Selective microbial degradation of saturated methyl branched-chain fatty acid isomers

Article abstract of DOI:10.1007/s11746-012-2092-0

Three strains of Pseudomonas (P.) bacteria were screened for their capabilities of degrading chemically synthesized saturated branched-chain fatty acids (sbc-FA). Mixtures of sbc-FA with the methyl-branch located at various locales along the fatty acid we

Full text of DOI:10.1007/s11746-012-2092-0

J Am Oil Chem Soc (2012) 89:1885–1893
DOI 10.1007/s11746-012-2092-0
ORIGINAL PAPER
Selective Microbial Degradation of Saturated Methyl
Branched-Chain Fatty Acid Isomers
•
•
Helen L. Ngo Richard D. Ashby Alberto Nun˜ez
Received: 5 October 2011 / Revised: 15 May 2012 / Accepted: 16 May 2012 / Published online: 14 June 2012
ꢀ AOCS (outside the USA) 2012
Abstract Three strains of Pseudomonas (P.) bacteria
were screened for their capabilities of degrading chemi-
cally synthesized saturated branched-chain fatty acids
(sbc–FA). Mixtures of sbc–FA with the methyl-branch
located at various locales along the fatty acid were used as
a carbon feedstock in shake-flask culture. Utilization (and
hence degradability) of the sbc–FA was monitored based
on positive bacterial growth, fatty acid recovery rates and
chromatographic (gas chromatography (GC) and GC-mass
spectroscopy (MS)) analysis of the recovered carbon
source. P. putida KT2442 and P. oleovorans NRRL
B-14683 were both able to grow on sbc–FA utilizing
35 wt% and 27 wt% of the carbon source, respectively
after 144 h. In contrast, P. resinovorans NRRL B-2649
exhibited the most efficient use of the carbon source by
utilizing 89 % of the starting material after 96 h resulting
in a cell dry weight (CDW) of 3.1 g/L. GC and GC–MS
analysis of the recovered carbon source revealed that the
bacterial strains selectively utilized the isostearic acid in
the sbc–FA mixture, and a new group of C10, C12, C14 and
C16-linear and/or branched-chain fatty acids (approxi-
mately 4–29 wt%) were formed during degradation.
Keywords Biodegradability Á Branched-chain
fatty acid isomers Á Isostearic acid Á Lubricants
Introduction
Environmental concerns have dictated a shift in focus to
the development of biocompatible substitutes for many
non-renewable materials. Historically, materials such as
fuels, polymers, lubricants and many other chemicals have
been produced from non-renewable petroleum-based
feedstocks. The dilemma has been that petroleum-based
materials, while generally cheaper to produce than bio-
based products, tend to resist the typical degradation pro-
cesses found in nature (e.g., photolysis: the chemical pro-
cess by which molecules are broken down into smaller
units through the absorption of light, and hydrolysis: the
chemical process in which molecules are broken-down by
the addition of water) resulting in problems in waste
management [1–3]. In addition, the little breakdown that
does occur often results in greenhouse gas emissions that
have been determined to contribute to ozone layer deple-
tion, as well as have other deleterious effects [4]. For this
reason, efforts are underway to develop sustainable
replacements for many of the petroleum-based materials
currently in use and to do it cost-effectively. Success in this
area will provide materials that can be produced from
readily available plant and animal materials, and that can
be naturally degraded in a carbon-neutral process.
H. L. Ngo and R. D. Ashby contributed equally to this work.
Mention of trade names or commercial products in this publication is
solely for the purpose of providing specific information, and does not
imply recommendation or endorsement by the U.S. Department of
Agriculture.
USDA is an equal opportunity provider and employer.
