Journal of Environmental Polymer Degradation, Vol. 1, No.

2, 1993

Cellulose Acetate Biodegradability upon Exposure to

Simulated Aerobic Composting and Anaerobic Bioreactor
Environments 1
Ji-Dong Gu, 2 D. T. Eberiel, 3 S. P. McCarthy, 4 and R. A. Gross 2,s

Cellulose acetate (CA) films with degree of substitution (d.s.) values of 1.7 and 2.5 were exposed
to biologically active in-laboratory composting test vessels maintained at approximately 53 C.
The CA 1.7- and 2.5-d.s. films (thickness values of - 0 . 5 - 1 . 0 and 2.0 rail, respectively) had
completely disappeared by the end of 7- and 18-day exposure time periods in the biologically
active bioreactors, respectively. The relatively small CA film weight loss observed in the poisoned
control test vessels allows the conclusion that CA film erosion during the composting exposures
resulted, at least in part, from biologically mediated processes. Under strictly anaerobic conditions, an active methanogenic inoculum was developed by acclimation of a sewage sludge to a
synthetic municipal solid waste (SMSW) mixture at 42C. The CA 1.7-d.s. film samples (0.5- to
1.0-mil thickness) were exposed in anaerobic serum bottles containing a 25% solids loading of
SMSW in which methanogenic activity was rapidly established after introducing of the developed
inoculum. For exposures of 30 days only small visually distinguishable fragments of the CA 1.7d.s. films were recovered. In contrast, exposure of the CA 1.7-d.s. film to a poisoned control test
vessel resulted in negligible weight loss. Therefore, degradation of the CA 1.7-d.s. films upon
exposure to the anaerobic bioreactors was due, at least in part, to biologically mediated processes.
KEY WORDS: Solid waste; composting; methanogenesis; degradation; cellulose acetate; biodegradability;
anaerobic bioreactor; biodegradation testing,

of the wastes (representing approximately 70% of the

solid waste stream) can be accomplished by using aerobic composting technology as well as by anaerobic bioreactors. The idea of 'biological recycling" can be applied to plastic waste by the design of plastics which can
be disposed and biologically degraded along with other
nonplastic organic wastes.
The loss of valuable resources by their burial in
sanitary landfills along with the decreasing availability
and increasing cost of landfill sites has resulted in an
increased interest in the aerobic composting of MSW.
The upsurge of interest in composting in recent years
has been nowhere more dramatic than in the United
States. There were more than 60 full-scale aerobic composting plants operating in 1983 with another 20 or so
in the design stage or under construction [1]. The number of solid waste aerobic composting plants had continuously increased through the 1990s [2]. Composting

INTRODUCTION
The treatment of disposed municipal solid wastes
(MSW) in biologically active environments is an important approach to solid waste reduction and utilization. This "biological recycling" of the organic fraction
Guest Editor: Dr. Graham Swift, Rohm & Haas.
2polymer Degradation Research Consortium, Department of Chemistry, University of Massachusetts at Lowell, Lowell, Massachusetts
01854.
3Polymer Degradation Research Consortium, Department of Biology,
University of Massachusetts at Lowell, Lowell, Massachusetts
01854.
4Polymer Degradation Research Consortium, Department of Plastics
Engineering, University of Massachusetts at Lowell, Lowell, Massachusetts 01854.
5To whom correspondence should be addressed at Department of
Chemistry, University of Massachusetts at Lowell, One University
Ave., Lowell, Massachusetts 01854.

143
1064-7564/93/0400-0143507.00i0 ' 1993PlenumPublishingCorporatmn

144

is simply a controlled process for the aerobic biological

stabilization of solid organic waste. In the composting
process, paper, yard, food, sludge, and new biodegradable plastic wastes can be decomposed by microorganisms with the evolution of carbon dioxide and heat,
resulting in a reduction in the bulk volume, elimination
of many pathogenic bacteria, and a change in the carbon
to nitrogen (C/N) ratio, thereby producing a highly humiffed organic-rich product. The stabilized compost
product can be used for land application and horticulture [31.
Although problematic, burial in landfills remains
the major route for the disposal of MSW. Landfilling,
which has previously been the method of choice for
many cities, is now at a crisis point for the reasons of
space availability, cost, and pollution. Pollution by
leachate contamination of the groundwater is of particular concern. Under landfill conditions, oxygen becomes depleted and facultative as well as strictly anaerobic microorganisms become dominant. When the redox
potential of the environment is less than - 3 5 0 mV,
methanogenesis takes place [4, 5]. Methanogenic bacteria, belonging to the family of Archaebacteria [6],
produce methane from the metabolic utilization of acetate, hydrogen, and carbon dioxide [5, 7, 8]. To degrade
complex molecules under such an environment, at least
three major groups of microorganisms must be present
forming syntrophic associations: hydrolytic and fermentative bacteria, obligate proton-reducing acetogenic bacteria, and methanogens [%9]. Although traditional
landfills are designed to minimize biodegradation, work
is currently in place to obtain enhanced biological activities in such disposal sites so that essentially a controlled
anaerobic bioreactor is created [10-12].
Plastic packaging and other plastic products are
components of MSW [13, 14], but the fate of plastics
placed in biologically active anoxic environments is still
unclear. Information about plastic biodegradation under
well-defined anaerobic conditions is essentially nonexistent but is crucial to our understanding of the potential for plastic biodegradability upon disposal in a suitably managed biologically active anaerobic "landfilltype" bioreactor.
Progress in biodegradable polymeric material science depends to a large extent on the development of
reliable, reproducible, and representative test methods
for the evaluation of material degradability in the relevant environment(s) of disposal. The test, or a specifically designated combination of tests, should provide
information on the rate and extent of material erosion
(weight loss) relative to other components of MSW, mineralization, and information on the toxicity of polymer

