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*
1Biology and Microbiology Department, South Dakota State University, Brookings, USA
2Agricultural and Biosystems Engineering Department, South Dakota State University, Brookings, USA
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 am-
monium hydroxide as a nitrogen source. Following the exponential phase, co-products of the biodiesel industry (soap-
stock 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 mono-
meric composition in the PHA formed was relatively uniform throughout incubation with 3-hydroxydecanoate domi-
nating. 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 composi-
tion of the polymer changed dramatically, with the 3-hydroxyoctanoate monomer becoming dominant and greater un-
saturation present in the PHA.
Keywords: Polyhydroxyalkanoate; Pseudomonas putida; Condensed Corn Solubles; Glycerol; Soap stock
1. Introduction
Polyhydroxyalkanoates (PHAs) are a class of biode-
gradable 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 break-
throughs 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). Produc-
tion 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 evalu-
ated for PHA production, but low p roductivities typically
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 gal-
lons 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 live-
stock 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, magne-
sium, sulfur, phosphate and nitrogen. Typically CCS is
deficient in nitrogen, but due to its otherwise rich nutri-
tional composition, CCS has been successfully used to
grow a range of bacteria and fungi [33-35].
The co-products of biodiesel production include fatty-
acids, soapstock, and glycerol water. Soapstock is ob-
*Corresponding a uthor.
C
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J. JAVERS ET AL. 242
tained from alkali refining of oilseeds and is composed of
phosphorus lipids, hydrateable and non-saponifiable
compounds, soaps of free fatty acids (FFA), vitamins A
and E, and carotenoids pigments [36-39]. Soapstock is
normally added back to oilseed meal for livestock feed-
ing or is disposed of with little or no economic compen-
sation [36].
Soapstock has been evaluated as a carbon and energy
source to produce value added products via fermentation.
Hesseltine and Koritala [40] evaluated a 2% soybean
soapstock medium for growth of 141 microorganisms,
including yeast, fungi, bacteria, and actinomycetes. A
non-disclosed Pseudomonas species grew at pH 8 - 11.
Later on Kaneshiro et al. [41] isolated a bacterium from
manure compost (tentatively identified as a Sphingobac-
terium and designated strain NRRL B-14797) that grew
on crude soybean soapstock and a soapstock extract, and
produced 10(R)-hydroxystearic acid. They found that
soapstock extracts were poor nutrients for growth, but
were utilized better for hydroxy acid bioconversions than
either crude soapstock or pure long chain fatty acids.
This is most likely due to removal of some nutrients fol-
lowing purification of the fatty acids. Benincasa et al.
[42,43] grew Pseudomonas aeruginosa LB1 aerobically
on a defined liquid salts medium with 2.5% sunflower
soapstock to produce rhamnolipids. Fed batch addition of
soapstock gave the most favorable results, with 16 g/L of
product after 54 h. Sunflower soapstock contains linoleic
acid 50%, oleic acid 25%, palmitic acid 7%, and stearic
acid 4%. Bednarski et al. [44] fed soapstock or post-re-
finery fatty acids to Candida antarctica and C.apicola to
synthesize surfactants (glycolipids). Soapstock resulted
in better glycolipid production than fatty acid s (7.3 - 13.4
g/L vs 6.6 - 10.5 g/L, respectively), which was similar to
the results observed in Pseudomonas aeruginosa LBI
[41].
According to National Biodiesel Board [45], biodiesel
production in the US has reached 1.1 billion gallons.
Glycerol is a primarybyproduct of biodiesel production.
Each gallon of biodiesel accompanies with 0.3 kg of
glycerol is equivalent to 10%, and this has triggered re-
search to develop alternative uses, including microbial
processes. For example, Flickinger and Perlman [46]
converted glycerol to dihydroxyacetone with a Glucono-
bacter strain. Du-Pont created a process to produce 1 - 3
propanediol that channels glucose through glycerol [47].
It is possible for this product to be created directly from
glycerol by microbial conversion via Clostridium strains
[48]. The fermentation of glycerol by E. coli to end
products such as ethanol, succinate, acetate, lactate, and
hydrogen w as f oun d to b e pH de pend ant [49]. Prod uctio n
of PHA from glycerol water has also been tested [21].