H. L. Ngo (&) Á R. D. Ashby Á A. Nun˜ez
U.S. Department of Agriculture, Agricultural Research Service,
Eastern Regional Research Center, 600 E. Mermaid Lane,
Wyndmoor, PA 19038, USA
The most effective means of breaking down any mate-
rial in nature is through biodegradation. The term ‘‘bio-
degradable’’ implies that a material is subject to microbial
e-mail: helen.ngo@ars.usda.gov
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J Am Oil Chem Soc (2012) 89:1885–1893
attack and mineralization to CO2, and water through the
action of specific enzymes present in a bioactive environ-
ment. Standards such as ASTM D 6400 [5], EN 13432 [6],
ISO 17088 [7] among others have been established as
benchmarks for determination of biodegradability and
although, each of these standards is unique, their funda-
mental requirements for determining complete biodegra-
dation are the microbial assimilation of the test material to
CO2, water and biomass whereas, 60–90 % of the carbon is
converted to CO2 in less than 180 days (depending on the
specific standard). In addition, the degradation rates should
approximate those of natural materials such as leaves,
paper, grass and food scraps, and the resultant compost
should have no negative impacts on plants. Many potential
bio-based materials derived from natural sources including
fats and oils for the lubricant industry [8], and certain
biopolymers such as polylactic acid (PLA; [9]), lignocell-
ulosics [10] and polyhydroxyalkanoates (PHA; [11, 12])
among others are being chemically modified to suit specific
applications. While it is generally assumed that materials,
whose origins are from natural sources are ‘‘environmen-
tally benign,’’ once chemical modifications occur there is
no guarantee that the newly synthesized materials will
exhibit the same biodegradable character as the parent
molecule, even in microbially active realms. Therefore, it
is imperative that these materials be tested to ascertain their
potential environmental impact upon disposal.
second strain, a Mycobacterium isolate used a-oxidation to
convert 3-MVA to 2-methylbutyrate, which was then
assimilated through the isoleucine pathway [16]. Lastly, an
Arthrobacter isolate metabolized 3-MVA via x-oxidation
to produce 3-methylglutarate that was degraded by the
3-hydroxy-3-methylglutarate pathway [16]. In each case,
unique bacterial strains utilized the methyl-branched sub-
strate demonstrating different metabolic variations that
could be used in degrading these molecules.
Isostearic acid (i.e., a mixture of saturated methyl
branched-chain fatty acid isomers) is a bio-based product
formed through a chemical process known as skeletal
isomerization of monounsaturated fatty acids [17–21].
Isostearic acid has a hydrophilic carboxylic acid head
group and a hydrophobic tail group with a methyl group
located at various positions along the alkyl chain
(Scheme 1, [2]). In contrast to stearic acid (Scheme 1, [3]),
a saturated linear chain fatty acid that is a solid at room
temperature, isostearic acid is a liquid at room temperature.
It has been found to possess good low-temperature prop-
erties, excellent lubricity, oxidative stabilities and a high-
viscosity index number [22]. Isostearic acid thus has a
potential for application in the lubricant, cosmetic, emol-
lient, and hydraulic fluid arenas. In this report, we describe
our efforts to demonstrate the biodegradable nature of is-
ostearic acid by utilizing 3 separate strains of Pseudomonas
that are known to have the capacity of using both saturated
and unsaturated linear and/or branched-chain fatty acids for
their metabolic processes specifically, as they relate to cell
growth and polyhydroxyalkanoate biosynthesis [23–25].
By monitoring bacterial growth, carbon source utilization
and content, we reveal selective utilization of isostearic
acid which is indicative of biodegradability under the
conditions employed. Conversely, we show that the bran-
ched chain c-stearolactones, linear chain c-stearolactones
and C36-dimer fatty acids also present in the carbon source
were resistant to bacterial breakdown under the conditions
employed.
Typically, linear fatty acids are catabolized through the
b-oxidation pathway 2 carbon units at a time; however, the
inclusion of methyl branches at different points along the
alkyl chain inhibits the typical b-oxidation pathway and
necessitates the presence of new or modified catabolic
pathways in order to degrade these materials. One strategy
that has been noted for the breakdown of methyl-branched
fatty acids is a-oxidation which is a process by which fatty
acids are shortened at the carboxyl-end by a single carbon
atom [13]. This pathway, while efficient is generally
associated with the peroxisomes of mammalian cells [14]
and not in the typical microbes that are naturally prevalent
in the environment. However, studies have been reported
that show the ability of certain bacterial strains to break
down methyl-branched saturated alkanoic acids. As early
as 1959 it was known that Pseudomonas (P.) aeruginosa
was capable of metabolizing certain branched alkanes by
simply monitoring bacterial growth [15]. Later, other
strains of bacteria were reported to have the capability of
breaking down methyl-branched alkanoic acids [16]. By
using 3-methylvaleric acid (3-MVA) as a growth substrate,
3 bacterial strains were discovered that possessed the
metabolic capability to catabolize 3-MVA. The first,
P. citronellolis utilized a b-methyl activation sequence
involving CO2 fixation, analogous to that seen in the
isovalerate pathway to break down 3-MVA [16] ,while the
Materials and Methods
Materials
All of the chemicals used in this work were obtained from
commercial sources and used without further purification.
