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Advances in Microbiology, 2012, 2, 241-251

http://dx.doi.org/10.4236/aim.2012.23029 Published Online September 2012 (http://www.SciRP.org/journal/aim)

Polyhydroxyalkanoate Production by Pseudomonas putida
KT217 on a Condensed Corn Solubles Based Medium Fed
with Glycerol Water or Sunflower Soapstock
Jeremy Javers1, William R. Gibbons1, Chinnadurai Karunanithy2*
1

Biology and Microbiology Department, South Dakota State University, Brookings, USA
Agricultural and Biosystems Engineering Department, South Dakota State University, Brookings, USA
Email: *karunanithy.chinnadu@sdstate.edu

2

Received February 16, 2012; revised April 30, 2012; accepted July 4, 2012

ABSTRACT
Pseudomonas putida KT217 was grown on a complex medium comprised of co-products of the ethanol and biodiesel
industries to assess the organism’s capability to produce medium-chain-length polyhydroxyalkanoate (mcl-PHA). The
growth phase was carried out in a medium containing 400 g/L condensed corn solubles (CCS), supplemented with ammonium hydroxide as a nitrogen source. Following the exponential phase, co-products of the biodiesel industry (soapstock and glycerin) were fed into the reactor to trigger PHA production. When glycerin was added to the bioreactor (75
g/L total addition), the final cell dry weight (CDW) and PHA content were 30 g/L and 31%, respectively. The monomeric composition in the PHA formed was relatively uniform throughout incubation with 3-hydroxydecanoate dominating. When a total of 153 g/L of sunflower soapstock was added to the bioreactor in a fed-batch manner, the final
CDW and PHA content were 17 g/L and 17%, respectively. Following addition of soapstock the monomeric composition of the polymer changed dramatically, with the 3-hydroxyoctanoate monomer becoming dominant and greater unsaturation present in the PHA.
Keywords: Polyhydroxyalkanoate; Pseudomonas putida; Condensed Corn Solubles; Glycerol; Soapstock

1. Introduction
Polyhydroxyalkanoates (PHAs) are a class of biodegradable polymers that have a wide range of physical
properties depending upon the monomeric composition
in the polymer [1-4]. For the past 30 years extensive
PHA research has been conducted. With recent breakthroughs in PHA production technology and soaring oil
prices, PHA and other biopolymers may be at the front of
true commercial integration [5]. Biopolymers such as 1 3 propanediol, polylactic acid, starch based polymers,
and PHA could capture as much as 1.5% - 4.8% of the
total plastics market (~260 million tonnes/year). Production of PHA from renewable biomass is providing the
“green” alternative to the pollution resulting from use on
non-degradable plastics.
The costs of producing and recovering biodegradable
polymers have been the key barrier to the marketplace.
Numerous methods have been attempted to reduce the
cost of extracting and purifying the polymer [6-13].
Similarly, several low cost substrates have been evaluated for PHA production, but low productivities typically
*

Corresponding author.

Copyright © 2012 SciRes.

result [14-30]. Solaiman et al. [31] reviewed the use of
vegetable oils, animal fats, dairy whey, molasses, and
meat and bone meal as feedstocks for PHA production.
The ethanol and biodiesel production industries also
generate large quantities of under-utilized byproducts
which could be used for PHA production.
According to Renewable Fuels Association [32], the
US ethanol production was 13507.9 million gallons from
204 plants and expected to increase by 522 million gallons on completion of 10 more plants under construction.
Each gallon of ethanol accompanies with 5% - 7% of
condensed solubles. The ethanol production co-product
condensed corn solubles (CCS) is normally used in livestock feeds. CCS contains carbon and energy sources
such as monosaccharides, oligosaccharides, organic acids,
and glycerol, as well as a range of micronutrients and
macronutrients such as zinc, iron, manganese, magnesium, sulfur, phosphate and nitrogen. Typically CCS is
deficient in nitrogen, but due to its otherwise rich nutritional composition, CCS has been successfully used to
grow a range of bacteria and fungi [33-35].
The co-products of biodiesel production include fattyacids, soapstock, and glycerol water. Soapstock is ob-

