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Yolanda Gonza´lez-Garcı´a1 Mario A. Rosales2 Orfil Gonza´lez-Reynoso2 ˜ as1 Rube´n Sanjua´n-Duen Jesu´s Co´rdova2
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Research Article
Polyhydroxybutyrate production by Saccharophagus degradans using raw starch as carbon source
1
Department of Wood, Cellulose and Paper, CUCEI, University of Guadalajara, carretera Guadalajara-Nogales. Zapopan, Jalisco, Mexico
2
Department of Chemical Engineering, CUCEI, University of Guadalajara, Blvd. Marcelino GarciaBarragan, Guadalajara, Jalisco, Mexico
Biosynthesis of poly(3-hydroxybutyrate) (PHB) from raw starch as the carbon source by the polysaccharide-digesting bacteria Saccharophagus degradans was investigated in a fed-batch culture. The production and properties of the PHB synthesized from starch were compared to those obtained using glucose as carbon source. In fed-batch cultures, S. degradans accumulated 21.35 and 17.46% of PHB, using glucose or starch as carbon source, respectively. The physical properties of the biopolymer produced from each carbon source were similar between them. Molecular mass, melting temperature and heat of fusion were 54.23 kDa, 165.611C and 59.59 J/g, respectively, using glucose; and 57.07 kDa, 174.311C and 67.66 J/g, respectively, using starch. This is the first work describing the capability of S. degradans to utilize raw starch as the sole carbon source for the production of PHB. Keywords: Biopolymers / Polyhydroxybutyrate / PHB / Saccharophagus / Starch Received: June 28, 2010; revised: September 1, 2010; accepted: October 1, 2010 DOI: 10.1002/elsc.201000118
1
Introduction
The polyhydroxyalkanoates (PHAs) are biopolyesters synthesized as carbon and energy reserves by a wide variety of microorganisms usually when cultured under nutrient unbalanced conditions in the presence of an available carbon source [1, 2]. The most widespread PHA is the polyhydroxybutyrate (PHB), which is composed of monomeric units of 3-hydroxybutyric acid. The PHB is fully biodegradable and has similar physical properties to that of polypropylene [3] and thus can be used for the manufacture of packing materials, disposable items and specialized devices for medical applications [4]. However, there are still economical limitations in replacing conventional plastics with PHAs. In fact, 40–50% of the total PHAs production cost is related to the raw material used as the substrate [5]. In order to achieve a cost-effective process, the use of bacterial strains able to utilize inexpensive carbon sources has become a focus of particular interest [6]. Therefore, in order to reduce manufacturing costs of PHAs production, agro-industrial residues or other waste materials (food scraps, wastewaters and wastewater sludge) are being considered as cheap substrates [7–9]. Nevertheless, prior to be used as a convenient carbon source for most of the known Correspondence: Dr. Yolanda Gonza´lez-Garcı´a (
[email protected]), Department of Wood, Cellulose and Paper, CUCEI, University of Guadalajara, Km. 15.5 carretera Guadalajara-Nogales. 45020 Zapopan, Jalisco, Mexico Abbreviations: PHA, polyhydroxyalkanoate; PHB, poly(3-hydroxybutyrate)
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PHAs-producing strains, nowadays the majority of the complex carbohydrates need a treatment such as hydrolysis [10–13]. This pretreatment step implies an extra cost and can cause environmental problems whether chemicals are used in the hydrolysis process. Since it is inexpensive and widely available, starch can be used as the carbon source for the production of PHAs. PHB production based on hydrolyzed starch has been explored using Ralstonia eutropha NCIMB 11599 [14], Halomonas boliviensis [15], Alcaligenes eutrophus [12], Bacillus sphaericus NCIM 5149 [16], recombinant Escherichia coli (containing the PHB synthase genes from Cupriavidus necator) [17], and Rhodobacter sphaeroides [18]. Interestingly, industrial starch wastewater was also used to produce PHB by cultivating fastgrowing rhizobia [19]. Regarding the use of non-hydrolyzed starch as the carbon source, the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Caldimonas taiwanensis from food-grade starches made from cassava, corn, potato, sweet potato and wheat, supplemented with valerate was investigated [20]. Few studies have been reported with regard to the production of PHA from raw starch, such as the use of corn of g potato starch to produce PHB by Bacillus cereus [21], Azotobacter chroococcum [22, 23], Haloferax mediterranii [24] and Aeromonas sp. [25]. Hence, strains degrading directly polysaccharides are of particular interest. Saccharophagus degradans was reported to consume untreated complex carbohydrates such as agar, alginate, xylan, cellulose and starch as the sole carbon and energy sources for growth [26]. Recently, the S. degradans capability
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for synthesizing PHB from glucose was investigated [27]; however, its ability to convert polysaccharides into PHB is still unknown. Thus, the aim of this study was to investigate the synthesis of PHB by S. degradans, utilizing raw potato starch as the carbon source. Likewise, the produced biopolyester was physically and chemically characterized and compared with that produced using glucose as the carbon source.