Oleic acid (91.2 wt% C18:1, 6.1 wt% C18:2, 2.7 wt% satu-
rated fatty acids) and methanol were purchased from the
Aldrich Chemical Company (Milwaukee WI). All salts
used as fermentation media components were purchased
from the Sigma Chemical Company (St. Louis, MO).
Sulfuric acid was obtained from the Mallinckrodt Baker
Co. (Phillipsburg, NJ). Pricat 9910 nickel (1 % w/w) on
123

J Am Oil Chem Soc (2012) 89:1885–1893
1887
Scheme 1
O
HO
Oleic acid [1]
1. Isomerization
2. Hydrogenation
3. Methylation
O
O
O
O
Methyl stearate [3]
Methyl isostearates [2]
Dashed lines indicate several possible
positions for the branching methyl group
O
O
O
O
Linear-chain- -stearolactone [5]
Branched-chain- -stearolactones [4]
Dashed lines indicate several possible
positions for the branching methyl group
O
O
O
O
C36-Dimer fatty acid methyl esters [6]
silica solid catalyst (Ni-SiO2) was a gift from Johnson
Matthey Incorporated (West Chester, PA). Ferrierite zeo-
lite (HSZ-720KOA, potassium (K?), 17.5 mol/mol SiO4/
AlO4) was from the Tosoh Co. (Tokyo, Japan). Methyl
tridecanoate (C13:0) and GLC 420-D11-A (C8:0-C18:3
FAME) reference standards were from Nu-Chek Prep, Inc
(Elysian, MN). P. resinovorans NRRL B-2649 and
P. oleovorans NRRL B-14683 were both obtained from the
National Center for Agricultural Utilization Research,
Agriculture Research Service, United States Department of
Agriculture (Peoria, IL). P. putida KT2442 was kindly
supplied by Professor Richard Gross, Herman Mark
Professor in the Department of Chemical and Biological
Science, Polytechnic Institute of New York University
(Brooklyn, NY).
high-pressure and high-temperature in the presence of
modified H-ferrierite zeolite, and a small amount of water
to give the unsaturated branched-chain fatty acid mixture
(ubc-FA). The ubc-FA intermediate was then pressurized
with 400 psi hydrogen gas in the presence of 1 % (w/w) Ni-
SiO2 at 170 ꢁC for 3 h. The reactor was then depressurized
and the Ni-SiO2 was separated from the sbc–FA by filtra-
tion. In the previous papers, Palladium on carbon catalyst
(Pd/C) was used to hydrogenate ubc–FA [17, 18]; however,
in this instance, we decided to use a nickel catalyst,
because of the large quantity of material needed and using
Pd/C would not have been cost-effective. The hydroge-
nated and methylated sbc–FA products were characterized
by GC–MS and, the relative concentration of the identified
FAME was determined by GC with a flame ionization
detector (FID) as previously reported [17, 18]. The relative
concentration was determined using an internal standard
(C13:0) and was based on the FID linear response for fatty
acid methyl esters (FAME) [26, 27].