AiM

5 g/L. fungi.apicola to synthesize surfactants (glycolipids).7). [42. while short term storage was on tryptic soy agar (TSA) slants. The fermentation of glycerol by E. Long term storage was via lyophilization.2.Trials were performed in an aerated benchtop reactor at the 2. and carotenoids pigments [36-39].13. respectively).242 J. The combination of co-products from the ethanol and biodiesel industries could be used to create a medium to Copyright © 2012 SciRes. [41] isolated a bacterium from manure compost (tentatively identified as a Sphingobacterium and designated strain NRRL B-14797) that grew on crude soybean soapstock and a soapstock extract.1 billion gallons. This is most likely due to removal of some nutrients following purification of the fatty acids. Our previous research revealed that Pseudomonas putida KT217 grew well on a medium comprised of CCS. Fed batch addition of soapstock gave the most favorable results. A one percent inoculum of a 24 h culture was used to inoculate aerated fed-batch bioreactor trials. Bacterial Strain. oleic acid 25%. Trials were conducted aerobically in a fed-batch mode using pH control and dissolved oxygen (DO) was monitored. The medium (~2.43] grew Pseudomonas aeruginosa LB1 aerobically on a defined liquid salts medium with 2. Sunflower soapstock contains linoleic acid 50%. [44] fed soapstock or post-refinery fatty acids to Candida antarctica and C. Subcultures were also transferred to shake flasks containing a CCS based medium (described below) at pH 7. Du-Pont created a process to produce 1 . In the glycerol trials. fed-batch additions of sterilize glycerol water (obtain from West Central Soy in Ralston. JAVERS ET tained from alkali refining of oilseeds and is composed of phosphorus lipids.3 . Experimental Design The basal CCS medium was prepared by mixing 1.3 kg of glycerol is equivalent to 10%. Glycerol is a primarybyproduct of biodiesel production. Each gallon of biodiesel accompanies with 0. supplemented with ammonium as a nitrogen source [50].48 h at 30˚C and 250 rpm.2 kg of CCS from a dry grind ethanol plant with deionized water to a volume of 3 L.6 . following inoculation the culture was incubated for 96 h at 30˚C. Benincasa et al. Flickinger and Perlman [46] converted glycerol to dihydroxyacetone with a Gluconobacter strain. The culture was routinely transferred in tryptic soy broth shake flasks incubated for 24 . Soapstock is normally added back to oilseed meal for livestock feeding or is disposed of with little or no economic compensation [36]. Production of PHA from glycerol water has also been tested [21]. Soapstock resulted in better glycolipid production than fatty acids (7. Materials and Methods 2. USA)and autoclaved. According to National Biodiesel Board [45]. and Inoculum Preparation The bacterium used was P. biodiesel production in the US has reached 1. CT.4 g/L vs 6. support growth and PHA accumulation. The medium pH was adjusted to 7. Hesseltine and Koritala [40] evaluated a 2% soybean soapstock medium for growth of 141 microorganisms.3 propanediol that channels glucose through glycerol [47].3 with 30% ammonium hydroxide (after autoclaving the medium pH typically dropped to 6. For pH control a saturated solution of sodium hydroxide and a 20% (v/v) solution of sulfuric acid were used. Soapstock has been evaluated as a carbon and energy source to produce value added products via fermentation. 2.35 L scale with soybean biodiesel-derived glycerin or sunflower-derived soapstock added fed-batch after 24 h to bolster PHA levels.5% sunflower soapstock to produce rhamnolipids. acetate. bacteria. vitamins A and E. They found that soapstock extracts were poor nutrients for growth. soaps of free fatty acids (FFA). In the soapstock trials the agitation and aeration had to be monitored to control foaming along with the antifoam. In the study reported herein we used this basal medium to test the effects of using a fed-batch feeding strategy with either glycerol water or sunflower derived soapstock to bolster PHA production. AL. succinate. and actinomycetes. including microbial processes. putida KT217. and hydrogen was found to be pH dependant [49]. After the initial 24 h growth phase. Maintenance. Enfield. For example. with aeration at 1 V/V/min and agitation at 500 rpm. The resulting mixture was centrifuged to remove suspended solids. palmitic acid 7%. Clerol FBA 3107 (Cognis) was used as an antifoam agent in these trials as needed.10. which was similar to the results observed in Pseudomonas aeruginosa LBI [41].1. but were utilized better for hydroxy acid bioconversions than either crude soapstock or pure long chain fatty acids. including yeast. hydrateable and non-saponifiable compounds. lactate. IA) or sunflower soapstock (obtained from AiM . with 16 g/L of product after 54 h. and stearic acid 4%. Later on Kaneshiro et al. Bednarski et al. and this has triggered research to develop alternative uses.35 liters each) was then dispensed into a 5 L Bioflo III bioreactor (New Brunswick Scientific. coli to end products such as ethanol. The liquid portion was then filtered through Whatman 113 filter paper to remove most of the remaining suspended solids and oils. and produced 10(R)-hydroxystearic acid. 2.11. It is possible for this product to be created directly from glycerol by microbial conversion via Clostridium strains [48]. A non-disclosed Pseudomonas species grew at pH 8 .