Gu, Eberiel, McCarthy, and Gross

degradation products or undegraded residue. In this paper, we present methodology's for studying the weight
loss degradation kinetics of cellulose acetate (CA) films
using simulated in-laboratory aerobic composting and
anaerobic bioreactor tests. CA is but one member of a
family of interesting and potentially useful polysaccharide esters.
The biodegradation of CA at different degrees of
substitution (d.s.) and other polysaccharide esters have
previously been discussed. Prior to 1992, it was generally assumed that CA with a d.s. of greater than 0.76
was not biodegradable [15]. Studies with fungi have
shown that species such as Pestalotiopsis westerdijkii
[161 and Rhodotorula mucilaginosa [17] produce esterases that allow them to grow on water-soluble CA with
d.s. values of 1.0 or less [18]. These esterases were inactive against water-insoluble (high-d.s.), high molecular weight CA such as cellulose triacetate. Acetylxylan
esterase activity exhibited by the thermophile Caldocellum saccharolyticum [19] as well as by R. mucilaginosa
[17] has also been reported, but it was unfortunately the
degree of substitution on acetyxylan was not documented in the study [19]. R. mucilaginosa is esterasepositive and xylan-negative so that the exoenzyme-catalyzed deacetylation observed is not due to concurrent
events of polysaccharide chain cleavage. Recently, investigators at Eastman Chemical Company and studies
carried out in our laboratory have indicated that CA of
d.s. 1.7 and 2.5 can be degraded by biological mechanisms [20-24]. Buchanan, Gardner, and White at the
Eastman Chemical Company have carried out studies on
the biodegradation of CA (1.7 and 2.5 d.s.) where the
acetate side groups were 14C-labeled [20]. They reported that a significant degree of mineralization of the
labeled side groups was observed, suggesting that deacetylation followed by cellulase activity was responsible for CA biodegradability. A preliminary report from
our laboratory on the degradation of CA [21, 23, 24] in
compost and anaerobic bioreactors also showed that CA
of substitutions greater than 1.0 was indeed biodegradable. In addition, we have recently isolated a number of
pseudomonads which are capable of growth on CA d.s.
1.7 and 2.5 as the sole carbon source [21, 22], The
growth characteristics of one of these CA degrading
pseudomonads, specifically Pseudomonas paucimobilis, has been reported [22].
In the present paper, detailed methods on carrying
out in-laboratory aerobic compost and anaerobic bioreactor simulation tests are presented. Various aspects
of experimental design to obtain statistically significant
measurements of polymer film weight loss as a function
of exposure time are described. These tests were used

145

Cellulose Acetate Biodegradability

to obtain a quantitative measure of highly substituted
(d.s. values of 1.7 and 2.5) CA film weight loss (erosion) and a qualitative assessment of film biodegradation.

EXPERIMENTAL
Films Used in the Aerobic and Anaerobic
Environmental Exposures
The Eastman Chemical Company (Kingsport, Tennessee) provided in granular form CA samples with d.s.
values of 1.7 and 2.5 CA d.s.-2.5 film was made by
casting onto Teflon from acetone (15% polymer w/v)
using a Gardner Knife. For CA 1.7-d.s., film was prepared in a similar manner as above but from acetonewater (1 : 1 ratio, 17% polymer w/v). The thickness of
the CA (1.7- and 2.5-d.s.) films were approximately
0.5-1.0 and 2.5 rail, respectively, after removal of the
solvent in vacuum (1 rail = 0.0254 ram). Cellophane
film, noncoated, with a thickness of approximately 1.6
rail, was obtained from E. I. Du Pont De Nemours &
Company (Wilmington, Delaware). The Films were cut
into dimensions of 2.54 x 2.54 cm and exposed to the
test environments. Molecular weights of these samples
were determined by gel permeation chromatography
(GPC) on a chromatographic system consisting of a Perkin Elmer Model 410 pmnp, a Waters Model 410 RI
detector, and a PE Nelson 2600 computerized data station. Two PL gel columns (300 x 7.7 ram; particle size,
5 ram; pore size, l0 s and 103 A ) were placed in series
and operated at a flow rate of 1 ml/min. The sample
concentration was 2 mg/ml and the injection volume was
20/zl. The analysis was performed using dimethyl formamide (DMF) containing 0.1% (w/v) LiBr as the eluent.
Molecular weights were calculated relative to polystyrene standards without further corrections. It was determined that the 1.7- and 2.5-d.s. samples had M w and
Mw/Mn values of 200,000 and 350,000, respectively,
and 1.7 and 1.8, respectively.

2.50% aluminum and steel shavings, 1.20% glass beads,

1.30% urea, 1.0% compost seed, and 40.0 ml water/
100 g of the compost mixture. The added water brought
the water content to 50 % of the water holding capacity.
With the exception of the cow manure, the components
were not dehydrated before use.

Bioreactor Design and Maintenance (See Fig. i).