2
Materials and methods
2.1
Microorganism and culture medium
S. degradans was bought from ATCC (43961) and activated in Difco marine broth by incubating at 301C for 24 h. Glycerol (15% v/v) was added to this culture, distributed in 2 mL microtubes and stored at 201C. The culture medium to promote bacterial growth was composed of (g/L): glucose or starch (raw, non-hydrolyzed), 20; NH4Cl, 5.5; yeast extract, 2.5; KH2PO4, 3.5; Na2SO4, 1.0; MgCl2, 1.5; NaCl, 23; KCl, 0.8; CaCl2, 0.35; trace element solution, 1 mL. The trace element solution contained (mg/L): H3BO3, 137; SrCl2 6H2O, 814; KI, 50; NiSO4, 13; ZnSO4 7H2O, 9; MnSO4 H2O, 2; CoSO4 7H2O, 0.3; CuSO4 5H2O, 0.3; FeSO4 7H2O, 8.4; EDTA, 8.5.
2.2
Inoculum
The inoculum was aseptically prepared by adding the cells contained in a stock microtube (2 mL) to 100 mL of culture medium in a 500 mL Erlenmeyer flask and incubating at 301C and 200 rpm for 24 h.
2.3
Fed-batch cultures of S. degradans for PHB production
Fed-batch cultures of S. degradans were performed for PHB production, using a 3 L bioreactor BIOFLO 3000 (New Brunswick Scientific) with control of pH (7.5) and temperature (301C). Dissolved oxygen was maintained over 50% of air saturation by aeration at 1 VVM and agitation at 400 rpm. Inoculum (100 mL) was added to 1.4 L of the above medium. A batch culture with no nutrimental limitations was initially established to promote the bacterial growth. After depletion of the initial carbon source, a sterile concentrated solution of glucose (200 g/L) or starch (100 g/L) was fed at a constant flow of 5 mL/h during 20 h for glucose, or 8.33 mL/h during 24 h for starch. Samples (2 mL) were centrifuged at 4000 rpm for 15 min and glucose, starch and ammonium were analyzed from the supernatant.
2.4
Quantification of carbon source, ammonium, biomass and PHB
Glucose concentration was quantified by the dinitrosalicylic acid method [28].
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To estimate the starch concentration, it was previously hydrolyzed as follows: 0.5 mL of 7.5 M HCl was added to 1 mL of sample and boiled in water bath for 20 min [23]. After cooling, 1 mL of 5 M NaOH was added and the glucose concentration was determined by the dinitrosalicylic acid method. Ammonium was quantified using the phenol–hypochlorite reaction as follows: 10 mL of water, 50 mL of 3 mM MnSO4 H2O, 0.6 mL of 1.25% (v/v) sodium hypochlorite solution (at pH 6.8) and 0.6 mL of the phenate reagent (2.5 g of NaOH and 10 g of phenol in 100 mL of water) were added to 10 mL of a sample properly diluted and the mixture was shaken in a vortex. After 10 min of reaction, absorbance (at 630 nm) was determined using a spectrophotometer Genesys 10. It was used a calibration curve of ammonium chloride as the standard (from 0.01 to 0.5 mg/L). Biomass was estimated by determining the cell protein content. The cell pellet from centrifuged samples was washed twice with water and resuspended in 3 mL of 0.3 M phosphoric acid. Samples were boiled in a water bath for 10 min and centrifuged at 4000 rpm for 15 min. The protein in the supernatant was determined by the Bradford method, using bovine serum albumin as the standard protein [29]. A standard curve was established by correlating dry cell weight and cell protein content. To quantify the cell content of PHB, 2 mL of sample were centrifuged at 4000 rpm for 15 min. The cell pellet was washed twice with distilled water and lyophilized (Labconco). The PHB was determined by GC analysis [30], using a Perkin & Elmer XL gas chromatograph equipped with a CP-Wax 52 CB capillary column (25 m 0.32 mm) and a flame ionization detector. The chromatographic conditions were: injection volume of sample, 1 mL; gas carrier, nitrogen; flow rate, 20 cm/s; injector and detector temperatures, 210 and 2201C; temperature ramp, 501C for 1 min, incrementing by 81C/min, and keeping at 1601C for 5 min. Methyl benzoate and polyhydroxybutyrate from Fluka were used as internal and external standards, respectively.
