ARTICLE
Utilization of Cellulosic Waste From Tequila
Bagasse and Production of Polyhydroxyalkanoate
(PHA) Bioplastics by Saccharophagus degradans
Luis Esteban Alva Munoz, Mark R. Riley
Agricultural and Biosystems Engineering, The University of Arizona, Tucson, Arizona;
telephone: 520-626-9120; fax: 520-621-3963; e-mail: riley@ag.arizona.edu
Received 1 August 2007; revision received 21 December 2007; accepted 11 February 2008
Published online 29 February 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21854
ABSTRACT: Utilization of wastes from agriculture is
becoming increasingly important due to concerns of
environmental impact. The goals of this work were to
evaluate the ability of an unusual organism, Saccharophagus
degradans (ATCC 43961), to degrade the major components
of plant cell walls and to evaluate the ability of S. degradans
to produce polyhydroxyalkanoates (PHAs, also known as
bioplastics). S. degradans can readily attach to cellulosic
fibers, degrade the cellulose, and utilize this as the primary
carbon source. The growth of S. degradans was assessed in
minimal media (MM) containing glucose, cellobiose, avicel,
and bagasse with all able to support growth. Cells were able
to attach to avicel and bagasse fibers; however, growth on
these insoluble fibers was much slower and led to a lower
maximal biomass production than observed with simple
sugars. Lignin in MM alone did not support growth, but did
support growth upon addition of glucose, although with an
increased adaptation phase. When culture conditions were
switched to a nitrogen depleted status, PHA production
commences and extends for at least 48 h. At early stationary
phase, stained inclusion bodies were visible and two chrono-
logically increasing infrared light absorbance peaks at
1,725 and 1,741 cm
1 confirmed the presence of PHAs.
This work demonstrates for what we believe to be the
first time, that a single organism can degrade insoluble
cellulose and under similar conditions can produce and
accumulate PHA. Additional work is necessary to more
fully characterize these capabilities and to optimize the
PHA production and purification.
Biotechnol. Bioeng. 2008;100: 882–888.
ß 2008 Wiley Periodicals, Inc.
KEYWORDS: lignocellulosic conversion; bioplastics pro-
duction; Saccharophagus degradans
Introduction
Tequila is a regional alcoholic beverage, obtained from
the distillation of fermented juice of Agave tequilana Weber,
Correspondence to: M.R. Riley
882 Biotechnology and Bioengineering, Vol. 100, No. 5, August 1, 2008
a succulent plant with bluish green leaves that grows in the
western regions of Mexico. This is a substantial commodity
for Mexico but consumes large resources as agave plants
require from 7 to 10 years to reach commercial maturity.
The tequila production process typically comprises four
steps: (1) cooking, to hydrolyze agavin into fructose and
glucose; (2) milling, to extract the sugars; (3) fermentation,
with one or several strains of Saccharomyces cerevisiae to
convert sugars into ethanol and other sensorial compounds;
and (4) two-step distillation process followed by a matura-
tion step. The milling step generates a lignocellulosic
byproduct called bagasse which is primarily the rind and
fibrovascular bundles dispersed throughout the interior
of the agave stalk (Iñiguez-Covarrubias et al., 2001). The
bagasse represents about 40% of the total weight of
the milled agave on a wet weight basis and has a typical
composition of cellulose, hemicellulose, and lignin com-
prising 85% by weight (Alva Muñoz, 2005).
Based on data from the Tequila Regulatory Council
(TRC), from 1995 to 2004 on average 3.3 L of tequila are
produced from one kilogram of agave. Approximately
22.6 thousands metric tons of bagasse was produced in 2004.
Currently the bagasse is composted, processed by chemical
and mechanical pulping processes, mixed with clay to make
bricks, sun dried to make furniture and packing materials,
and as feed for animals. None of these uses has been
considered to be economically or environmentally produc-
tive and therefore, efforts are required in the search for
improved uses of the bagasse (Cedeño, 1995).