Each composting vessel containing the synthetic waste
and plastic samples was constructed from a 127-ramdiameter x 260.4-ram-long acrylic cylinder, aluminum
top and bottom bulkhead plates (152.4 x 152.4 ram),
and four threaded bolts assembled as shown in Fig. 1.
There is an exit port (diameter, 6.4 ram) for air discharge in the top bulkhead plate and an entrance port
(diameter, 6.4 ram) for air feed into the bioreactor at
25.4 mm from the column bottom. A perforated separation plate was positioned approximately 50.8 mm from
the bottom of the aluminum bulkhead above which the
SMSW-plastic mixture was placed. Humidified air was
pumped into the vessels through the bottom chamber
which eventually passed through the perforations in the
separation plate using a central air pump (General Electric, Powerpal, Model MT500402; maximum, 100 psi).
The desired flow rates were maintained by flow meters
which were placed inqine for each test vessel.
The air which was used to provide O2 to the hiereactors was pretreated by passage through three 500ml Ehrlenmeyer flasks containing 400 ml of 2 M NaOH

outlet
Wing nut
Top aluminum
bulkhead
O-rings

Threads

Aerobic Compost Simulations

Simulated or Synthetic Municipal Solid Waste
(SMSW) Mixture. Simulated waste [13, 14, 25] consisted of 28.50% shredded maple and oak leaves (1 : 1
ratio), 3.85% cow manure, 1.45% saw dust, 11.75%
shredded news and computer paper (1:1 ratio), and
4.80% meat waste (mixture of dog and cat food in a
ratio of 1 : 1), 12.80% food waste (mixture of peas, corn,
and carrots pureed using blender and frozen until use),

infer

aluminum
bulkhead

Bottom

Fig. 1. Illustration of the tempesting bioreactor constructed.

146
to remove CO 2 and was finally humidified by passage
through a diffusion stone at the bottom of a 500-ml Ehrlenmeyer flask containing 350 ml deionized water. Since
the aqueous solutions through which the air was passed
were equilibrated to 53C, the air was warmed by this
process before entering the bioreactors to avoid cooling
of the reactor contents. The air flow rate for each vessel
was maintained at 100 ml/min, and the bioreactor was
equilibrated to the temperature of the environmental incubation chamber, which was maintained at approximately 53C throughout the degradation exposure.
The plastic film samples were prepared a minimum
of 2 weeks prior to their use. Triplicate plastic samples
were used for each anticipated sampling time point (approximately every 5 days). The experiments were carried out in triplicate bioreactors along with a poisoned
control test vessel. The poisoned vessel was used to determine the degree to which nonbiologically mediated
degradation processes take place. The poisoned control
vessels were prepared and maintained identically to the
nonpoisoned active vessels with the exception that for
the poisoned vessels, the compost mixtures were autoclaved for 45 min after which KCN was dissolved in
water and added (2/100 g of compost). After loading the
bioreactors (and the poisoned control vessels) and carrying out test sample exposures for the desired time periods, triplicate film samples were randomly selected
from each of the three replicate vessels and one poisoned control test vessel. The withdrawn samples were
gently cleaned using a moist tissue, dried under vacuum, and the remaining material was analyzed.
During the period of composting simulation, the
compost mix within the bioreactors, as well as the poisoned control, was monitored for moisture content, total
dry weight of the mix, pH, and internal temperature.
Approximately 2 g of the compost mixture was withdrawn from the bioreactors periodically (normally at 5day intervals), the pH of the sample was measured by
placing a pH strip (accurate to _+0.2 pH unit) in contact
with the moist sample, and the moisture content was
obtained by measuring the loss in sample weight after
drying in an oven at 105C for approximately 16 h to a
constant weight. The total dry weight of the remaining
bioreactor mix was then calculated by measuring the total wet weight and subtracting the moisture content. The
internal temperature of the bioreactor was monitored at
the time of sampling using a digital display temperature
pyrometer. Mixing of the vessel contents was carried
out by hands after events of film sampling.
The plastic component of the waste mix in the
aerobic composting bioreactors used to test the biodegradability of the CA 1.7-d.s. and cellophane film sam-

Gu, Eberiel, McCarthy, and Gross

pies consisted of only these two plastics. For compost
exposures carried out to study the biodegradability of
CA 2.5-d.s. films, the bioreactors contained other films
in addition to the CA 2.5-d.s. films. These films included polyvinyl alcohol (PVOH), poly ~3-hydroxybutyrate-co-/3-hydroxyvalerate [P(HB-co-HV)], polycaprolactone (PCL), and various P(HB-co-HV)/starch and
P(HB-co-HV)/PCL blends. For the purpose of this paper, the results presented herein are limited to that for
the degradation of CA (1.7- and 2.5-d.s.) and cellophane films.

Anaerobic Bioreactor Simulations

Simulated or Synthetic Municipal Solid Waste
(SMSW) Mixture. The synthetic waste used in this study
consisted of 24.0% shredded maple and oak leaves (1 : 1
ratio), 46.0% shredded news and computer paper (1 : 1
ratio), 15% meat (dog and cat food) and food waste,
9% saw dust, and up to 5 % plastics [25].
Anaerobic Salts Medium. The medium consisting
of 0.138 g KH2PO4, 0.176 g K2HPO4, 0.0200 g
(NH4)2HPO4, 0.2000 g NHaC1, 0.600 g MgC12 6H20,
0.2000 g FeCI2 4H20, 0.1000 g KC1, 0.1000 g CaC12,
0.0100 g KI, 0.0040 g MnC12 4H20, 0.0040 g CoC12
6H20, 0.0005 g NiCI2 6H20, 0.0005 g CuCI2,
0.0005 g ZnC12, 0.0005 g H3BO 3, 0.0005 g Na2MoO4
H 2 0 , 0.0005 g NaVO3, 0.0001 g Na2SeO3 and 1 ml
of0.1% (w/v) resazurin per liter, was autoclaved for 15
min and put under a continuous flow of nitrogen, which
was previously passed through hot copper filings
(300C) to remove traces of oxygen. When cooled,
4.2 g of NaHCO 3 and 0.25 g Na2S 9H20 were added
to the medium and the pH of the medium was adjusted
to 7.2 with HC1.
SMSWAcclimation. Serum bottles (550-ml internal
volume) containing 30 g of SMSW were purged with
nitrogen for approximately 2 rain before the above-described anaerobic medium (250 ml) was transferred into
the serum bottle under a deoxygenated nitrogen stream.
The serum bottles were sealed with butyl rubber stoppers/aluminum crimp seals and inoculated with 20 ml
of an active fermenting microbial consortium aseptically
using a syringe under a deoxygenated nitrogen stream.
The 20-ml inoculum aliquot containing microorganisms
derived from sewage sludge (Rockland, Massachusetts)
was first acclimated (by multiple transfers) to a 2 % (w/v)
solids loading of SMSW. Then this 2 % culture was further acclimated to a 5 and 10% SMSW. The 10% (w/v)

