteriology 1984, Hasanuzzaman et al. 2004). Based on the above results, we next focused on studying PHA production from soy molasses by P. corrugata. P. corrugata was cultured in 500 ml and 1l E* medium supplemented with 2% (w/v) and 5% (w/v) soy molasses. Consumption of the three major soy sugars (i.e., Su, Ra, and St), cell-mass and PHA yields, and the repeatunit composition of PHA at the end of the 3-day fermentation runs were determined. The clear spent culture medium was analyzed by HPLC to determine the consumption of soy sugars. The results showed that the sucrose was completed consumed by P. corrugata in both the 2% and 5% soy molasses cultures for both the 0.5 and 1l cultures. As found in the survey described above, ra?nose and stachyose were not consumed by the organism under the large-scale (1l) shake?ask growth conditions. We observed that cellmass yields were proportional to the amounts of soy solubles present in the medium. Table 2 shows that the amounts of cellular materials produced in the 5% soy molasses medium were 2.1– 2.4 times those obtained with 2% soy molasses.

Yields of 3.2–3.6 g CDW/l obtained with the 5% soy-molasses cultures were higher than those obtained with P. corrugata grown on E* medium supplemented with glucose (1.52 g CDW/l) or oleic acid (1.62 g CDW/l) (Solaiman et al. 2002). However, since the soy molasses-containing medium was not treated (e.g., by ?ltration or centrifugation) to remove insoluble substances that might be present in the feedstock, the apparently higher cell yields with soy molasses as substrate cannot be de?nitively ascertained. Table 2 also lists crude PHA yields of P. corrugata grown on soy molasses. Because of the low yields (5–17% CDW), we did not attempt to purify the PHAs by solvent precipitation. We previously had obtained crude PHA yields of 31 and 61%-CDW from P. corrugata grown on glucose or oleic acid, respectively (Solaiman et al. 2002). The low PHA yields obtained with P. corrugata on soy molasses re?ect the general observation that unrelated fermentation feedstocks support lower mcl-PHA production than fatty-acid substrates. As a high nitrogen content of growth medium might also a?ect total mcl-PHA accumulation (Solaiman et al. 2003), the nitrogenous substances in soy molasses (up to 5% w/v) could contribute to the observed low yields of this study. The effect of feedstock on PHA repeat-unit composition has been the subject of several investigations. Table 3 shows that 3-hydroxydecanoic acid is the most prominent repeat-unit monomer of the mcl-PHA extracted from P. corrugata grown on soy molasses. This was followed by 3-hydroxyoctanoic acid, 3-hydroxydodecanoic acid, and 3-hydroxytetradecenoic acid. This repeat-unit composition is similar to

Table 3. b-Hydroxyalkanoate repeat-units of mcl-PHA from P. corrugata grown on various feedstocks. Feedstock Composition (mol %)e C6 Soy molasses (2%)a Soy molasses (5%)a Glucose (0.5%)b Oleic acid (0.5%)c Glycerol (5%)d
a b

C8 17 20 10 42 14

C10 40 49 56 30 46

C12:0 14 21 11 10 9

C12:1 6 Tr n.d.e Tr 29

C14:0 3 Tr 2 Tr 1

C14:1 17 12 9 14 Tr

3 Tre 2 4 1

? Data were averages of values obtained from 500 ml and 1 l experiments where no signi?cant di?erence was observed for each entree From Solaiman et al. (2002). c From Solaiman et al. (2002, 2005). Values represent the averages of those obtained in the two separate studies described in these references. d From Ashby et al. (2005). The mcl-PHA was produced by P. corrugata 388 in a mixed-culture fermentation with P. oleovorans NRRL B-14682, a scl-PHA-producing organism. e The values typically have standard errors of <10% due to the crude nature of the preparations. Tr, trace amounts detected ( ? 0.5 mol %). N.d., not detected. C6 3-hydroxyhexanoate, C8 3-hydroxyoctanoate, C10 3-hydroxydecanoate, C12:0 3-hydroxydodecanoate, C12:1 3-hydroxydodecenoate, C14:0 3-hydroxytetradecanoate, C14:1 3-hydroxytetradecenoate.

that of PHA obtained from P. corrugata grown on glucose (Table 3). Glycerol also directed the production of mcl-PHA with 3-hydroxydecanoic acid as the predominant repeat-unit monomer (Table 3). These observations suggest that mclPHA produced in P. corrugata via the de novo fatty acid biosynthesis pathway contains the 10-carbon hydroxy fatty acid as its most prominent repeat-unit monomer. These results conform to the long-held observation that unrelated substrates such as glucose and gluconate yielded mcl-PHA having 3-hydroxydecanoate as the predominant monomer (see Steinbuchel 1991). In contrast, the polymer ¨ produced by P. corrugata from oleic acid via the b-oxidation pathway contains 3-hydroxyoctanoate as the predominant monomer, followed closely by 3-hydroxydecanoate (Table 3).

inexpensive soy molasses to the value-added mcl-PHA.

Acknowledgement The authors thank Nicole Crocker, Marshall Reed and Bun-Hong Lai for their technical assistance.

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Summary We have demonstrated in this study that soy molasses can be used as a feedstock for the fermentative production of mcl-PHA using P. corrugata as the producing strain. The repeatunit composition of the PHA polymer was determined and compared to those obtained from other feedstocks. Further study to manipulate the fermentation conditions and to genetically engineer the producing strain could result in a high-yield production system for converting the

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