This research investigated the use of Saccharophagus
degradans strain 2–40 (ATCC 43961) in the bioremediation
scheme of tequila manufacture’s lignocellulosic waste and its
potential for the production of bioplastics as a value added
product. The use of S. degradans for these purposes is
attractive mainly because of its non-pathogenesis, its highly
specialized multienzymatic complexes acting in concert,
which allows it to depolymerize and metabolize a wide
variety of insoluble complex polysaccharides (ICPs), and
the genomic identification of three key enzymes for
ß 2008 Wiley Periodicals, Inc.
polyhydroxyalkanoate (PHA) synthesis. S. degradans has
been proposed as a model for ICP (Weiner et al., 1998) and
indeed, this specie is the most versatile carbohydrate
degrader yet identified (Ekborg et al., 2005).
S. degradans is periphytic marine/estuarine gammapro-
teobacteria isolated in the middle of the 1980s growing
on salt marsh grass located in the lower Chesapeake Bay,
Virginia and deposited in the American Type Culture
Collection (ATCC 43961). It is a gram-negative, hetero-
trophic, aerobic, oligotrophic, saprophyte, motile by a single
polar flagellum, which forms pleomorphic rods (Marx,
1986). When growing in media containing ICPs, cells can
be pleomorphic and produce surface protuberances
and vesicles (Howard et al., 2004). It has an absolute
requirement for sodium ions, does not grow below 10 g L
1
sea salt, and grows optimally in 23 g L
1 sea salt, although it
grows at 100 g L
1. It grows at temperatures within 4–378C,
with an optimal temperature of 308C and pH of 7.5,
although 4.5–10.0 are feasible (González and Weiner,
2000; Howard et al., 2004). Sequence analysis has revealed
more than 130 putative proteins involved in polysaccharide
depolymerization, a large number of proteases, sugar
transporters, carbohydrate binding proteins, and some
genetic duplication in several of the complex polysaccharide
degrading systems (Howard et al., 2004). S. degradans has
been shown to degrade cellulose and lignin (Ekborg et al.,
2005; González and Weiner, 2000; Howard et al., 2004;
Whitehead, 1997). Recently, Kalia et al. (2003) published the
sequence analysis of 123 bacterial genomes including
13 presumed, but not necessarily confirmed PHA producers
which included S. degradans.
PHAs are biodegradable polymer materials synthesized
and stored in the cell cytoplasm as water insoluble inclusions
(typically 0.2–0.5 mm in diameter), of at least 75 different
genera of gram-positive and gram-negative bacteria. These
macromolecules are accumulated as granules to levels as
high as 90% of the cell dry weight (Lee, 2006). A wide range
of bacteria synthesize them when a carbon source is
provided in excess and one essential growth nutrient is
limited (Wu et al., 2003). PHA acts as a bacterial carbon and
energy reserve (Madden et al., 1999) due to its low solubility
and high molecular weight, which exerts negligible osmotic
pressure to the bacterial cell (Sudesh et al., 2002).
The goals of the present study were to assess the capability
of S. degradans to metabolize the main components of plant
cell walls and to synthesize PHAs.
Materials and Methods
The lyophilized strain (ATCC 43961) was maintained in
sterile Difco 2216 marine broth (MB) at 308C. MB consists
of 37.4 g L
1 of a mixture of 5.0 g Bacto peptone, 1.0 g Bacto
yeast extract, 0.1 g ferric citrate, 19.4 g sodium chloride, 5.9 g
magnesium chloride, 3.24 g sodium sulfate, 1.8 g calcium
chloride, 0.55 g potassium chloride, 0.16 g sodium
bicarbonate, 0.08 g potassium bromide, 0.034 g sodium
strontium chloride, 0.022 boric acid, 0.004 g sodium silicate,
0.0024 g sodium fluoride, 0.0016 g ammonium nitrate, and
0.008 g disodium phosphate (Marx, 1986).
In this first set of experiments, the batch growth
kinetics of S. degradans (ATCC 43961) was determined
when growing in D-glucose or D-cellobiose as a sole
carbon source in minimal media (MM) comprised of sea
salts (23 g/L); EZMixTM yeast extract (1 g/L); ammonium
chloride (0.5 g/L); Trizma1-HCl (1M, pH 7.6, 50 mL/L);
carbon source (2 g/L). Stock solutions of D-glucose
(10%, w/v) and D-cellobiose (5%, w/v) were filter sterilized
in 20 mM pH 6.8 Pipes buffer and added to cooled media to
produce a final concentration of 11.10 mM. At each time
point optical density was determined two culture samples
were centrifuged to determine microbial protein accumula-
tion and cell dry weight. Protein was measured by a
modification of the Bradford dye method (Bradford, 1976).