Cellulose Acetate Biodegradability

loading of solids acclimated culture was used in setting
up the 25 % solid loading serum bottles. The inoculated
serum bottles were incubated at 42C in the dark. The
total gas production was measured periodically by displacement of acidified (pH 2) water and the percentage
methane was determined on a gas chromatography (see
below for detaiIs on the gas analysis).

Culture Conditions. Serum bottles (120-ml internal

volume) were used for testing the degradability of CA
1.7 d.s. and cellophane. Weight SMSW (approximately
9 g) and the plastic samples (10 x 40-mm film sample
dimensions, total weight of plastic equal to approximately 45 rag) were added into each serum bottle. Following the procedure above to maintain anoxic and
aseptic conditions, approximately 22 ml of the anaerobic salts medium (see above for composition) was then
added to the serum bottle so as to obtain a 25 % SMSW
solids loading. These serum bottles were then inoculated by transferring aseptically to each 5 ml of the 10%
SMSW solids loading fermentation, described above,
while it was in the active methane producing phase.
Triplicate film samples for each time point investigated
were placed in each microcosm serum bottle, and four
microcosm serum bottles which included three replicate
biologically active test bottles (to evaluate vessel to vessel reproducibility) and one poisoned control bottle were
used for each plastic type. The poisoned control vessels
were obtained using the same procedure as above with
the addition of 5 M KCN (5 ml). An additional set of
three biologically active serum test bottles was initiated
with a 25 % SMSW solids loading where no plastic was
added.
Analysis. Periodically, the gas produced in the
serum bottles was released and collected by displacing
acidified water (pH 2) in an enclosed graduated cylinder
where the volume was measured. The composition of
the gas collected was analyzed by gas chromatography
(GC) using a Perkin Elmer Model 8500 GC equipped
with a 10-port (1/16-in.) zero-volume sample valve fitted with a 0.5-ml sample loop. A Carbosieve SII column (100/120 mesh, 3.05 m long 3.2 mm in. internal
diameter) and a thermal conductivity detector, both
maintained at 150C throughout the analysis were used.
The flow rate of the carrier gas, helium, was 30 ml/min.
External gas standards were used to generate calibration
curves for methane and carbon dioxide quantitation. The
detector response for these gases was linear over a range
0 to 100%. Measured gas volumes were corrected for
the presence of water vapor and adjusted to atmospheric
pressure and ambient temperature. This allowed the cal-

1147
culation of the percentage dry methane produced by the
bioreactors.
Liquid supematant samples (1 ml, not filtered) were
withdrawn weekly from the serum bottles, the pH was
measured, and then the samples were stored frozen in
glass vials for chemical oxygen demand (COD) analysis. If the pH dropped below 5.5, an alkali solution containing an equal volume mixture of 2 M sodium carbonate and 2 M potassium carbonate was used to raise the
pH to approximately 7.0. For COD analysis, culture
samples were thawed, centrifuged (13,000 g), and filtered through Gelman (Ann Arbor, Michigan) 0.2->m
Acrodisc membrane filters. The resulting filtrate was
used for the COD analysis following the dichromate reflex procedure and titrimetric method described previously [26, 27].

RESULTS AND DISCUSSION

Surface Area and Statistical Considerations in

Polymer Film Weight Loss Kinetics
The weight loss values for the exposed polymer
films are reported (see Figs. 3, 4, and 8) normalized to
the exposed surface area (including both sides of a film).
In other words, the film weight loss values were calculated by measuring the gravimetric weight loss and dividing this by the initial film surface area (units of ~/
mm:). For a sample density of 1 g/ml, a 12.5-/xg/mm 2
normalized film weight loss value corresponds to a loss
in fihn thickness of approximately I mil (0.0254 ram).
In carrying out this analysis of the film weight loss, it
is assumed that the degradation processes involved (such
as enzyme catalyzed transformations and subsequent
dissolution of degradation products) occur exclusively
at the surface of the films.
A statistical analysis of the film weight loss kinetics for identical polymer film samples within a vessel
and in different vessels has been evaluated herein. The
standard deviation values shown in Figs. 3, 4, and 8
were calculated for experiments using three biologically
active test vessels and one poisoned control vessel.
Three replicate film samples were studied for each time
point in each vessel. Therefore, the average weight loss
of polymer films and the associated average standard
deviation values reported in Figs. 3, 4, and 8 were calculated for each sampling time point based on nine film
samples for the biologically active test vessels and three
film samples for the poisoned control vessels. It was
observed that the statistical variability was rather small,
allowing for a meaningful evaluation of the weight loss

148

Gu, Eberiel, McCarthy, and Gross

results. Furthermore, a comparison of the average standard deviation for film weight loss between vessels, as
opposed to samples withdrawn from the same vessel,
resulted in rather similar statistical values. This suggests
that the methodology used herein for our simulated
compost and anaerobic bioreactors provided rather similar biological activity in different locations within a
vessel and in replicate vessels.

