Cellulose Degradation by Three Strains of Bacteria Found in the Gut of Zootermopsis angusticollis

Item

Title

Eng Cellulose Degradation by Three Strains of Bacteria Found in the Gut of Zootermopsis angusticollis

Date

1995

Creator

Eng Peterson, Shane

Subject

Eng Environmental Studies

extracted text

Cellulose Degradation by Three Strains of Bacteria Found in the Gut of
Zootermopsis angusticollis.

by

Shane Peterson

a thesis
submitted in partial fulfillment
of the requirements for the degree of
Master of Environmental Studies
The Evergreen State College
September 1995

This Thesis for the Master of Environmental Studies Degree by

Shane Peterson
has been approved for The Evergreen State College
by

Richard Cellarius

James Neitzel

Terry B. White

Abstract
Cellulose Degradation by Three Strains of Bacteria Found
in the Gut of Zootermopsis angusticollis.
by
Shane Peterson
Cellulose is the most abundant renewable organic molecule in the world,
making it a huge resource of renewable energy. However, cellulose, along with
lignin and hemicellulose, the other components of lignocellulosic materials, is
very resistant to biological degradation. Lignocellulosic by-products constitute a
large and growing waste stream during industrial conversion of plant materials
into refined goods. This waste stream is currently disposed of by land filling,
dilution, or combustion. With disposal costs rising, other strategies must be
developed to handle the lignocellulosic materials.
Biological digestion of lignocellulosic materials can convert waste streams
into valuable products. Most biological conversions are aimed at changing
cellulose into ethanol to replace fuels currently supplied by petroleum. Cellulases,
a family of enzymes, digest cellulose into glucose units. Cellulases are found in
niches where lignocellulosic materials are abundant. The gut of wood-eating
termites contain symbiotic organisms that digest cellulose. In this study, three
different strains of bacteria were isolated from Zootermopsis angusticollis, the
damp wood termite. These isolates were tested for their ability to digest cellulose
using carboxymethylcellulose and crystalline cellulose as indicators.
These isolates, identified as members of the Bacillus genus, contained
carboxymethylcellulase activity, as measured by a reduction in viscosity and an
increase in reducing ends in media containing carboxymethylcellulose.
Additionally, two of the isolates demonstrated the ability to digest crystalline
cellulose as measured by dye release from dyed cellulose and metabolism of
microfine crystals of cellulose. These assays demonstrated that two of the isolates
had complete cellulolytic systems capable of digesting crystalline and modified
cellulose. The results from these assays, which establish the capabilities of the
cellulase systems, show potential industrial value for these two isolates.

Table of Contents
Page
List of Tables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
List of Figures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cellulose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Microbial cellulases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Industrial importance of cellulase. . . . . . . . . . . . . . . . . . . . . . . . .
Research goals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1
1
3
5
8

Materials and Methods.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Termites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Collections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Dissections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Isolations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Inocula. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Controls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Screening. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Results and Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation of bacteria. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cellulolytic screening. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Identification of candidate organism genus. . . . . . . . . . . . . . . . .
Growth rates.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantitative assays.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Carboxymethylcellulose assays. . . . . . . . . . . . . . . . . . . . . . . . . .
Crystalline cellulose assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary of results. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

16
16
16
16
20
20
22
26
29

Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Assays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Comparisons to other cellulase producers. . . . . . . . . . . . . . . . . .
Industrial value.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

32
32
32
34
35

iii

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Appendix A. List of Abbreviations and Acronyms. . . . . . . . . . . . . . . . . . . . . . . 43
Appendix B. List of Microbiological Assays.. . . . . . . . . . . . . . . . . . . . . . . . . . . 44

iv

List of Tables
Page
Table 1. Bacterial Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Table 2. Physical Characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Table 3. Growth Response.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

List of Figures
Page
Figure 1. Line Drawing of Cellulose. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Figure 2. Hydrogen Bonding Within Cellulose. . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Figure 3. Schematic Diagram of Trigger Circuit. . . . . . . . . . . . . . . . . . . . . . . . . 13
Figure 4. Growth Curves. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Figure 5. Change in Viscosity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Figure 6. Change in Reducing Ends.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Figure 7. Digestion of Crystalline Cellulose. . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

v

Acknowledgments
I was encouraged to begin this project by Terry White and Burt Guttman,
both from The Evergreen State College (TESC). They saw my interest in the
subject of biodegradation and bioremediation and removed barriers to my progress
in attempting a biochemical study of cellulose degradation by bacterial symbionts
isolated from the damp wood termite. Working without significant funding, I was
forced to develop low cost strategies and experiments. In this vein, Dr. White and
Dr. Guttman helped me utilize existing supplies and equipment to achieve my
goals.
As additional thanks, I recognize Betty Kutter, in whose lab I learned
microbiological and biochemical techniques as an undergraduate student and lab
technician. She always provided me a place to work and good words of
encouragement. I should also thank the science instructional staff at TESC, Lynne
Taylor, Marty Beagle, and Peter Robinson, without whose help I could not have
completed this project.
My thanks go to the MES faculty for their unending patience and support.
I know that without Ralph Murphy's encouragement, I would not have managed to
finish those pesky "loose ends." I also need to profusely thank my readers, Terry
White, Jim Neitzel , and especially Richard Cellarius, who in their extraordinary
willingness to help me continue this project, pushed me to finish.
Finally I wish to thank all those outside people who helped me with the
other details of life. To that end I thank my wife, Janet Peterson, my friends and
family who never failed to ask if I was done yet, Bonnie Prange, Janine Bogar and
especially Amy Morgan for her encouragement and critical reviews of my work.
All together they gave me the support and encouragement I needed.