Proteins are the major component of prokaryotic cells
contributing 40–60% (w/w) of the normal gross cell
composition (Kneipp et al., 2000). Supernatant, recovered
by centrifugation for dry weight determinations, was diluted
and analyzed using the 3,5-dinitrosalicylic acid reducing
sugar assay (DNSA) in order to determine consumption
rates for the tested carbon sources. The phenol-hypochlorite
reaction was used to quantify ammonia in the culture broth,
which served as the chief nitrogen source.
Lignin alkali low sulfonate content (SIGMA 471003) was
utilized as a lignin model compound to determine the
capability of S. degradans to tolerate and/or degrade lignin
under optimal culture conditions. For this set of experi-
ments, lignin as a sole carbon source and two lignin
concentration levels supplemented with 0.2% (w/v) glucose
were utilized.
In order to evaluate S. degradans lignocellulosic degrading
capabilities, its batch growth kinetics on A. tequilana
bagasse and Avicel (microcrystalline cellulose) as sole
carbon sources was monitored. This presents a challenge
in determining cell concentration as cells adhere strongly to
fibers and so absorbance or dry weight measurements are
not feasible. Total solution protein content (as quantified
after hydrolysis of fiber samples) was taken as a measure
of bacterial protein content. In non-fiber tests, a strong
correlation was obtained between Bradford protein
measurement and optical density (not shown).
A sample (30 mL) was centrifuged (8,000g for 15 min
at 48C) and washed twice with 2.3% (w/v) NaCl. The pellet
was resuspended in 2 mL of 0.2 N NaOH, and this
suspension was placed in a boiling water bath for 10 min.
After cooling, the hydrolyzed sample was centrifuged as
described above and the supernatant was diluted in 2 mL of
0.2 N NaOH as well as crystalline bovine serum albumin
(BSA), which was used as the protein standard.
The microorganism was grown in MM containing
1% (w/v) of Avicel1 PH-101 (particle size 50 mm) or
A. tequilana bagasse (sun-dried, grounded to pass a 425 mm
sieve and be retained on a 250 mm sieve). Supernatant and
cell pellet were separated and stored at
208C until their
Alva Munoz and Riley: Utilization of Cellulosic Waste 883
Biotechnology and Bioengineering
analysis. Scanning electron micrographs of late stationary
phase cells grown on avicel and A. tequilana bagasse as the
sole carbon source were obtained using standard methods.
Two PHA induction strategies were developed:
1. Natural depletion of nitrogen. For this group of
experiments, bacterial suspensions in MB were trans-
ferred to flasks containing 62.5 mL of MM supplemented
with D-glucose. A stock solution of D-glucose (20%, w/v)
was filter sterilized and added to cooled media to produce
a final concentration of 4% (w/v).
2. Cell transfer into low nitrogen media. Bacterial cells were
harvested by centrifugation on late logarithmic/early
stationary phase and transferred to the homologous
culture media lacking a nitrogen source.
FT-IR analysis was performed on microbial cultures
collected and centrifuged. The growth medium was removed
by washing-resuspension in filter-sterilized nanopure water.
The final washed pellet, raw bacteria suspension, and first
supernatant solution were deposited onto polyethylene (PE)
IR-cards from Thermo Fisher Scientific, Inc. (Waltham,
MA). Card deposits were dried for 24 h before spectra were
acquired. PE served as background.
Spectra were collected using a Nicolet model Magna-IR
560 FT-IR spectrometer with a liquid N2-cooled InSb DTGS
detector. Absorbance spectra were collected at wavenumber
values between 4,000 and 650 cm
1 with spectral resolution
of 4 cm
1 with 32 co-added scans. All spectra were water
corrected.