CA and Cellophane Degradation in Simulated

Aerobic SMSW Composting Bioreactors
The moisture content, compost dry weight loss,
pH, and polymer weight loss (erosion) were monitored
for a 7-day composting incubation period. The compost
mixture used was that described under Experimental
with the exception that the steel shavings and glass beads
were not included in this particular experiment. The
polymers investigated in this study were CA (d.s. of
1.7, film thickness of approximately 0.5-1 mil) and cellophane (film thickness of approximately 1.6 mil). We
observed that the moisture level in all of the bioreactors
remained at approximately 60 %, the pH increased from
7.4 +_ 0.2 to 8.2 +_ 0.2, and the compost dry weight
decreased from 78 _+ 43.3 to 42 +_ 3.8 g (a 46% loss)
from day 0 to day 8 (see Fig. 2). A prolonged time of
composting simulation, from 8 to 25 days, where the
moisture content was maintained at approximately 60 %,
showed an additional compost mixture dry weight loss
to 32 _+ 4 g (a 59% decline) and a pH increase to 8.8
+_ 0.2. Furthermore, the internal temperature of the

100

[~

Moisture C~ntent (%)

Compost Dry Weight (g)
pH value

compost material in the vessels remained equilibrated

with the external environmental chamber at 53 +_ 2C
throughout the incubation. It is important to note that
the pH of composting environments normally increases
from near neutral to alkaline as the composting proceeds
[28, 29]. Furthermore, the reduction in dry mass of
composting material is quite variable and dependent on
the material components presented in the process and
homogeneity of distribution [30, 31]. Moisture content
and air-flow rate are convenient and useful process control parameters in composting, but they are poor means
of comparing the waste status of dissimilar organic materials as this relates to microbial activity [32]. A moisture content between 50 and 60 % is favorable, whereas
70% is considered too high [31]. Although of great
complexity, the interaction of microbiological activities
and associated chemical and physical changes involved
in composting show remarkable consistency during repeated process operation if certain key environmental
parameters are controlled.
The weight loss values for CA 1.7-d.s. and cellophane films are shown in Fig. 3. About 35 % of the film
weight loss were observed on day 4 in the biologically
active bioreactors, while there was almost no change for
the films in the poisoned reactor. Both samples had
completely disintegrated at the end of a 7-day exposure
period and were no longer recoverable. Similar film
weight loss values for 7-day exposure periods were observed for biologically active test vessels which contained the steel shavings and glass beads components
(see Experimental) of the SMSW mixture (data not

30

12

Biere~etor ~
1.7 d.s.)
Poisoned (CA 17 d.s.)
Biore~ctor (Cellophane)
Poisoned (Cellophane)

80

.E

I
~I

! 2
0 I2

o~
60

40

o
D

E
E

20

o_
or,
-~

"5

~w

10

~
1.2

20

1,6

'~

<x

0
0

4
Time (dciys)

Fig. 2. Moisture content, dry weight of the compost, and pH during

degradation of CA ( 1.7-d. s.) and cellophane films. Standard deviation
values are indicated by vertical lines.

N
0

Time (doys)
Fig. 3. Weight loss of solution-cast 1.7-d.s. CA and cellophane films
in composting bioreactors and poisoned control. Standard deviation
values are given above the bars.

Cellulose Acetate Biodegradability

149

30

CA ( 2 5

2.2

E
E

1
[~

20

cleaving the glycosidic linkages between sugar residues

[33], then it must take a significantly extended time period for the 2.5-d.s. CA relative to the 1.7-d.s. CA to
achieve the degree of deacetylation required for cellulase attack.

d.s.)

Active Bioreactors
Poisoned Bioreactors

2
o

CA and Cellophane Degradation in Simulated

MSW Anaerobic Bioreactors

2.3
o~

7=

10

1.4

1.8

10

15

2O

Time ( d a y s )

Fig. 4. Weight loss of solution-cast 2.5-d.s. CA films in composting

bioreactors and poisoned controls. Standard deviation values are given
above the bars.

shown). In contrast, the samples exposed in the poisoned control vessels (see Experimental) showed comparatively small weight loss values (see Fig. 3). Therefore, it may be concluded that the disappearance of these
materials over the 7-day exposure period was due in
large part to biologically mediated processes.
Cellulose acetate of relatively higher degree of substitution was also exposed to the compost bioreactors.
The weight loss of CA 2.5-d.s. films (film thickness,
- 2 rail) was observed periodically over an 18-day exposure period (see Fig. 4). The study was carried out in
an identical manner to that for the CA 1.7-d.s. and cellophane films except that the steel shavings and glass
beads components to the SMSW recipe were included
(see Experimental and above). By the end of the 18-day
exposure period, the weight loss was 23.5 + 2 #g/ram 2,
which corresponded to essentially complete disappearance of the polymer films. In the poisoned reactors, CA
2.5-d.s. films showed negligible weight loss over an 18day exposure period. Once again, by comparison of the
biologically active and poisoned reactor results, it may
be concluded that the disappearance of CA 2.5 d.s. can
be attributed in large part to biologically mediated processes. In addition, as would be expected, the 1.7-d.s.
CA films showed much more rapid degradation kinetics
relative to the 2.5-d.s. film samples. If deacetylation is
indeed the first step in the biodegradation of CA of d.s.
<0.8 as suggested by Reese (see Ref. 15 and the discussion above), and accepting the idea that a critical degree of deacetylation of CA is required (to a d.s. of approximately 0.8) before cellulase enzymes are active in