The combination of co-products from the ethanol and
biodiesel industries could be used to create a medium to
support growth and PHA accumulation. Our previous
research revealed that Pseudomonas putida KT217 grew
well on a medium comprised of CCS, supplemented with
ammonium as a nitrogen source [50]. In the study re-
ported herein we used this basal medium to test the ef-
fects of using a fed-batch feeding strategy with either
glycerol water or sunflower derived soapstock to bolster
PHA production.Trials were performed in an aerated
benchtop reactor at the 2.35 L scale with soybean bio-
diesel-derived glycerin or sunflower-derived soapstock
added fed-batch after 24 h to bolster PHA levels.
2. Materials and Methods
2.1. Bacterial Strain, Maintenance, and
Inoculum Preparation
The bacterium used was P. putida KT217. Long term
storage was via lyophilization, while short term storage
was on tryptic soy agar (TSA) slants. The culture was
routinely transferred in tryptic soy broth shake flasks
incubated for 24 - 48 h at 30˚C and 250 rpm. Subcultures
were also transferred to shake flasks containing a CCS
based medium (described below) at pH 7. A one percent
inoculum of a 24 h culture was used to inoculate aerated
fed-batch bio re act or tria l s.
2.2. Experimental Design
The basal CCS medium was prepared by mixing 1.2 kg
of CCS from a dry grind ethanol plant with deionized
water to a volume of 3 L. The medium pH was adjusted
to 7.3 with 30% ammonium hydroxide (after autoclaving
the medium pH typically dropped to 6.7). The resulting
mixture was centrifuged to remove suspended solids. The
liquid portion was then filtered through Whatman 113
filter paper to remove most of the remaining suspended
solids and oils. The medium (~2.35 liters each) was then
dispensed into a 5 L Bioflo III bioreactor (New Bruns-
wick Scientific, Enfield, CT, USA)and autoclaved.
Trials were conducted aerobically in a fed-b atch mode
using pH control and dissolved oxygen (DO) was moni-
tored. For pH control a saturated solution of sodium hy-
droxide and a 20% (v/v) solution of sulfuric acid were
used.
In the glycerol trials, following inoculation the culture
was incubated for 96 h at 30˚C, with aeration at 1
V/V/min and agitation at 500 rpm. Clerol FBA 3107
(Cognis) was used as an antifoam agent in these trials as
needed. In the soapstock trials the agitation and aeration
had to be monitored to control foaming along with the
antifoam.
After the initial 24 h growth ph ase, fed-batch add itions
of sterilize glycerol water (obtain from West Central Soy
in Ralston, IA) or sunflower soapstock (obtained from

J. JAVERS ET AL. 243
Cargill) were delivered through the septum port on the
fermentor with a sterile disposable 60 mL syringe. Glyc-
erol water could be added as is, however the viscosity of
soapstock was too high to deliver directly via syringe.
Therefore, 40 g of Sunflower soapstock was diluted with
deionized water to 150 mL total volume and autoclaved
prior to feeding. Glycerol water was fed based upon
HPLC data, to maintain glycerol levels between 5 and 3 5
g/L during incubation. In total 240 mL of glycerol water
was added between 24 and 70 h. Soapstock was fed in
response to dissolved oxygen levels rising above 10%
following 24 h of cultivation, with 1350 mL (360 g) of
soapstock solution added between 24 and 90 h.
2.3. Cell Dry Weight and Colony Forming Unit
Measurement
Cell dry weights (CDW) were obtained by harvesting 30
- 50 mL samples and centrifuging at 4000 rpm for 30
min. Cell pellets were washed with 30 mL deionized
water, re-centrifuged, and dried to a constant weight in a
60˚C oven. Colony forming units (CFU) were deter-
mined by serial dilution and plating in triplicate on TSA.
2.4. HPLC Analysis
A Waters HPLC system (Waters Scientific, Milford, MA,
USA) with refractive index detector was used to quantify
sugars, organic acids, and glycerol in the culture samples.
Prior to analysis samples were filtered through 0.2 µm
filters, then 50 µl injections were made. The mobile
phase was helium-degassed 4 mM sulfuric acid at 0.6
mL/min, through a Biorad HPX-87H (Biorad, Hercules,
CA, USA) organic acid analysis column operated at 65˚C.