Results
The goal of these studies was to evaluate the ability of
S. degradans to grow on multiple carbon sources and to be
able to produce PHA. A summary of growth parameters on
carbon sources glucose, cellobiose, avicel, and bagasse are
shown in Table I.
Growth of S. degradans with glucose as a sole carbon
source (Fig. 1) displays the prototypical exponential growth
and plateau reached upon exhaustion of glucose between
12 and 14 h. At that time, consumption of the nitrogen
source ammonium ceased (Fig. 1). Growth of microorgan-
isms was quantified both through optical density measure-
ments (O.D. at 600 nm) and with microbial protein
accumulation (BSA equivalents). These methods showed
identical profiles with an O.D. value of 0.87 being equivalent
Table I. Summary of batch growth parameters.
Carbon source m (h
1) TD (h)
D-glucose 0.368 1.88
D-cellobiose 0.266 2.60
Avicel 0.051 13.56
A. tequilana bagasse 0.041 16.78
884 Biotechnology and Bioengineering, Vol. 100, No. 5, August 1, 2008
to 255 mg/mL of BSA, the maximum amount of biomass
produced from the initial 11.1 mM glucose MM.
Growth on alternate carbon sources display consistent,
but slower growth than on glucose (Fig. 2, Table I). Growth
profiles on cellobiose are similar to that with glucose.
A higher maximal biomass is achieved with cellobiose (by
7%), but doubling times are longer (2.6 h with cellobiose
compared to 1.9 h with glucose). Metabolism of cellobiose is
less efficient with lower yields of biomass from carbon
source or nitrogen source compared to growth on glucose.
Growth of S. degradans on the crystalline avicel was
significantly slower than that on glucose with doubling
times sevenfold longer. After 56 h of growth, a plateau had
not definitively been reached, but growth had slowed
considerably. As avicel was the only carbon source available
to the bacteria it was consumed although at a slow rate.
S. degradans was able to attach to avicel crystals (Fig. 3a–c)
and slow degradation of the avicel was observed.
S. degradans was also able to attach to A. tequilana bagasse
fibers (Fig. 3) and degrade the bagasse for utilization as
a carbon source. Bacterial growth required a lag phase of at
least 12 h and then was much slower than on other carbon
sources evaluated. The maximum biomass produced was
less than 40% that achieved with glucose as the carbon
source. Despite the slow growth on bagasse, the ability to
utilize this material as a carbon source is encouraging.
The A. tequilana bagasse fibers consist of cellulose,
hemicellulose, and lignin. Lignin has previously been shown
to inhibit or prevent the growth of a number of organisms.
To test whether lignin was responsible for the slow growth
on bagasse, lignin was added to cultures in a glucose MM.
Figure 4a shows the growth curves for no lignin and two
levels (low ¼ 0.076%, w/v, and high ¼ 0.155%, w/v). The
presence of lignin increases the time of the lag phase, slowing
the onset of exponential growth. Once the cultures reach
the exponential phase, growth in lignin containing media
is nearly as rapid as without lignin. Effectively the same
maximum biomass level is reached regardless of lignin
concentration. Most likely lignin is not being utilized as a
carbon source since the profiles of glucose consumption are
quite similar except for the 10-h delay in onset of growth
(Fig. 4b). Most importantly, the presence of lignin does not
significantly limit the growth or metabolic activity.
Next the ability of S. degradans to produce PHA was
evaluated with two methods to initiate onset of production.
The strategy named natural depletion resulted in easier and
more reproducible results than did the cell transfer strategy.
Maximum biomass
YBSA/C.S. YBSA=NH4 Cl produced (BSA mg mL
1)
0.130 0.725 254.93
0.074 0.547 272.43
0.043 0.563 218.26
n/d 0.275 88.60

Figure 1. Biomass production reaches plateau due to depletion of glucose and
not due to depletion of nitrogen.