5:7. Nitrogen and Phosphorus Analysis Samples were assayed for nitrogen and phosphorus using Hach test kits and a Hach DR/2010 colorimeter (Hach. Phosphorus was measured as free phosphates using of the Hach HCT 122 Unicel kit. PHA Extraction Standards are not readily available for each of the monomers present in the mcl-PHA produced by P. 2. followed by an ethanol modified step to extract PHA. The digests of cell samples were then analyzed and the percentage of PHA contained within determined.5 % v/v) at 100˚C for 4 h. USA). PHA was dissolved in hot chloroform and precipitated by addition of ten volumes of cold methanol. Prior to analysis samples were filtered through 0. PHA Analysis The created standards were analyzed on Thermo-Finnigan Trace GC/MS 2000.2 µm filters. then washing and re-centrifuging the cells. using benzoic acid as an internal standard [54-56].6 mL/min. Colony forming units (CFU) were determined by serial dilution and plating in triplicate on TSA.7. The mobile phase was helium-degassed 4 mM sulfuric acid at 0. Soapstock was fed in response to dissolved oxygen levels rising above 10% following 24 h of cultivation. 0. Samples were filtered through 0. CA. propionic acid. lactic acid.5 µm df). 2. PHA Hydrolysis and Derivitization Samples of lyophilized cells or purified PHA (10 .J. Loveland.3.50 mL samples. Hercules. and glycerol in the culture samples.60 mg) were digested in chloroform:methanol:sulfuric acid (50: 42.0. acetic acid. Glycerol water was fed based upon HPLC data. The total areas of the monomers present were correlated to the amount of PHA contained in three different levels of standard and normalized to the methyl ester of benzoic acid as an internal standard to create a calibration curve (R2 = 0. Cell Dry Weight and Colony Forming Unit Measurement Cell dry weights (CDW) were obtained by harvesting 30 . In the first step. 2. Therefore.25 mmID. then diluted 1:100 with double distilled water. neat supercritical carbon dioxide was used to extract nonPHA lipids. PHA Analysis by NMR Spectroscopy Samples obtained from SFE were dissolved in deuterated AiM . Nitrogen was measured as ammonia using the Hach HCT 102 Unicel kit. Cell pellets were then lyophilized and homogenized prior to PHA extraction. however the viscosity of soapstock was too high to deliver directly via syringe. centrifuging at 4500 rpm at 10˚C for 30 min. Milford. To overcome this.10. The FID detector was subsequently utilized for quantitative analysis of the PHA present in the samples. Following centrifugation at 4500 rpm to remove excess ethanol.5. In total 240 mL of glycerol water was added between 24 and 70 h. 243 2. 30 meter. PHA was utilized from culture samples to create quantitative standards. organic acids.9. then 50 µl injections were made. through a Biorad HPX-87H (Biorad. to maintain glycerol levels between 5 and 35 g/L during incubation. This procedure creates methyl esters of the 3-hydroxy acids present in the PHA. The column utilized was an RTx-5Sil MS (Restek. JAVERS ET Cargill) were delivered through the septum port on the fermentor with a sterile disposable 60 mL syringe.6. 40 g of Sunflower soapstock was diluted with deionized water to 150 mL total volume and autoclaved prior to feeding. AL. succinic acid and glycerol were used for calibration.97 .4.8. Following digestion the mixture was washed with distilled water to remove excess sulfuric acid and methanol from the chloroform phase. For determination of the fractional analysis of PHA monomers. HPLC Analysis A Waters HPLC system (Waters Scientific. MA. the area of the individual monomers was divided by the total area of the monomers. 2.99). Lyophilized cell samples from the final culture sample of the soapstock fed trial were extracted with a two-step supercritical fluid extraction [53].50 mL samples and centrifuging at 4000 rpm for 30 min. 2. [51] and Kim [52]. This procedure of dissolving in chloroform and precipitation in cold methanol was repeated three times to purify the PHA.2 µm filters. PHA Analysis PHA concentrations were determined by first removing 30 . glucose. CO. following the methods describe by Foster et al. re-centrifuged. 2. 0. and dried to a constant weight in a 60˚C oven. Cell pellets were washed with 30 mL deionized water. USA) organic acid analysis column operated at 65˚C. These standards were prepared by hydrolyzing and derivatizing the methylesters of the respective monomers included in the PHA. The MS detector was utilized first for qualitative analysis of the monomers present in the samples. Copyright © 2012 SciRes. 2. A portion of the purified PHA was dissolved in chloroform and used as a stock solution to prepare quantitative standards. putida KT217. USA) with refractive index detector was used to quantify sugars. with 1350 mL (360 g) of soapstock solution added between 24 and 90 h. Standard solutions of maltose. Glycerol water could be added as is.