2.5 2.5.1
Characterization of the PHB produced by S. degradans Polymer extraction
PHB was extracted from cells by mixing 3–5 g of lyophilized biomass with 50 mL of chloroform/hypochlorite (1:1, v/v), stirred during 5 h and then centrifuged at 8000 rpm for 10 min [31]. The chloroform phase was recovered with a pipette and poured into 100 mL cold methanol while stirring. The precipitated PHB was recovered from the methanol by centrifugation at 8000 rpm for 15 min, and air dried.
2.5.2
Molecular mass determination
The polymer molecular mass was determined by gel permeation chromatography using a Waters HPLC 600, equipped with two serially connected Styragel columns (HR1 and HT 6E, 7.8 300 mm) and an RI detector 2410. The chromatographic
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conditions were: injection volume of sample (1 mg/mL), 50 mL; temperature, 401C; mobile phase, toluene; flow rate, 1 mL/ min. Polystyrenes with molecular masses ranging from 0.4 to 650 kDa were used as standards.
2.5.3
Monomeric composition
The polymer monomeric composition was determined by GCMS, using a Varian Saturn 3800 gas chromatograph coupled to a Varian Saturn 2000 Mass spectrometer, equipped with an FFAP 25Mx capillary column (25 m 0.32 mm). The chromatographic conditions were: injection volume of sample, 1 mL; carrier gas, helium; flow rate, 1 mL/min; temperature of injector and detector, 230 and 2751C, respectively; temperature ramp, 801C for 1 min, incrementing by 81C/min, and 2201C for 12 min.
2.5.4
Thermal properties
The glass transition temperature (Tg), the melting point (Tm) and the heat of fusion (DHm) of the polymers were measured by using a differential scanning calorimeter (Perkin-Elmer, model DSC7). The PHA samples were heated at a rate of 101C/min from 4 to 2001C.
3
Results
Fed-batch cultures were performed using either glucose or raw starch, in order to compare the fermentative performances of S. degradans and the characteristics of the synthesized polymers using both carbon sources. Further details of the kinetics of growth, PHB production, as well as carbon source and ammonium consumption by S. degradans are described in Fig. 1. The production of PHB started during the exponential growth phase of the batch step, nevertheless, the amount of polymer accumulated at the end of this stage was low (0.58 g/L at 20 h for glucose and 0.53 g/L at 24 h for starch). At these culture times, residual concentrations of carbon source and ammonium were respectively, 0.61 and 0.78 g/L, for glucose fermentation and 0.83 and 0.93 g/L for starch fermentation. The feeding of carbon source solution allowed to maintain glucose or starch in a low concentration along the culture (around 0.5 and 1.5 g/L, respectively), avoiding substrate accumulation and providing evidence that the feeding rate was well coupled with the uptake rate. After starting the feeding of carbon source, the production of PHB increased exponentially until the end of the fed-batch step, reaching 2.71 g/L (21.3% of dry cell weight) for glucose and 2.04 g/L (17.42% of the dry cell weight) for starch. The final amount of products formed and substrate consumed, as well as the kinetic parameters of the S. degradans fed-batch culture from each carbon source are shown in Tables 1 and 2 respectively. The values obtained for both substrates were quite similar, except for PHB production, PHB productivity and PHB accumulation percentage, which were slightly higher when using glucose than raw starch. It is worth to note that acetic acid was produced either from glucose or starch.