Methane is the terminal product of a series of biologically mediated reactions involved in solid waste decomposition under anaerobic conditions such as those
observed in moist landfills [10-13]. The percentage of
methane in the total gas is a direct indicator of the methanogenic activities. In other words, the percentage
methane indicates that the required balance of microbial
populations has developed (such as fermentative, acetogenic, and methanogenic species) to sustain efficielit
waste decomposition. In addition, rapid and sustained
total gas production values, which show a corresponding and characteristic increase, and the chemical oxygen
demand (COD) values, which show a decrease for specific stages of the gas production profile, are useful indicators that the desired biological activity was obtained
[10, 27, 34].
The biological activity was immediately initiated
when methanogenic conditions were created in the serum
bottle which contained 10% SMSW solids loading (see
Experimental) and inoculum derived from sewage
sludge. This is evident since there was an apparent lag
phase for neither the biogas production or the percentage methane (see Fig. 5). The total gas production
reached about 60 L/kg within a 90-day time period, and
the percentage methane reached approximately 60 % by
day 30. Therefore, successful methanogenic conditions
for SMSW degradation were obtained in this test vessel.
This fermentation at day 24 was used to inoculate the
25% SMSW solids loading test bottles containing the
plastic samples.
The COD values (see Fig. 6) for the 25 % SMSW
solids loading serum bottles increased from 6000 to
40,000 mg/L in 24 days and then rapidly decreased for
extended fermentation. The initial increase in the COD
is expected and corresponds to the production of soluble
organic substrates by fermentative and acetogenic microorganisms. This results in the production of large
quantities of biogas (see Fig. 7A). In addition, the percentage methane reaches maximal values at the same
time when the maximum COD value was measured (Fig.
7B). Subsequent decreases in the COD values correspond to the utilization of the soluble organics and a
declaration of the total gas production. These general

150

Gu, Eberiel, McCarthy, and Gross

,40

O No p l a s t i c added

0 Total gas
~ % Methane

120

.....

~f{--'~{~"

Cellophane

80

CA

(t.7, d.s.)

JA4
2

>
t,,
to
<
(D
_J

4o*

&

eft

20

0~

20<

4oj 1
0

20

40

60

80

100

TIME (days)

Fig. 5. Total gas production and percentage methane in inoculumacclimation microcosms containing 10% solid loading of SMSW. Arrow indicates the time at which the fermentation was used as an inoculum for the 25 % solid loading SMSW serum bottles containing

O~ g

20

40

60

I
80

100

tIME (days)
100

plastics.

50000

0 No plastic a d d e d
Cellophane
40000

V CA (1.7, d.s.)

60

-~',

'~'

40

[ ~
~ //"

"~" 50000

0 No plastic a d d e d
Cellophane

f~C~/

V CA (1.7, d.s.)

2o {

S
o 20000

0..~

10000

(B)
l

20

40

60

80

100

TIME (days)
I
10

20

I
30

i
4O

5O

Time (deys)

Fig. 7. Total gas production (A) and percentage methane (B) in plastic degradation microcosms. All curves shown are the average o f triplicate serum bottles.

Fig. 6. Chemical o x y g e n demand (COD) change in plastic degradation microcosms over time. The values presented are measurements
made on a selected serum test bottle containing cellophane, CA (1.7
d.s.), or no plastic.

characteristics have been observed in other laboratories

during the study of anaerobic organic digestion processes [10, 11] and are consistent with the establishment
of a healthy anaerobic biological reactor.
The methane concentration reached the 30% level
for the 25 % SMSW solids loading test vessels within a
5-day period during the fermentation and subsequently

remained at this level for about 7 days. This lag phase

may have resulted from a pH decrease which is associated with the initiation of the SMSW degradation when
carbohydrates and soluble organic carbon become available to the microorganisms. Similar observations by
other workers have established that a pH decrease can
inhibit gas production when it drops too low without
proper adjustment [10, 11, 35]. The percentage methane
reached between 60 and 70% by day 25, then oscillated
around 70% for extended fermentation times (see Fig.
7B). In addition, a large gas production was rapidly es-