the first of four glycerol additions was made (Figure 1).E+09 20 1. These additions maintained glycerol levels between 8 . glycerol cdw AL. putida KT217 on a CCS medium with fed-batch glycerol addition. 3. lactic (4. cdw. For example. Madison. USA) at a probe temperature of 27˚C. Growth on a CCS Medium with Fed-Batch Addition of Glycerol Water P. Copyright © 2012 SciRes..5 g/L/h at the end of fermentation. At 28 h. 34.g.E+10 30 25 1. and 62 h. Only after these carbon sources were depleted (at approximately 12 h) did P. 48. and perhaps the increased salt concentration from the glycerol water. putida begin metabolizing glycerol.1 g/L). the utilization rate was maintained between 1 2 g/L/h. Viable cell counts increase more rapidly than CDW due to the lower mass of individual cells during exponential growth.0 g/L). and Figure 2 shows the glycerol utilization rate. and propionic acids (1. ammonia phosphate PHA%CDW cfu/ml 1. However. acetic (1. as cells filled with PHA. is shown in Figure 1. Growth and PHA production of P. the latter of which had accumulated to ~2%.E+12 45 40 1. PHA (g/L) ammonia. The CDW included both cell mass and PHA. Results and Discussion 3. the glycerol utilization rate peaked at 2. The 400 g/L CCS formulation had previously been identified as being optimal for rapid production of cell mass [50]. This CDW yield was slightly higher than theoretical and may be attributed to utilization of other medium components in CCS that were not detected by HPLC (e.1. amino acids).4 g/L at 28 h. putida KT217 was grown in an aerated bioreactor on a 400 g/L CCS-based medium supplemented with 2.E+06 100 Time (h) Figure 1. JAVERS ET 244 chloroform and 13C-NMR spectra were recorded on a Bruker 400 MHz GRX NMR spectrometer (Bruker AXS. During the subsequent additions of glycerol. The CDW was produced from ~42 g of carbon.E+07 5 0 0 10 20 30 40 50 60 70 80 90 1. P. after the stationary phase was reached.35 g/L.3 g/L initially to 8. for a CDW yield of ~0. posphate (dg/L) 35 10 1. in a phenol red broth medium with glucose as the carbon source P. Average of two fermentations with error bars representing standard deviation.E+08 15 viable cell population (cfu/ml) glycerol. which corresponded to the plateau in cell population (6. which fell from 39. putida was able to use proteins and oils in CCS as carbon sources. putida KT217 was found to catalyze deamination of media proteins. Inc. CDW increased as cells filled with PHA.4e10 CFU/mL) and CDW (~23 g/L).9 g/L). (Figure 1). This lower rate was likely due to several factors including limitations of nitrogen and oxygen.E+11 1. oligosaccharides.2 g/L ammonium hydroxide. approaching 0.55 g CDW/g carbon utilized. in which glycerol water was added at 29.0 g/L) within 20 h (data not shown). succinic (2. putida KT217 first utilized the glucose (2. The average growth curve from two replications.87 g/L/h. It is possible that P.J.6 g/L). AiM . The glycerol utilization rate continued to decline. WI. At 28 h. Of the nutrients initially present in the CCS medium. Ammonia was exhausted by approximately 28 h.