Standard solutions of maltose, glucose, lactic acid, acetic
acid, propionic acid, succinic acid and glycerol were
used for calibration.
2.5. Nitrogen and Phosphorus Analysis
Samples were assayed for nitrogen and phosphorus using
Hach test kits and a Hach DR/2010 colorimeter (Hach,
Loveland, CO, USA). Samples were filtered through 0.2
µm filters, then diluted 1:100 with double distilled water.
Nitrogen was measured as ammonia using the Hach HCT
102 Unicel kit. Phosphorus was measured as free phos-
phates using of the Hach HCT 122 Unicel kit.
2.6. PHA Analysis
PHA concentrations were determined by first removing
30 - 50 mL samples, centrifuging at 4500 rpm at 10˚C for
30 min, then washing and re-centrifuging the cells. Cell
pellets were then lyophilized and homogenized prior to
PHA extraction.
2.7. PHA Extraction
Standards are not readily available for each of the
monomers present in the mcl-PHA produced by P. putida
KT217. To overcome this, PHA was utilized from cul-
ture samples to create quantitative standards, following
the methods describe by Foster et al. [51] and Kim [52].
Lyophilized cell samples from the final cultur e sample of
the soapstock fed trial were extracted with a two-step
supercritical fluid extraction [53]. In the first step, neat
supercritical carbon dioxide was used to extract non-
PHA lipids, followed by an ethanol modified step to ex-
tract PHA. Following centrifugation at 4500 rpm to re-
move excess ethanol, PHA was dissolved in hot chloro-
form and precipitated by addition of ten volumes of cold
methanol. This procedure of dissolving in chloroform
and precipitation in cold methanol was repeated three
times to purify the PHA. A portion of the purified PHA
was dissolved in chloroform and used as a stock solution
to prepare quantitative standards. These standards were
prepared by hydrolyzing and derivatizing the methyl-
esters of the respective monomers included in the PHA.
2.8. PHA Hydrolysis and Derivitization
Samples of lyophilized cells or pu rified PHA (10 - 60 mg)
were digested in chloroform:methanol:sulfuric acid (50:
42.5:7.5 % v/v) at 100˚C for 4 h, using benzoic acid as an
internal standard [54-56]. Following digestion the mix-
ture was washed with distilled water to remove excess
sulfuric acid and methanol from the chloroform phase.
This procedure creates methyl esters of the 3-hydroxy
acids present in the PHA.
2.9. PHA Analysis
The created standards were analyzed on Thermo-Finni-
gan Trace GC/MS 2000. The column utilized was an
RTx-5Sil MS (Restek, 30 meter, 0.25 mmID, 0.5 µm df).
The MS detector was utilized first for q ualitativ e analysis
of the monomers present in the samples. The FID detec-
tor was subsequently utilized for quantitative analysis of
the PHA present in the samples. The total areas of the
monomers present were correlated to the amount of PHA
contained in three different levels of standard and nor-
malized to the methyl ester o f benzoic acid as an internal
standard to create a calibration curve (R2 = 0.97 - 0.99).
The digests of cell samples were then analyzed and the
percentage of PHA contained within determined. For
determination of the fractional analysis of PHA mono-
mers, the area of the individual monomers was divided
by the total area of the monomers.
2.10. PHA Analysis by NMR Spectroscopy
Samples obtained from SFE were dissolved in deuterated

J. JAVERS ET AL.
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244
chloroform and 13C-NMR spectra were recorded on a
Bruker 400 MHz GRX NMR spectrometer (Bruker AXS,
Inc, Madison, WI, USA) at a probe temperature of 27˚C.
The CDW was produced from ~42 g of carbon, for a
CDW yield of ~0.55 g CDW/g carbon utilized. 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.g., oligosac-
charides, amino acids). For example, in a phenol red
broth medium with glucose as the carbon source P.
putida KT217 was found to catalyze deamination of me-
dia proteins. It is possible that P. putida was able to use
proteins and oils in CCS as carbon sources. Viable cell
counts increase more rapidly than CDW due to the lower
mass of individual cells during exponential growth.