Cellulose Acetate Biodegradability

tablished (see Fig. 7A). The gas production for these
serum bottle test reactors followed induction-acceleration-deceleration-acceleration-deceleration phases. A
similar trend was previously reported by this laboratory
for an altemative vessel design and experimental method
[11]. However, the microbiological activity was enhanced using the methodology reported herein relative
to previous reports for in-laboratory anaerobic bioreactor SMSW digestion simulations [11, 12]. From the
above, it therefore may be concluded that the biological
reactors containing 25 % SMSW solids loading showed
substantial gas production and percentage methane values, suggesting that they did indeed obtain a balance
microbial consortium capable of achieving fermentation
and subsequently the methanogenesis of the SMSW.
Cellophane and CA 1.7-d.s. films were exposed to
the 25 % SMSW solids loading serum bottle microcosms
for 30- and 60-day periods. The weight loss for these
films as a function of the exposure time is shown in Fig.
8. For a 30-day exposure, only visually distinguishable
fragments of the films were recovered. This corresponds
to weight loss values for the CA and cellophane films
(film thickness of approximately 0.5-1.0 and 1.6 rail,
respectively) of 8.5 2 and 20 2 vg/mm 2, respectively. Further incubation of these films for an additional 30 days resulted in the complete visual disappearance of these samples. In contrast, exposure of the
CA 1.7-d.s. and cellophane films to the poisoned control vessels (see Experimental) resulted in weight loss
values of 0.5 0.5 and 1 _+ 0.5 t~g/mm 2, respectively.
It is important to note that no biogas was produced by
the poisoned bottles so that any degradation observed in
these bottles would measure that which may be attributed to non-biologically mediated processes. From the
above analysis, it appears that both the cellophane and
the CA 1.7-d.s. films, when exposed to the anaerobic
bioreactor microcosm, were degraded in large part by
biologically mediated processes.
Cellulose, a natural polymer widely present in
plants, can be degraded under aerobic as well as anaerobic conditions by cellulase enzymes produced by pure
and mixed bacterial cultures [36, 37]. Xylan, a polysaccharide built from xylose units, occurs in association
with cellulose in higher plants. Recently, acetylxylan
esterase activity, associated predominantly with the cells
and not the culture fluid, has been found for the anaerobic bacterial species Butyrivibrio fibrisolvens with an
acetylated xylan (11% acetyl, w/w) [38]. These bacteria, ubiquitously distributed in the gastrointestinal tracts
of mammals, are saccharolytic. An individual species is
capable of fermenting cellulose, xylan, starch, and other
plant cell polysaccharides. Although the precise roles of

151
50

Cellophane

[ ~ ] CA (!.7, d.s.)
2
(h44)

E 2oP

~I

-~ ~0

30

60

Time (,doys)

Fig. 8. Weight loss of cellophane and CA (1.7 d.s.) over time of

incubation. Numbers above the bars represent the corresponding standard deviation vatues.

the acetylxylan esterase and other esterase activities of

B. fibrisolvens strains in the degradation of plant ceil
walls, hemicellulose, and xylans are not clear at this
time, it seems that these activities complement the xylanase activity to enhance overall polymer degradation.
From the above, and considering the wide variety of
esterase activity in complex mixed microbial environments, it is anticipated that, similar to the aerobic degradation of CA, anaerobic CA degradation proceeds by
deacetylation and subsequent cellulytic steps to achieve
the complete degradation of the substrate.

SUMMARY OF RESULTS
In-laboratory simulation of an SMSW aerobic compost process was developed and used to study the biodegradability of CA films. The replicate test bioreactors
were maintained at approximately 53 C throughout the
study by equilibration to an external environmental
heated enclosure. The desired moisture content and
C/N ratios were initially established in the test reactors
and the moisture, internal vessel temperature, compost
dry weight loss, and film weight loss were monitored
during exposure time intervals. The exposure of CA
(1.7- and 2.5-d.s.) and cellophane films to biologically
active and poisoned in-laboratory composting test vessels clearly demonstrated that these materials were biodegradable under controlled composting conditions. In
other words, the film weight less observed resulted, at
least in part, from biologically mediated processes.

152
The development o f an experimental method to test
plastic degradation in serum bottle anaerobic bioreactors
which contain S M S W was presented. Using this approach, methanogenic activity in serum bottles which
contain a 25 % S M S W solids loading was rapidly established. The characteristic gas and C O D profiles observed demonstrated that active anaerobic microcosms
were formed. F r o m experiments where C A 17-d.s. and
cellophane films were exposed to these microcosms and
to corresponding poisoned control test bottles, it is clear
that the weight loss observed in the biologically active
test bottles was due in large part to biologically mediated processes.
The biodegradability of cellophane film in the
above exposure environments was, o f course, anticipated since cellophane is simply regenerated cellulose.
In contrast, the rapid degradation o f C A films in the
aerobic and anaerobic in-laboratory biodegradation test
methods was not expected and serves to demonstrate the
excellent potential o f C A for biodegradable plastic applications. However, the methods used above limit the
conclusions which may be reached with regard to C A
biodegradability. F o r example, since film weight loss
was the only criterion used to establish biodegradability
for the environmental simulation studies presented
herein, it is not possible from this measurement alone
to conclude the degree to which the polymers were mineralized. It may be that non-biologically mediated processes such as chemical degradation, physical disintegration, and dissolution o f degradation products
contributed to the weight loss observed during these exposures. Nevertheless, the fact that little to no degradation o f the C A films was observed in the poisoned
control vessels in combination with the expectation that
the primary biodegradation products from C A will be
acetate and cellulose (possibly with a low degree o f
acetylation) suggests that C A erosion was due in large
part to biologically mediated processes. Finally, since
the run-to-run (as opposed to between vessels within a
given run) statistical evaluation of the test methods has
not been carried out as yet, the exact values reported for
the weight loss may differ when averaged over additional trials. Future work will be carried out to address
the run-to-run reproducibility o f these test methods and
to evaluate C A mineralization during the composting and
anaerobic bioreactor processes.
W o r k is currently in progress in our laboratory to
study the mechanism of C A biodegradation using microbial isolates and their corresponding enzymes [22].
It is expected that by the careful investigation of C A
degradation mechanisms, our understanding o f p o l y m e r

Gu, Eberiel, McCarthy, and Gross

biodegradation structure property relationships will be
significantly extended.

ACKNOWLEDGMENTS
We are grateful for the financial support received
from 3M (Division of Environmental Engineering and
Pollution Control, support for J . - D . G . ) and the Army
Research Office, Biological Sciences Division, under
Grant DAAL03-90-G-0111. In addition, we wish to
thank Drs. Robert Gardner, Charles Buchanan, and Alan
White from Eastman Kodak for their encouragement in
pursuing biodegradation studies on C A and for results
which they shared with us on C A degradability.