This represents 31% of the total CDW. putida KT217 grown on a CCS medium with fed-batch glycerol addition. putida KT217 could utilize the components in soapstock as a carbon and energy source during aerated shake flask trials in a defined medium (data not shown). The foaming problem in these trials was consistent with previous reports [42. Ammonia depletion occurred at 32 h. at 6 . the average glycerol utilization rate was 1. These conclusions are supported by 13C-NMR data shown in (Figure 4). and propionic acids) followed the same pattern as observed in Figure 1. When glycerol fell to 10 g/L (~28 h) sunflower soapstock additions were initiated. Viable cell counts plateaued at 15 h. This caused oxygen saturation to drop to near zero. JAVERS ET AL. 3-hydroxytetradecanoate. Since the viable cell population remained constant during this time period.5 2 1. This is greater than the total increase in cell mass following limitation and may indicate that there was significant turnover of cell mass in the medium during this time resulting in a cell mass with varying PHA content. corresponds to a CDW yield of 0. Therefore when the dissolved oxygen saturation rose above 10%. oxygen saturation gradually increased.11 g/g glycerol (CDW is sum of PHA and cell mass). Because soapstock was viscous.5 3 2. [60] previously reported that dissolved oxygen levels can be used to monitor carbon levels.5 0 0 20 80 100 time (h) Figure 2.4 . Lee et al. putida KT217 was grown on 400 g/L CCS-based meCopyright © 2012 SciRes. Between 15 and 35 h. Additionally. Soapstock also increased foaming in the aerated AiM .44e10 CFU/mL by 40 h. glycerol utilization began. and 3-hydroxyhexadecanoate were all detectable. This corresponded to an increase in CDW from ~11 to 24 g/L over the same period. Glycerol consumption continued until depletion at 39 h. 150 mL of culture broth was removed prior to the diluted soapstock addition. which corresponded to the peak CDW level.6e10 CFU/mL to 9.75 g/L/h. Carbon sources initially utilized for growth (glucose.2. 3hydroxyoctanoate.glycerol utilization rate (g/L/hr) J. Glycerol utilization rate for P. but in very small concentrations (Figure 3). Polymer composition was dominated throughout by 3-hydroxydecanoate which consistently represented more than 60% of the polymer.2. and as it was consumed. The overall concentration of 3-hydroxydodecanoate and 3-hydroxydodecenoate in the polymer decreased as the incubation proceeded. After 28 h. soapstock additions were made.58. since it is a complex of several lipids.43]. whereas the amount of 3-hydroxyhexanoate. and when these were depleted (~20 h).10 h intervals through 86 h.5 1 0. which was comparable to the average rate observed (1.9 g/L/h) in the previous trials in the same time frame. and 3-hydroxydecanoate all increased (Figure 3). The various processes resulting in the utilization of carbon source for maintenance energy have been reviewed [57]. 3. This lower yield indicates that the majority of the glycerol was being used for maintenance energy in the culture. The increase in CDW of 7 g/L from use of ~65 g/L of glycerol. 40 g of soapstock was blended with water to a volume of 150 mL prior to each addition. The composition of the PHA produced by P. Growth on a CCS Medium with Fed-Batch Addition of Sunflower Soapstock P. the increase of 7 g/L in CDW was primarily due to the accumulation of PHA. Chemical shifts are similar to previously reported data for PHA analyzed in this method [15. an increase of PHA of 8 g/L was measured by gas chromatography.35 L. acetic. However. but rose from 4. succinic.59]. To compensate for this added volume. dium supplemented with ammonia as a nitrogen source (Figure 5). 40 60 245 3. It was not possible to monitor the level of soapstock. lactic. theCDW continued to increase to around 30 g/L at 85 h. We had previously observed that P. The concentration of 3-hydroxytetradecenoate. Following glycerol depletion in the medium the CDW decreased until 80 hours. A total of 360 g of sunflower soapstock was added in a total volume of 1. putida KT217 on a CCS medium fed with glycerol water changed with time as evidenced by gas chromatographic analysis (GC-MS and GC-FID).