However, after the stationary phase was reached, CDW
increased as cells filled with PHA.
3. Results and Discussion
3.1. Growth on a CCS Medium with Fed-Batch
Addition of Glycerol Water
P. putida KT217 was grown in an aerated bioreactor on a
400 g/L CCS-based medium supplemented with 2.2 g/L
ammonium hydroxide. The 400 g/L CCS formulation
had previously been identified as being optimal for rapid
production of cell mass [50]. The average growth curve
from two replications, in which glycerol water was added
at 29, 34, 48, and 62 h, is shown in Figure 1. At 28 h, the first of four glycerol additions was made
(Figure 1). These additions maintained glycerol levels
between 8 - 35 g/L, and Figure 2 shows the glycerol
utilization rate. At 28 h, the glycerol utilization rate
peaked at 2.87 g/L/h. During the subsequent additions of
glycerol, the utilization rate was maintained between 1 -
2 g/L/h. This lower rate was likely due to several factors
including limitations of nitrogen and oxygen, and per-
haps the increased salt concentration from the glycerol
water. The glycerol utilization rate continued to decline,
approaching 0.5 g/L/h at the end of fermentation, as cells
filled with PHA.
Of the nutrients initially p resen t in th e CCS medium, P.
putida KT217 first utilized the glucose (2.1 g/L), suc-
cinic (2.9 g/L), lactic (4.6 g/L), acetic (1.0 g/L), and
propionic acids (1.0 g/L) within 20 h (data not shown).
Only after these carbon sources were depleted (at ap-
proximately 12 h) did P. putida begin metabolizing glyc-
erol, which fell from 39.3 g/L initially to 8.4 g/L at 28 h.
(Figure 1). Ammonia was exhausted by approximately
28 h, which corresponded to the plateau in cell popula-
tion (6.4e10 CFU/mL) and CDW (~23 g/L). The CDW
included both cell mass and PHA, the latter of wh ich had
accumulated to ~2%.
glycerol
0
5
10
15
20
25
30
35
40
45
0 102030405060708090100
Time (h)
gly cer o l, c d w, PHA ( g /L )
ammonia, posphate (dg/L)
1.E+06
1.E+07
1.E+08
1.E+09
1.E+10
1.E+11
1.E+12
viable c ell pop ula t ion ( cfu/ml)
cdw ammonia phosphate PHA%CDW cfu/ml
Figure 1. Growth and PHA production of P. putida KT217 on a CCS medium with fed-batch glycerol addition. Average of
two fermentations with error bars representing standard deviation.

J. JAVERS ET AL. 245
0
0. 5
1
1. 5
2
2. 5
3
3. 5
0 2040608
time (h )
glycerol utilization rate (g/L/hr)
0100
Figure 2. Glycerol utilization rate for P. putida KT217 grown on a CCS medium with fed-batch glycerol addition.
After 28 h, theCDW continued to increase to around
30 g/L at 85 h. Since the viable cell popu lation remained
constant during this time period, the increase of 7 g/L in
CDW was primarily due to the accumulation of PHA.
The increase in CDW of 7 g/L from use of ~65 g/L of
glycerol, corresponds to a CDW yield of 0.11 g/g glyc-
erol (CDW is sum of PHA and cell mass). This lower
yield indicates that the majority of the glycerol was being
used for maintenance energy in the culture. The various
processes resulting in th e utilization of carbon source for
maintenance energy have been reviewed [57]. Addition-
ally, an increase of PHA of 8 g/L was measured by gas
chromatography. This represents 31% of the total CDW.
This is greater than the total increase in cell mass fol-
lowing limitation and may indicate that there was sig-
nificant turnover of cell mass in the medium during this
time resulting in a cell mass with varying PHA content.