Although on some occasions PHA production was identified
following the cell transfer strategy, the appropriate harvest-
ing time could not be accurately defined. Improper
transference resulted in stressed cells that tended to reduce
in size and not accumulate PHA. The following results show
positive PHA accumulating cells of S. degradans in MM
supplemented with D-glucose 4% (w/v). The logic followed
in this strategy is based on a nutrient depletion profile in
which the exhaustion of the carbon source (D-glucose 0.2%,
w/v) seemed to impact cell growth while ammonium
chloride was still detected in the culture media. As
mentioned above, PHA induction usually occurs when a
carbon source is provided in excess and one essential growth
nutrient is limited. With the natural depletion strategy,
Figure 2. S. degradans growth kinetics in four carbon sources.
carbon source surplus was ensured and likely the scarcity of
at least one essential growth nutrient was achieved as is
shown with positive PHA accumulating S. degradans cells
stained with Nile red (Fig. 5). Within the cell cytoplasm,
separated inclusion bodies can be seen as cells presented
from one to five inclusions.
Once potential PHA induction conditions based on Nile
red staining were identified, the more quantitative approach
of FT-IR analysis was used to identify PHA accumulation
within S. degradans cells. At each time point raw cells,
expended media (supernatant) and washed cells were used.
The inverse second derivative of raw samples (data not
shown) using the spent media as background showed a
chronological increment in the area suggested primarily for
PHB (wavenumber value 1,735 cm
1). The average peak
was located at 1,742 cm
1, giving strong evidence of the
presence of PHA-like substances within the cells.
The following IR spectra were obtained from the same
experiment. Figure 6 depicts S. degradans spectra of cells
under PHA accumulating conditions at different time
points. In this case, the PHA average peak was located at
1,732 cm
1, and the characteristic amide I (cellular protein)
peak on average was located at 1,656 cm
1. The amide I peak
was used as means to normalize the data based on biomass
since protein content is relatively constant.
In order to minimize the presence of spectral artifacts,
the inverse second derivative analysis was developed to the
group of spectra shown in Figure 6. The PHA feature
(1,720–1,750 cm
1) appears to be the combination of two
peaks at 1,724 and 1,743 cm
1, referred to as PHA 1 and
PHA 2, respectively. Fourier self-deconvolution (FSD
performed on the Omnic software) was used to establish
the relationship between the area under the PHA and
amide I peaks. This relationship served to identify the
behavior of the PHA peak compared with the biomass
intrinsic growth.
The maximum PHA accumulation per biomass (amide I)
occurred from 24 to 30 h (Fig. 7). The maximum PHA
accumulation rate occurred at early stationary phase,
determined by microbial protein accumulation, expressed
as microgram BSA equivalents per milliliter.
FSD analysis identified four stages during PHA accumu-
lation in S. degradans. During the first stage, less than 18 h,
there was no significant peak around the PHA suggested
spectral location. In the second stage, at 18 h, the PHA
2 feature appeared. At this time, the relationship between
PHA 2 and Amide I area was less than one, suggesting that
the concentration of PHA within the cells is less than the
accumulated microbial protein. The third stage, at 24 h, the
PHA 1 peak started to emerge. At this stage, the relationship
between PHA peaks and Amide I still was less than one. On
the fourth stage, from 30 to 60 h, the area under both
PHA peaks exceed the area under the curve of amide 1 peak
suggesting that intracellular PHA accumulation was
occurring.
In an attempt to relate PHA induction to nitrogen
scarcity, Figure 7 also shows ammonium chloride depletion
Alva Munoz and Riley: Utilization of Cellulosic Waste 885
Biotechnology and Bioengineering
Figure 3. a: Live cell stain and optical micrograph of S. degradans in avicel minimal media. One large rectangular avicel fiber is shown near the top of this image and displays
significant bacterial attachment. b: Photomicrograph of S. degradans degrading avicel. c: Scanning electron micrograph of S. degradans on avicel crystalline cellulose. d: Scanning
electron micrograph of attachment of S. degradans to a bagasse fiber.

with PHA accumulation. In both cases, the accumula- Discussion

tion appears to be induced at levels around 2 mM
ammonium chloride. Under this condition there was
surplus of carbon source (D-glucose quantification data
not shown) and scarcity of at least one essential nutrient
required for growth.
The amount of PHA produced can be approximated from
the area under the curves for PHA and protein (amide I)
along with the protein analysis (Figure 7a) which shows a
maximum of 375 mg/mL protein was produced. The area
under the PHA 1 feature at 1,724 cm
1 is 4 times that of the
amide I feature, indicating that the cell population contains
4 times the PHA as protein. Thus the concentration of PHA
may reach as high as 1.5 mg/mL.