80% 70% 60% percent of PHA 50% C6% C8% C10% C12-1% C12% C14-1% C14% C16% 40% 30% 20% 10% 0% -10% 0 10 20 30 40 50 60 70 80 90 100 time (h) Figure 3. putida KT217 on a syrup medium fed with glycerol water. ppm 200 175 150 125 100 75 50 25 0 13 Figure 4. Time profile of monomeric composition determined by gas chromatography in PHA derived from fed-batch fermentation of P. Copyright © 2012 SciRes.J. C-NMR of the PHA extracted from the final sample of the glycerol water fed batch fermentation. AiM . JAVERS ET 246 AL.

eventually rising to 7 g/L. posphate (dg/L) 35 10 1.J.hydroxytetradecanoate.E+08 viable cell population (cfu/ml) glycerol. PHA accumulated at a slow rate throughout incubation.E+06 100 time (h) Figure 5. bioreactor.E+12 45 40 1. reaching a concentration of 2.E+11 1. explaining the increase in PHA. thus diverting some acetyl-CoA to acetic acid. cdw. 13C-NMR analysis of the final PHA produced during the incubation supported the information derived from GC-MS and GC-FID analysis showing an increase in unsaturation in the polymer (Figure 7). This build up in acetyl-CoA may have activated the fatty acid synthesis pathway as well. Growth and PHA production of P. the composition favored 3-hydroxydecanoate monomers due to the utilization of glycerol in the CCS.150 ppm which has been shown previously [15. 4. PHA (g/L) ammonia. the broth removed prior to soapstock additions contained cells at least partially filled with PHA.E+10 30 25 1. coupled with the decreased aeration rate and hence reduced acetic acid being excreted from the cells. polymer composition shifted towards 3-hydroxyoctanoate monomers. CDW decreased. compared to the more dilute soapstock (50% FFA) [37]. Conclusions PHA production was observed over 100 h of aerated incubation when P. Following soapstock addition. The composition of the PHA Copyright © 2012 SciRes. JAVERS ET glycerol cdw acetic acid AL.E+07 5 0 0 10 20 30 40 50 60 70 80 90 1. It is likely that the Krebs cycle could not process acetyl-CoA at the same rate it was being produced. representing 17% of the total CDW. Arrows indicate addition of 40 grams of sunflower soapstock. putida KT217 was grown on a basal medium of 400 g/L CCS (wet basis) and 2. changed dramatically during incubation (Figure 6). This was likely due to fatty acid degradation into acetyl-CoA. even with antifoam addition. putida KT217 on a CCS medium with fed-batch sunflower soapstock addition. Acetic acid levels began to accumulate after 60 h. Therefore we reduced the aeration rate from 1 vvm to 0.5 vvm. and these were replaced by newly formed cells that didn’t contain as much PHA. After soapstock additions began.8 g/L at the end. ammonia 247 phosphate PHA cfu/ml 1.2 g/L ammonium hydroxide. Foaming was a problem during soapstock trials. This did not occur in the prior glycerol fed-batch process because only 240 mL of the concentrated (85%) glycerol water was added.55].E+09 20 15 1. even though viable cell populations remained steady. The final PHA contained 3-hydroxytetradecanoate and 3. This is evident by examining the peaks in the chemical shift range of 120 . Evidently. Initially. but was of less concern when using glycerol AiM . supplemented in fed-batch mode with either biodiesel co-products glycerol water (240 g) or soapstock (390 g). Average of two fermentations with error bars representing standard deviation.

Copyright © 2012 SciRes. ppm 200 175 150 125 100 75 50 25 0 13 Figure 7. C-NMR of the PHA extracted from the final sample of the sunflower soapstock fed batch fermentation. JAVERS ET 248 AL. 80% 70% percent of PHA 60% 50% C6% C8% C10% C12-1% C12% C14-1% C14% C16% 40% 30% 20% 10% 0% 0 10 20 30 40 50 60 70 80 90 100 time (h) Figure 6. putida KT217 on a syrup medium fed with sunflower soapstock. Time profile of monomeric composition determined by gas chromatography in PHA derived from fed-batch fermentation of P. AiM .J.

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