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Figure 1. Kinetics of growth and PHB production of S. degradans cultured on fed batch using glucose (A) or raw starch (B) as the carbon source. J – glucose or starch, m – ammonium, & – biomass, & – PHB. Biomass represents the dry cell weight not considering the PHB weight. Data represent the mean and standard deviation of two assays from two independent cultures.
Table 1. Comparison between amount of products formed, and substrate consumed at the end of the fermentations, using glucose or starch as the carbon source Variable Biomass (g) PHB (g) Carbon source consumed (g) Acetic acid (g) Volume (L) Time (h)
Glucose
Starch
20.0171.06 4.3970.86 69.0672.76 3.3570.77 1.65 40
19.4771.06 3.4671.06 67.0471.06 4.2771.06 1.75 48
Data represent the mean and standard deviation of two assays from two independent cultures.
The monomeric composition of the biopolyesters produced by S. degradans either from glucose or starch as carbon source was 3-hydroxybutyric acid. The molecular mass and thermal properties of the polymer produced from glucose were similar to those that are produced from starch (Table 3).
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Table 2. Kinetic parameters of S. degradans cultured on glucose or raw starch as the carbon source for the production of PHB Parameter
1
m (h ) YX/S (g/g) YP/S (g/g) PHB accumulation (% DWC) PHB productivity (g/l h)
Glucose
Starch
Culture step
Culture step
Batch
Fed batch
Batch
Fed batch
0.1870.05 0.3670.11 0.03170.002 7.1872.70 0.02970.003
0.01770.001 0.2870.12 0.1770.02 21.3572.42 0.1170.01
0.1670.04 0.3570.09 0.02870.003 7.1272.34 0.02270.002
0.01970.008 0.26370.10 0.1470.02 17.4672.73 0.0670.01
m 5 Growth rate, YX/S 5 Yield of biomass on substrate, YP/S 5 Yield of PHB on substrate. Data represent the mean and standard deviation of two assays from two independent cultures.
Table 3. Thermal properties and molecular mass of the PHB produced by S. degradans using glucose or raw starch as the carbon source Parameter
Glucose
Starch
Glass transition temperature (Tg) (1C) Melting temperature (Tm) (1C) Molecular mass (MW) (kDa) Heat of fusion (DHm) (J/g)
37.9576.01 40.6874.42 165.6175.50 174.3178.18 54.2376.23 57.07714.23 59.5972.20 67.6673.80
Data represent the mean and standard deviation of two assays.
4
Discussion
The goal of this work was to prove the conversion of raw starch into PHAs by S. degradans. Additionally, the production and characteristics of PHA from starch were compared to those produced from glucose as carbon source. Since the synthesis of PHA in most bacteria is triggered by nutrimental unbalanced growth, this condition was provoked by feeding a concentrated solution of carbon source (glucose or starch) to the culture. Fed-batch cultures were started in a batch modus to stimulate bacterial growth (m 5 0.18 and 0.16 h 1 for glucose and starch, respectively). Although this step was nutritionally balanced, the production of PHB occurred slightly. This fact indicated that the PHB synthesis route is permanently active in S. degradans, which is a common feature of others PHB producer strains [32]. Once glucose or starch was depleted in the batch cultures (20 and 24 h, respectively), the feeding of carbon source started and the polymer accumulation in S. degradans increased remarkably, suggesting that the carbon flux was better oriented toward the PHB production under unbalanced nutrimental conditions as occurs in most of the PHA producing strains. Biomass (PHB content not considered) was synthesized during the fed-batch culture; this fact indicated that the reached nutrimental condition was insufficiently unbalanced to stop the bacterial growth. Related to this, residual ammonium was detected during the fed batch (Fig. 1). It is worth to notice that only a few microorganisms (bacteria and archaea) are able to use directly raw starch to produce PHAs. For instance, A. chroococcum, H. mediterranii, and B. cereus accumulated 46, 60 and 48% of PHB, respectively [4, 2, 24] when cultivated on starch and valerate [20].