Synthesis of Crude Saturated Branched-Chain
Fatty Acid Isomers (sbc–FA)
The detailed procedure used to convert the unsaturated
linear chain fatty acid (oleic acid) to the crude saturated
branched-chain fatty acid isomeric mixture (sbc–FA) was
reported previously [17, 18]. The crude sbc–FA were
synthesized in a two step process, starting from commer-
cially available oleic acid at quantitative yield for each step
(Scheme 1). First, the oleic acid was isomerized under
Shake-Flask Parameters
Three separate bacterial strains P. resinovorans, P. oleo-
vorans, and P. putida were utilized in this work. Cryo-
genically preserved stock cultures of each organism were
prepared as described previously [28]. All degradation
123

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J Am Oil Chem Soc (2012) 89:1885–1893
experiments were conducted on the shake-flask scale in
500 mL of Media E* [29] (starting pH 7.0) in 1-L unbaf-
fled Erlenmeyer flasks. Media E* salts and carbon source
(sbc–FA synthesis described above) were prepared result-
ing in growth media containing 1 % (w/v) sbc–FA. The
media was then autoclaved to sterilize. Each of the afore-
mentioned bacterial strains was tested for its ability to
degrade sbc–FA as a function of time. The inocula for each
of the shake-flask experiments were prepared by inoculat-
ing 50 mL of Luria–Bertani (LB) broth (1 % tryptone,
0.5 % NaCl, 0.5 % yeast extract), with 1.5 mL of bacteria
from frozen stock cultures and incubating the cultures at
30 ꢁC, 250 rpm for 24 h. After 24 h, the LB cultures were
removed from the shake incubator and 0.5 mL of LB
bacterial culture was transferred into each 500-mL test
flask, and each flask placed back into the shake incubator at
the previously noted conditions. At the appropriate times,
flasks were harvested as described below at either 24 h or
48 h intervals.
total) and excess sodium sulfate (Na2SO4) was added to
remove any residual water, after which the Na2SO4 was
removed by filtration through Whatman #2 filter paper. The
organic fractions were then placed into separate tared
round-bottom flasks and the hexane was removed by rotary
evaporation leaving the residual carbon source behind. The
round-bottom flasks were placed under vacuum overnight
to remove any residual hexane and were then reweighed.
The amount of residual carbon source was determined
gravimetrically by difference. The residual carbon sources
were analyzed by GC and GC–MS as described above to
determine preferential carbon source utilization, within the
sbc–FA isomeric mixtures. The aqueous layers were cen-
trifuged (8,0009g, 4 ꢁC for 15 min) to pellet the bacterial
cells. The cells were washed once in deionized water and
re-centrifuged under the same conditions, after which the
cells were frozen and lyophilized to dryness (*24 h).
Once dry, the CDW for each of the cultures were deter-
mined gravimetrically.
Shake-Flask Harvest Conditions
Results and Discussion
Single flasks were harvested in a manner that would allow
re-isolation of residual sbc–FA and/or any fatty acid
derivatives, and allow the gravimetric determination of
bacterial cell dry weight (CDW) over time. To harvest, the
entire culture was placed into a large separatory funnel and
extracted twice with 300 mL of hexane. After each
extraction, the hexane/organic fraction (containing the
residual carbon source) and the aqueous fraction (con-
taining the bacterial cells) were separated. The organic
layers from the two extractions were combined (*600 mL
The objective of this work was to determine, if fatty acids
maintained biodegradability after chemical modification to
sbc–FA (methyl-branching), in order to gain a better
understanding of the potential impact of sbc–FA upon dis-
posal. One of the easiest methods to determine, whether a
specific bacterial strain can utilize and hence degrade a
particular material is by monitoring its growth, when the
material in question is used as the sole carbon source. In this
instance the crude sbc–FA mixture was used as a feedstock
Table 1 Carbon source content of the recovered saturated branched chain-FA after exposure to bacterial growtha
Entry
Bacterial strain
Time
[h]
Percentage composition from GC
Methyl
isostearates
[2b]
Methyl
stearate
[3b]
c-Stearolactones
[4b & 5b]
C
36-dimer
FAME
Unknowns
C10,C12,C14,C16-
branched-FAME
[6b]
1
2
3
4
5
6
7
8
9
None
0
48
75.4
62.4
48.0
51.3
40.4
40.4
71.5
65.9
46.0
2.1
1.7
1.7
1.9
1.8
1.5
2.3
2.2
1.9
8.6
11.0
17.0
10.1
10.8
11.0
8.8
13.9
22.0
22.4
18.2
17.6
18.1
13.7
14.2
16.3
0
2.9
10.9
0
0
P. resinovorans
P. resinovorans