The composition of the PHA produced by P. putida
KT217 on a CCS medium fed with glycerol water
changed with time as evidenced by gas chromatographic
analysis (GC-MS and GC-FID). The overall concentra-
tion of 3-hydroxydodecanoate and 3-hydroxydodece-
noate in the polymer decreased as the incubation pro-
ceeded, whereas the amount of 3-hydroxyhexanoate, 3-
hydroxyoctanoate, and 3-hydroxydecanoate all increased
(Figure 3). Polymer composition was dominated through-
out by 3-hydroxydecanoate which consistently repre-
sented more than 60% of the polymer. The concentration
of 3-hydroxytetradecenoate, 3-hydroxytetradecanoate, and
3-hydroxyhexadecanoate were all detectable, but in very
small concentrations (Figure 3). These conclusions are
supported by 13C-NMR data shown in (Figure 4). Ch-
emical shifts are similar to previously reported data for
PHA analyzed in this method [15,58,59].
3.2. Growth on a CCS Medium with Fed-Batch
Addition of Sunflower Soapstock
P. putida KT217 was grown on 400 g/L CCS-based me-
dium supplemented with ammonia as a nitrogen source
(Figure 5). Carbon sources initially utilized for growth
(glucose, lactic, succinic, acetic, and propionic acids)
followed the same pattern as observed in Figure 1, and
when these were depleted (~20 h), glycerol utilization
began. Viable cell counts plateaued at 15 h, but rose from
4.6e10 CFU/mL to 9.44e10 CFU/mL by 40 h. This cor-
responded to an increase in CDW from ~11 to 24 g/L
over the same period. Between 15 and 35 h, the average
glycerol utilization rate was 1.75 g/L/h, which was com-
parable to the average rate observed (1.4 - 2.9 g/L/h) in
the previous trials in the same time frame. Glycerol con-
sumption continued until depletion at 39 h, which corre-
sponded to the peak CDW level. Ammonia depletion
occurred at 32 h. Following glycerol depletion in the
medium the CDW decreased until 80 hours.
When glycerol fell to 10 g/L (~28 h) sunflower soap-
stock additions were initiated. Because soapstock was
viscous, 40 g of soapstock was blended with water to a
volume of 150 mL prior to each ad dition. To compensate
for this added volume, 150 mL of culture broth was re-
moved prior to the diluted soapstock addition. It was not
possible to monitor the level of soapstock, since it is a
complex of several lipids. However, Lee et al. [60] pre-
viously reported that dissolved oxygen levels can be used
to monitor carbon levels. Therefore when the dissolved
oxygen saturation rose above 10%, soapstock additions
were made. This caused oxygen saturation to drop to
near zero, and as it was consumed, oxygen saturation
gradually increased. A total of 360 g of sunflower soap-
stock was added in a total volume of 1.35 L, at 6 - 10 h
intervals through 86 h.
We had previously observed that P. 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
[42,43]. Soapstock also increased foaming in the aerated

J. JAVERS ET AL. 246
-10%
0%
10%
20%
30%
40%
50%
60%
70%
80%
0 102030405060708090
time (h)
percent of PHA
C6%
C8%
C10%
C12-1%
C12%
C14-1%
C14%
C16%
100
Figure 3. Time profile of monomeric composition determined by gas chromatography in PHA derived from fed-batch fer-
mentation of P. putida KT217 on a syrup medium fed with glycerol water.
p
pm 200 175 150 125 100 75 50 25 0
Figure 4. 13C-NMR of the PHA extracted from the final sample of the glycerol water fed batch fermentation.

J. JAVERS ET AL. 247
0
5
10
15
20
25
30
35
40
45
0 102030405060708090
time (h)
glycerol, cdw, PHA (g/L)
ammonia, posphate (dg/L)
100
1.E+06
1.E+07
1.E+08
1.E+09
1.E+10
1.E+11
1.E+12
viable cell population (cfu/ml)
glycerol cdwacetic acidammonia phosphate PHA cfu/ml
Figure 5. Growth and PHA production of P. putida KT217 on a CCS medium with fed-batch sunflower soapstock addition.
Arrows indicate addition of 40 grams of sunflower soapstock. Average of two fermentations with error bars representing
standard deviation.
bioreactor, even with antifoam addition. Therefore we
reduced the aeration rate from 1 vvm to 0.5 vvm.
After soapstock additions began, CDW decreased,
even though viable cell populations remained steady.