This work demonstrates that S. degradans has capabilities to
hydrolyze crystalline cellulose, showed the effect of lignin in
growth kinetics, and quantified S. degradans growth rate in
plant matter monoculture. Lignin does have a substantial
effect on growth kinetics which is demonstrated primarily as
a longer time to reach the onset of exponential growth, not
an inhibition on the total biomass produced. S. degradans
can accumulate PHA with a trigger of nitrogen depletion.
The PHA amount reaches approximately 1.5 mg/mL of total
biomass.
Results of these and other experiments suggest a complex
relationship between nitrogen source switching methods,

886 Biotechnology and Bioengineering, Vol. 100, No. 5, August 1, 2008

Figure 4. a: Growth of S. degradans in the presence of lignin delays onset of
exponential growth, but does not alter the maximum biomass produced. b: Depletion of
glucose by S. degradans in the presence of lignin.
glucose and nitrogen availability, and induction medium.
Only experiments performed without pH control showed
PHA production. PHA production was increased by
minimal availability of glucose after the switch of medium.
In a one-step fermentation, PHA production was increased
by inoculation of cells from MM rather than from marine
media; however the correlation between nitrogen source
prior to or after the switch and PHA production is not clear.
In a two-stage fermentation, PHA production was greatest
when an intermediate media were used to transition cells
from the initial medium of yeast extract with added
ammonium into the nitrogen free medium. An intermediary
medium of peptone and yeast extract prior to introduction
to nitrogen free medium most often was successful in
stimulating PHA production.
Together these results indicate that Saccharophagus
species has a unique capability to degrade cellulose and
Figure 5. Nile read staining PHA producing cells after 24 h (a) and 36 h (b) from
incubation.
can be induced to produce bioplastic PHA’s. This ability
has the potential to reduce the economic hindrance to
production of bioplastics on a large scale since inexpensive
precursors could be utilized without the need for extensive
hydrolysis or other fiber reducing procedures. PHA ac-
cumulation can be stimulated by a natural depletion of
nitrogen source while in the presence of sufficient carbon.
Growth on cellulose or baggasse was slower than on soluble
sugars, as anticipated due to the lower accessibility of sugars.
This is not a major hindrance since culture conditions can be
altered to increase accessibility and potentially to impact the
composition of the PHA produced. S. degradans has strong
capabilities for use in monoculture processing of waste
agricultural cellulose to produce bioplastics.
To best utilize the capabilities of S. degradans to degrade
insoluble cellulose and produce bioplastics requires a more
in depth analysis of the nutritional requirements of this
Alva Munoz and Riley: Utilization of Cellulosic Waste 887
Biotechnology and Bioengineering

Figure 6. Baseline-corrected FT-IR spectra of washed cells with a background
of polyethylene.
bacterium and to develop an efficient large scale process.
It may be feasible to cycle operating conditions from
growth inducing (with sufficient nitrogen present) to
growth inhibited bioplastics production (with insufficient
nitrogen). A balance must be reached in delivery of nutrients
to maximize productivity.
Conclusions
This work demonstrates that S. degradans has capabilities to
hydrolyze crystalline cellulose, showed the effect of lignin in
growth kinetics, and quantified S. degradans growth rate in
plant matter monoculture. In addition, this demonstrates
Figure 7. Microbial growth (protein), ammonium chloride depletion, and PHA
production.
888 Biotechnology and Bioengineering, Vol. 100, No. 5, August 1, 2008
that S. degradans accumulates PHA. Work is still required to
optimize production of PHA and for purification.
This work was supported by The USAID sponsored project, ‘‘Devel-
opment of Human Resource Training Capabilities in Emerging Areas
of Agricultural Production and Processing in Mexico.’’ We gratefully
acknowledge the assistance of Dr. Delia Aideé Orozco Hernández,
Tequila Herradura, for providing tequila bagasse and Dr. Jesus
Nungaray, University of Guadalajara, for technical assistance and
discussion on bioplastics production.
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