P. putida
0
96
0
48
18.5
29.4
29.0
3.8
8.6
25.8
P. putida
96
0
P. putida
144
48
0
P. oleovorans
P. oleovorans
P. oleovorans
0
96
9.1
0
144
10.0
0
FAME fatty acid methyl esters, FA fatty acids, P Pseudomonas
a
Starting fatty acids are sbc–FA. Products were methylated and then analyzed by GC. Results were compared against internal standard methyl
tridecanoate (C13:0)
b
Referring to Scheme 1
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J Am Oil Chem Soc (2012) 89:1885–1893
1889
for 3 specific strains of Pseudomonas, whose growth and
sbc–FA consumption was monitored in order to assess the
relative rates of carbon source utilization. Table 1, entry 1
lists the product distribution obtained by GC analysis for
the crude sbc–FA before exposure to bacterial growth. The
parental material included both monomeric (75.4 wt%)
isostearic acid [2], Scheme 1; 2.1 wt% stearic acid [3],
Scheme 1; and 8.6 wt% c-stearolactones [4], [5], Scheme 1
as well as oligomeric (13.9 wt% dimer fatty acids [6],
Scheme 1) products. GC–MS analysis showed that the
monomeric components had the following molecular ions
(M?•) at m/z ([2] M?• = 298; [3] M?• = 298; [4]
M?• = 282; [5] M?• = 282). In our previous work [17,
18, 22], we reported that the branched-chain mixtures
contained small amounts of hydroxy-methyloctadecano-
ates. However, after further analysis of the mixture, we
found that the product was in fact c-branched-chain ste-
arolactones (Scheme 1, [4]), but not hydroxy-methylocta-
decanoates. Matrix-assisted laser desorption/ionization-
time of flight (MALDI-ToF) showed that the dimer
(Scheme 1, [6]) had a molecular weight of 595. We used
this carbon source mixture in the present study, because as
mentioned previously, this mixture has excellent lubricity
and selected properties [22]. Fig. 1a, b show the relative
rates of carbon source utilization and the CDW among the
3 bacterial strains tested. P. putida and P. oleovorans uti-
lized 35 % and 27 % of the starting sbc–FA carbon source
after 144 h resulting in CDW of 0.76 g/L and 0.74 g/L,
respectively. These results demonstrated that these 2 bac-
terial strains both had the ability to utilize at least a portion
of the carbon substrate, but the utilization was accompa-
nied by a relatively long lag phase resulting in prolonged
incubation times. In contrast, P. resinovorans utilized 89 %
of the sbc–FA substrate in 96 h resulting in a CDW of
3.1 g/L. These data showed that P. resinovorans was better
equipped genetically to handle growth on the crude sbc–FA
mixture resulting in more efficient carbon source utilization
and higher CDW. This was not entirely surprising in that
one of our previous reports showed the ability of P. resi-
novorans to convert isostearic acid to methyl-branched
medium-chain-length (mcl-) polyhydroxyalkanoate bio-
polymers [23].
120
a
In order to monitor preferential, carbon source utiliza-
tion and degradation products, the recovered carbon source
from each of the specific bacterial strains was analyzed by
GC and GC–MS. Table 1 shows the results of those anal-
yses. Based on those results, it was concluded that sbc–FA
can be effectively degraded by each of the bacterial strains
tested albeit to varying degrees. Table 1, entries 2–9 list the
sbc–FA product distribution in the recovered carbon source
after incubation for 48 h–144 h with each of the bacterial
strains. The results showed that the sbc–FA mixture in
particular, the isostearic acid was selectively utilized by
each of the bacterial strains. For example, in the presence
of P. resinovorans at 30 ꢁC for 48 h, the concentration of
isostearic acid was reduced by 17 % from 75.4 wt% to
62.4 wt% (Table 1, entry 2). Prolonging the incubation
time to 96 h led to a further significant decrease of isos-
tearic acid to 48 wt% yield (a total decrease of 36 %;
Table 1, entry 3). P. putida and P. oleovorans were also
capable of utilizing the isostearic acid fraction (Table 1,
entries 4–9). Interestingly, for all three strains the per-
centage of isostearic acid steadily dropped initially, but
leveled off at *40 % after 144 h. Although, based on
cellular growth and carbon source recovery, all three
strains could degrade isostearic acid, it was determined that
P. resinovorans was actually more equipped to utilize
isostearic acid at a faster rate. Thus subsequent work
focused on this strain. As shown in Table 2, the time-
dependent degradation studies clearly indicated that
P. resinovorans degraded isostearic acid from 72.4 wt% to
39.3 wt% (entries 1–7) after it was incubated for 168 h.