Evidently, the broth remove d prior to so apsto ck add itio ns
contained cells at least partially filled with PHA, and
these were replaced by newly formed cells that didn’t
contain as much PHA. This did not occur in the prior
glycerol fed-batch process because only 240 mL of the
concentrated (85%) glycerol water was added, compared
to the more dilute soapsto ck (50% FFA) [37]. Acetic acid
levels began to accumulate after 60 h, eventually rising
to 7 g/L. This was likely due to fatty acid degradation
into acetyl-CoA, coupled with the decreased aeration rate
and hence reduced acetic acid being excreted from the
cells. It is likely that the Krebs cycle could not process
acetyl-CoA at the same rate it was being produced, thus
diverting some acetyl-CoA to acetic acid. This build up
in acetyl-CoA may have activated the fatty acid synthesis
pathway as well, explaining the increase in PHA.
PHA accumulated at a slow rate throughout incubation,
reaching a concentration of 2.8 g/L at the end, represent-
ing 17% of the total CDW. The composition of the PHA
changed dramatically during incubation (Figure 6). Ini-
tially, the composition favored 3-hydroxydecanoate mo-
nomers due to the utilization of glycerol in the CCS. Fol-
lowing soapstock addition, polymer composition shifted
towards 3-hydroxyoctanoate monomers. The final PHA
contained 3-hydroxytetradecanoate and 3- hydroxytet-
radecanoate. 13C-NMR analysis of the final PHA pro-
duced 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
is evident by examining the peaks in the chemical shift
range of 120 - 150 ppm which has been shown previ-
ously [15,5 5] .
4. Conclusions
PHA production was observed over 100 h of aerated in-
cubation when P. putida KT217 was grown on a basal
medium of 400 g/L CCS (wet basis) and 2.2 g/L ammo-
nium hydroxide, supplemented in fed-batch mode with
either biodiesel co-products glycerol water (240 g) or
soapstock (390 g). Foaming was a problem during soap-
stock trials, but was of less concern when using glycerol

J. JAVERS ET AL. 248
0%
10%
20%
30%
40%
50%
60%
70%
80%
0 102030405060708090
time (h )
percent of PHA
C6%
C8%
C10%
C12-1%
C12%
C14-1%
C14%
C16%
100
Figure 6. Time profile of monomeric composition determined by gas chromatography in PHA derived from fed-batch fer-
mentation of P. putida KT217 on a syrup medium fed with sunflower soapstock.
ppm 200 175 150 125 100 75 50 25 0
Figure 7. 13C-NMR of the PHA extracted from the final sample of the sunflower soapstock fed batch fer mentation.

J. JAVERS ET AL. 249
water. Variations observed during replicate trials were
likely caused by slight differences in aeration. P. putida
was more efficient in using glycerol for cell growth (30
g/L CDW) and PHA content (31%), with PHA primarily
composed of 3-hydroxydecanoate generated by the de
novo fatty acid synthesis pathway. This phenomenon was
reported by Solaiman et al. [61] who observed that P.
corrugata grown on soybean molasses carbohydrates
also resulted in 3-hydroxy acyl-CoA monomers of this
chain length. In contrast, sunflower soapstock resulted in
only 17 g/L CDW and 17% PHA content, with PHA
composition shifting from 3-hydroxydecanoate to 3-hy-
droxyoctanoate over time due to β-oxidation, de novo
fatty acid synthesis and fatty acid elongation pathways.
This phenomenon was also noticed with P. corrugata
when grown on a biodiesel co-product where the FFA’s
were preferentially utilized [62]. So apstock derived PHA
also showed an increase in unsaturation likely due to the
unsaturation in the FFA contained in the soapstock.
The maximum concentration of PHA in cell mass that
we observed was only 31%, following a lengthy incuba-
tion when glycerol water was used as the carbon source.
A majority of the carbon source fed to the P. putida was
evidently used for maintenance energy (or possibly other
products not detected). This low PHA level would likely
lead to poor extraction economics, as current industrial
processes target a 90% PHA level. However these pro-
cesses are primarily based on PHB-PHHx copolymers,
and utilize starch-derived sugars for growth. Use of
lower costs carbon sources may lead to more economi-
cally PHA production processes, however additional re-
search is needed to improve microbe performance and
feeding strategy.
5. Acknowledgements
Funding for this research was provided by the South
Dakota Corn Utilization Council.
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