REFERENCES
1. G. B. Willson and D. Dalmat, BioCycle 24, 20-23 (1983).
2. J. Glenn and R, Spencer, in BioCycle 32, 34-84 (1991).
3. J. H. Crawford, in Biotechnology: Applications and Research,
P. N. Cheremisinoff and R. P. Ouellette, ed. (Technomic, Lancaster, PA, 1985), pp. 68-77.
4. R. E. Hungate, Method. Microbiol. 3B, 117-132 (1969).
5. B. Schink, in Biology of Anaerobic Microorganisms, A. J. B.
Zehnder, ed. (John Wiley and Sons, New York, 1988), pp. 771846.
6. C. R. Woese, Microbiol Rev. 51,221-27l (1987).
7. M. T. Wolin and T. L. Miller, in Biology oflndustrial Microorganisms, A. L. Demain and N. A. Solomon, eds. (Butterworths, Boston, 1985), pp. 189-221.
8. J. G. Ferry, J. BacterioL 174, 5489-5495 (1992).
9. W. Gujer and A. J. B. Zehnder, War. Sci. Technol. 15, 127-167
(1983).
10. M. A. Barlaz, D. M. Schaefer, and R. K. Ham, AppL Environ.
Microbiol. 56, 56-65 (1989).
11. G. P. Smith, B. Press, D. Eberiel, R. A. Gross, S. P. McCarthy,
and D. L. Kaplan, Polym. Mat. Sci. Eng. 63, 867-871 (1990).
12. G. P. Smith, B. Press, D. Eberiel, S. P. McCarthy, R. A. Gross,
and K. L. Kaplan, Polym. Mat. Sci. Eng. 63,862-866 (1990).
13. J. M. Suflita, C. P. Gerba, R. K. Ham, A. C. Palmisano, W. L.
Rathje, and J. A. Robinson, Environ. Sci. Technol. 26, 14861495 (1992).
14. M. Z. A. Khan and Z. H. Abu-Ghararah, J. Environ. Eng. 117,
376-380 (1991).
15. E. T. Reese, Ind. Eng. Chem. 49, 89-92 (1957).
16. K. M. Downing, C. S. Ho, and D. W. Zabriskie, Biotechnol.
Bioeng. 29, 1086-1096 (1987).
17. H. Lee, R. J. B. To, R. K. Latta, P. Biely, and H. Schneider,
AppL Environ. MicrobioL 53, 2831-2834 (1987).
18. O. Fuchs, in Polymer Handbook, J. Brandrup and E. H. Immergut, eds. (John Wiley & Sons, New York, 1989) pp. Vll/400.
19. E. Luthi, N. B. Jasmat, and P. L. Bergquist, AppL Microbiol.
Biotechnol. 34, 214-219 (1990).
20. C. Buchanan, R. M. Gardner, and A. White, presented at the
Conference on Biodegradable Materials and Packaging, Natick,
MA, June (1992).
21. R. A. Gross, J.-D. Gu, D. T. Eberiel, M. Nelson, and S. P.
McCarthy, in Fundamentals of Biodegradable Materials and
Packaging, D. Kaplan, E. Thomas, and C. Ching, eds. (Technomic, Lancaster, PA, 1992).

Cellulose Acetate Biodegradability

22. M. Nelson, S. P. McCarthy, and R. A. Gross, Polym. Mat. Sci.
Eng. 67, 139-I40 (t992).
23~ J.-D. Gu, S. P. McCarthy, G~ P. Smith, D. Eberiel, and R. A.
Gross~ Polym. Mat. Sci. Eng. 67, 230-231 (1992).
24. J.-D. Gu, M. Gada, G. Kharas, D. Eberiel, S. P. McCarthy, and
R. A. Gross, Polym. Mat. Sci. Eng. 67, 351-352 (1992).
25. H. B. Goatoas, Composting Sanitary Disposal and Reclamation
of Organic Waste (World Health Organization, Geneva, 1966)~
26. Anonymous, J. Water Pollut. Control Fed. 36, 1479-1481
(1964).
27. K. Gocke and H. G. Hoppe, in Microbial Ecology o f a Brackish
Water Environment, G. Rheinheimer, ed. (Springer-Verlag, New
York, 1977) pp. 6t-70.
28. A. M. Fogarty and O. H. Tuovinen, Microbiol. Rev. 55, 225233 (1991)~
29. P. F. Strom, AppL Environ. Microbiol. 50, 899-905 (1985).

153
30. R. T. Haug and W. F. Elisworth, BioCycle 32, 56-62 (t99l).
31. D..L Suler and M. S. Finstein, AppL Environ. Microbi~f. 33,
345-350 (! 977).
32. T. D. Brock, AppL MicrobioL 29, 495-.501 (1975).
33. W. G. Glasser, B. K. McCartney, and G. Samaranayake, Presented at the American Chemical Society meeting, Washington,
DC, Aug. (1992).
34. E. Senior and M. T. M. Balba, in Microbiology of Landfill Sites,
E. Senior, ed. (CRC Press, Boca Raton, FL, 1990), pp. 17-58.
35. P. L. McCarty, Public Works Sept,, 107-112 (1964).
36. S. Soundar and T. S. Chandra, J. lndust. MicrobioL 2, 257-265
(1987).
37. S. Sonndar and T. S. Chandra, J. Indust. Microbiol. 5, 269-276
(1990).
38. R. B. Hespell and P. J. O'Bryan-Shah, Appl. Environ. Microbiol. 54, 1917-1922 (1988).