These results imply that the isostearic acid was only
100
P. oleovorans
80
60
40
20
0
P. putida
P. resinovorans
0
20
40
60
80
100
120
140
160
Time (h)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
b
P. resinovorans
P. putida
P. oleovorans
0
20
40
60
80
100
120
140
160
Time (h)
Fig. 1 Carbon source recovered a and cell dry weights b from the
cultures containing P. resinovorans, P. putida and P. oleovorans as a
function of time
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J Am Oil Chem Soc (2012) 89:1885–1893
partially utilized by P. resinovorans, under the conditions
tested. Beta-oxidation is the most well known means by
which fatty acids are broken-down; however, 3-methyl-
substituted fatty acids cannot undergo b-oxidation, because
the 3-methyl-group prevents the formation of the 3-keto
substituent in the dehydrogenation step, which impedes
further b-oxidation. In the present study, the isostearic acid
carbon source was a mixture, where the methyl group was
located at different positions along the carbon backbone,
thus it was probable that some b-oxidation did occur ini-
tially, but as the fatty acids were shortened and converted
to 3-methyl-branched derivatives, b-oxidation stopped
leaving approximately 40 wt% of the starting isostearic
acids as unutilized at 96 h and above. The sbc–FA mixture
used as carbon source in this study (Table 2, entry 1) was
slightly different from the material reported in Table 1,
mainly due to experimental variation in sample preparation
and data analysis. For all subsequent studies, two important
points should be noted: first, the bacterial strains also
degraded the stearic acid present in the carbon mixture, but
to a much smaller extent than the isostearic acid; second,
when the percentages of isostearic acid and stearic acid
decreased due to degradation, the percentages of c-stearo-
lactones (Scheme 1, [4] and [5]) and dimer products
(Scheme 1, [6]) which were not utilized by the bacterial
strains showed an increase. It was not, because the bacterial
strains produced more of these materials.
carbon mixture from P. oleovorans was comparable to that
from P. putida, so is not shown). The mass spectral anal-
ysis showed 4 new types of fatty acids being formed. The
peaks labeled A are consistent with C10-branched-chain
FAME isomers with M?• at m/z 186, B: C12-branched-
chain FAME isomers with M?• at m/z 214, D: C14-bran-
ched-chain FAME isomers M?• at m/z 242, and E: C16-
branched-chain FAME isomers M?• at m/z 270. These
results were compared against a standard which contained
C10–C18 saturated linear chain FAME (data not shown).