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Although, the PHB accumulated in S. degradans (21.35 and 17.46%, respectively, for glucose and starch fed-batch cultures) was low compared to other producer strains, this aspect is quite common in wild strains under not optimal culture conditions [33]. Part of the carbon source could be used for cellular maintenance purposes. In fact, maintenance coefficients for microorganisms growing under nutrient limited conditions are higher [34]. Another reason for the low PHB accumulation in S. degradans might be the deviation of the carbon flux toward the synthesis of organic acids and exopolymers. It is important to note that acetic acid was produced at similar amounts as PHB (Table 1). Furthermore, during the fed-batch culture of S. degradans, it was observed the increase in the medium viscosity due to the production of an exopolymer. The production of exopolymers in marine bacteria, such as S. degradans, is a common feature since these compounds confer to the cells the ability of adhering to surfaces, which enhance their survival in aquatic environments [35]. The simultaneous production of exopolymers and PHB has been reported in some bacterial strains [36, 37]. This finding in the fed-batch culture of S. degradans is outstanding since bacterial exopolymers have interesting properties for industrial applications as gelling agent, emulsifier, texture enhancer, heavy metal remover and as a source of specific monosaccharides [38]. Work is ongoing to study the type and characteristics of the exopolymer produced by S. degradans and the culture conditions influencing its production. Particularly, studies are being oriented to establish the conditions that trigger the carbon flux toward the synthesis of PHB or exopolymer in S. degradans. Owing to the increase in the culture medium viscosity, the oxygen transference became restricted and the desirable dissolved oxygen concentration (50% of air saturation) was not possible to be supplied to the system. For this reason, fedbatch cultures had to be stopped at 20 and 24 h after the feeding was initiated, for glucose and starch, respectively (at the higher level of PHB production). In order to increase the accumulation of PHB in this strain it would be necessary to optimize the culture conditions and evaluate the effect of other variables such as limitation by different nutrients. Furthermore, it could be also possible to apply genetic engineering strategy such as random mutagenesis, or gene dosage for increasing the copy number of phb genes, which had presented good results regarding PHB
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accumulation in other microorganisms [39]. As mentioned earlier in S. degradans cultures there were produced other compounds besides PHB. Thus, another strategy to direct the carbon flux toward PHB synthesis could be to shut down the pathways leading to the production of organic acids and exopolymeric substances. Nevertheless, it must be first necessary to perform an extensive study of this strain in order to evaluate its metabolic capabilities, and use metabolic engineering tools in order to elucidate the best strategy to increase the PHB biosynthesis from starch. Molecular masses and thermal properties of PHB obtained by culturing S. degradans with glucose or starch were similar, suggesting that the carbon source did not have influence on these characteristics (Table 3), and confirming that the PHA molecular mass is an inherent characteristic for each given strain [2]. Thus, the PHB molecular mass of S. degradans (56 kDa) was similar to that produced by some Pseudomonas or Methylobacterium strains, whose values range from 50 to 300 kDa and it was low compared to Azotobacter and Ralstonia strains which accumulate PHAs whose molecular masses range from 600 to 2000 kDa [2]. It is worth noting that the PHA molecular mass is also influenced by the extraction technique employed; indeed, neutral solvents yield higher values than alkaline hypochlorite treatment [31]. Since the second technique was used in this study, the extraction procedure could have affected the molecular mass. Values of melting temperatures and heats of fusion were in the range of those reported for PHB produced by other marine bacteria, 162.3–177.01C and 32.4–65.8 J/g, respectively [40] (Table 3).
5
Concluding remarks
S. degradans is able to utilize raw starch as the sole carbon source for the production of PHB. This is an interesting feature considering that starch could be a renewable, cheap and widely available carbon source. Nevertheless, the polymer content within the cell, reached under the conditions utilized in this work, is low in comparison with other bacteria, thus it would be important to optimize the fermentation process or implement a metabolic engineering strategy in order to increase the production of PHB. During the culture of this strain it was observed the production of exopolymeric substances, which could represent a new field of study in this microorganism. Yolanda Gonzalez-Garcia and Mario A. Rosales acknowledge the grants received from CONACYT. The authors have declared no conflict of interest.
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