The retention times for the linear chain FAME versus
branched-chain FAME of various chain lengths were very
similar. These results further supported, we had generated
C10, C12, C14 and C16-branched-chain fatty acid isomers
during the incubation process. In addition, we suggested
that these were short chain fatty acid isomers, because the
molecular ions obtained by the MS were the same. For
example, the mass spectra of the peaks within group B of
Fig. 2 were different, but they all showed the same
molecular ion, indicating their isomeric nature Fig 3. In
addition, fragment m/z 183 corresponds to the loss of
CH3O• (M-31), which is expected for FAME products and
the ion at m/z 74 is a result of a rearrangement of the fatty
acid known as the McLafferty rearrangement. The frag-
mentations further supported that the new materials formed
during degradation are short chain FAME isomeric prod-
ucts. On the other hand, the new group of products gen-
erated by P. resinovorans observed eluting with a retention
time of approximately 19.3 min for the more abundant
constituent, and representing between 4 % and 10 % of the
chromatographic profile (Fig. 4, B) was found to be quite
challenging to identify. These products were observed for
methylated samples only with very similar spectra, show-
ing a high mass ion at m/z 250, and an ion series
From the GC and GC–MS results, we identified the new
materials generated by P. putida and P. oleovorans
(Table 1, entries 4–9), but were having difficulty identi-
fying the new materials generated by P. resinovorans
(Table 1, entries 2, 3). For instance, Fig 2 shows the GC
chromatogram of the recovered crude sbc–FA from
P. putida after methylation (data for recovered crude
Table 2 Carbon source content of the recovered saturated branched chain-FA after exposure to P. resinovorans for various timesa
Entry
Bacterial strain
Time
[h]
Percentage composition from GC
Methyl
isostearates
[2b]
Methyl
stearate
[3b]
c-Stearolactones [4b & 5b]
C36-dimer
FAME
[6b]
Unknowns
1
2
3
4
5
6
7
None
0
24
72.4
72.2
63.5
48.1
41.2
39.2
39.3
3.2
3.0
3.4
4.1
4.6
4.5
5.3
13.2
10.8
14.3
16.4
20.3
18.6
18.4
11.2
14.0
16.6
20.3
21.0
25.8
26.8
0
P. resinovorans
P. resinovorans
P. resinovorans
P. resinovorans
P. resinovorans
P. resinovorans
0
48
2.2
72
11.1
12.9
11.9
10.2
96
120
168
FAME fatty acid methyl esters, FA fatty acids, P Pseudomonas
a
Starting fatty acids are sbc–FA. Products were methylated and then analyzed by GC. Results were compared against internal standard methyl
tridecanoate (C13:0)
b
Referring to Scheme 1
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J Am Oil Chem Soc (2012) 89:1885–1893
1891
F
pA
C
I
G
H
D
B
E
A
Time--> 0
20
4
6
8
10
12
14
16
18
22
24
26
28
30
32
Fig. 2 GC-FID chromatogram of the methylated sbc–FA recovered
from P. putida. C10 methyl-branched-chain FAME isomers (A), C12
methyl-branched-chain FAME isomers (B), C13:0 internal standard
(methyl tridecanoate; C), C14 methyl-branched-chain FAME isomers
(D), C16 methyl-branched-chain FAME isomers (E), C18 methyl-
branched-chain FAME isomers (methyl isostearates; F), C18 linear
chain FAME (methyl stearate; G), branched-chain-c-stearolactones
(H), linear chain-c-stearolactone (I). FA fatty acids, FAME fatty acid
methyl ester
74
D
Average of 11.830 to 11.860 min.
pA
F
101
A
55
87
183
157
199
140
115
214
C
40
60
80
100
120
140
160
180
200
220
B
74
Average of 12.022 to 12.058 min.
E
0
87
4
8
12
16
20
24
28
32
Time-->
55
138
Fig. 4 GC chromatogram of methylated sbc–FA recovered from
P. resinovorans. C13:0 internal standard (methyl tridecanoate; A),
unknowns (B), C18 methyl-branched-chain FAME isomers (methyl
isostearates; C), C18 linear chain FAME (methyl stearate; D),
branched-chain-c-stearolactones (E), linear chain-c-stearolactone
(F). FA fatty acids, FAME fatty acid methyl esters
101
115
171
214
157
183
199
50
70
90
110
130
150
170
190
210
87
Average of 12.094 to 12.118 min.
74
55
degradation products from the carbon source or the dead
bacterial cells.
157
141
115
101
183
128
214
199
m/z-->
40
60
80
100
120
140
160
180
200
220
In an attempt to identify further the materials generated
by the bacterial strains, control experiments were carried
out with the unsaturated form of the branched-chain fatty
acid mixture (ubc-FA). Table 3 lists the results of the
ubc-FA incubated with P. resinovorans under similar
conditions as the sbc–FA mixtures. The fatty acids were
recovered, hydrogenated and methylated to facilitate the
analysis. The results showed that P. resinovorans utilized
the unsaturated fatty acid mixture in a similar fashion to the
sbc–FA mixture. Interestingly, the new fatty acids gener-
ated by P. resinovorans (which was approximately 10 %
after incubation for 192 h) using ubc-FA were similar to
the ones produced by P. putida and P. oleovorans from
sbc–FA. The GC chromatogram of the recovered ubc-FA
oils after hydrogenation and methylation also showed the
formation of 4 new types of fatty acids with molecular ions
Fig. 3 Mass spectral traces of the saturated C12-branched-chain fatty
acid methyl ester isomers from B in Fig. 2
characteristic of an alkyl chain at 14 Da apart and the base
peak at m/z 67 or 95. The retention time and the spectrum
of these products were not altered by silylation (i.e.,
addition of trimethylsilyl to hydroxyl group), showing the
absence of a hydroxyl group in the structure. The fact that
they were methylated products indicated carboxylic acid
functionality associated with an alkyl chain, but the low-
concentration and the lack of diagnostic ions in the high
mass region of the spectra made it difficult to characterize
their structure. These products are reported as unknowns in
Tables 1 and 2 and currently cannot be recognized as
123

1892
J Am Oil Chem Soc (2012) 89:1885–1893
Table 3 Carbon source content of the recovered saturated branched chain-FA after exposure to P. resinovorans for various timesa
Entry
Bacterial strain
Time
[h]
Percentage composition from GC
Methyl
isostearates
[2b]
Methyl
stearate
[3b]
c-Stearolactones
[4b & 5b]
C
36-dimer FAME
C10,C12,C14,C16-
branched FAME
[6b]
1
2
3
4
5
6
7
None
0
24
74.6
72.7
67.4
61.7
57.2
55.7
53.9
7.8
6.5
3.3
2.3
1.4
2.4
1.6
8.9
7.1
8.8
9.4
0
P. resinovorans
P. resinovorans
P. resinovorans
P. resinovorans
P. resinovorans
P. resinovorans
4.3
7.6
13.5
13.1
9.5
9.9
48
10.4
9.6
11.4
12.9
14.0
16.0
18.1
72
96
14.3
16.4
16.5
168
192
FAME fatty acid methyl esters, FA fatty acids, P Pseudomonas
a
Starting fatty acids are sbc–FA. Products were methylated and then analyzed by GC. Results were compared against internal standard methyl
tridecanoate (C13:0)
b
Referring to Scheme 1
5. ASTM standardization news what’s next for biodegradable and
corresponding to C10, C12, C14, and C16-branched-chain
compostable plastics? http://www.astm.org/SNEWS/APRIL_
fatty acid isomers (Table 3). Unfortunately, this informa-
2001/mojo_apr01.html.(accessed May 2012)
6. Vincotte requirements of the EN 13432 standard. http://www.
tion still did not help us identify the fatty acids generated
by P. resinovorans in the sbc–FA system.
okcompost.be/data/pdf-document/Doc-09e-a-Requirements-of-
norm-EN-13432.pdf (accessed May 2012)
Although, we were unable to definitively identify all the
breakdown products from P. resinovorans in the sbc–FA
system, we were able to show that P. resinovorans,
P. putida and P. oleovorans do have the capability of
biodegrading the isostearic acid fraction of the sbc–FA
mixtures used in this study. P. resinovorans demonstrated
the fastest isostearic acid degradation, thus subsequent
work will focus on this strain. By demonstrating the
degradable nature of these sbc–FA, it is envisioned that
these materials will be aided in their potential for ‘‘envi-
ronmentally friendly’’ applications.
7. International Organization for Standardization ISO 17088:2008
http://www.iso.org/iso/catalogue_detail.htm?csnumber=43373
(accessed May 2012)
8. Salimon J, Salih N, Yousif E (2010) Biolubricants: raw materials,
chemical modifications and environmental benefits. Eur J Lipid
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9. Lee S-H, Wang S (2006) Biodegradable polymers/bamboo fiber
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(2007) Thorough chemical modification of wood-based ligno-
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12. Zinn M, Hany R (2005) Tailored material properties of polyhy-
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Acknowledgments We thank Mr. Kerby Jones for help on the
analytical procedures used in this study. We thank Mr. Bun Hung Lai
and Ms. Heather Vanselous for experimental help.
13. Casteels M, Foulon V, Mannaerts GP, Van Veldhoven PP (2003)
Alpha-oxidation of 3-methyl-substituted fatty acids and its thia-
mine dependence. Eur J Biochem 270:1619–1627
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