vi

Introduction

Cellulose. Cellulose is one of the most abundant organic macromolecules in the
ecosystem (Ross, Mayer, & Benziman, 1991; Tewari, 1990). Technically,
cellulose is a polymer of D-glucose units linked by $-1,4 glucosidic bonds that
form large crystalline fibrils. In lay terms, cellulose is a long chain of carbon rings
connected through an oxygen atom between each glucose molecule (Figure 1).
When these chains are laid down next to each other, they form hydrogen bonds
which hold the chains together to form large fibers (Figure 2).

Figure 1. Line drawing of cellulose showing $ 1-4 linkages between individual
glucose units (Solomons, 1988).
Cellulose is produced by a variety of plants and bacteria, plus a few
animals (Ross, Mayer & Benziman, 1991). It is found both as a homopolymer
and in lignocellulosic material, a crystalline glucose polymer in a matrix with
lignin and hemicellulose in an approximate mass ratio of 2:1:1 (OTA, 1995).
Lignin is a polymer of three similar monolignol precursors, p-coumaryl, coniferyl,
and sinapyl alcohols (Sarkanen et al., 1991). These alcohols include a benzene

1

Figure 2. Line drawing showing potential hydrogen bonds (dashed lines) within
and between cellulose molecules (Ross, 1991).

ring resistant to chemical and biological degradation. Hemicellulose is a group of
non-glucose sugar polymers found in lignocellulosic material. The main
components of hemicellulose are xylose, mannanose, and arabinose. This
combination of cellulose, lignin, and hemicellulose is very resistant to enzymatic
degradation (Wood & Kellogg, 1988a, 1988b). After products like sugars or
gums are extracted from agricultural plants, cellulose, along with lignin and
hemicellulose, remains a significant waste product, disposal of which poses an
increasing cost to many manufacturers.

2

Microbial cellulases. Cellulase is a family of cellulolytic enzymes that degrade
cellulose. One measure of how many cellulase systems are produced by
organisms is the number of taxonomic families found in DNA sequences. There
have been more than 30 different cellulase genes identified which have been
categorized into nine families (Saddler, 1993). As more cellulose degrading
systems are isolated and quantified, this number will grow. Complete and
incomplete cellulose degradative systems are synthesized by many
microorganisms, including fungi, actinomycetes, gliding bacteria, and true
bacteria. Incomplete cellulase systems are those collections of proteins that are
missing one enzyme, or have reduced functionality in cellulose digestion.
Cellulose degradation occurs in three general steps (Bisaria & Mishra,
1989; Klyosov, 1990). First, the long chain polymer is degraded into random
lengths of 4 to 6 glucose units by an endoglucanase, which is often exported to the
environment outside the cell. Second, an exoglucanase cleaves the shortened
chains into dimers. This enzyme is exported or held in close association with the
outer membrane. The final step is cleavage of the dimer into glucose by a $glucosidase, most commonly located within the cell.
Microorganisms with cellulase systems inhabit many different niches, but
thrive mainly in areas of higher concentrations of cellulose, such as soils, plants,
and in the guts of plant-eating animals and insects. Termites host a large variety
of symbiotic microorganisms in their gut that includes several cellulose degrading
aerobic and anaerobic bacteria (Bignell, Oskarsson & Anderson, 1980; Eutick,
3

O'Brien & Slaytor, 1978; Thayer, 1976, 1978). The damp wood termite,
Zootermopsis angusticollis (Banks & Snyder, 1920), is a common termite of the
west coast of the United States (Thorne et al., 1993) and contains a diverse gut
flora. However, little is known about that flora.
There is some controversy in the meager literature about termite symbionts
as to the role bacteria play in digesting cellulose. Slaytor (1990) in his review
claims that, "there is no evidence that bacteria are involved in cellulose digestion
in termites and cockroaches." But many authors have found cellulolytic bacteria
in both lower and higher Order termites. Indeed Breznak (1982) found that, "a
number of bacterial isolates have been found to possess Cx-type1 noncrystalline
cellulase activity." The argument of whether bacteria produce cellulases
necessary for termite survival could hinge on a semantical position of whether
bacteria play a ancillary or insignificant role in cellulose digestion. Lower Order
termites produce cellulases themselves and possess cellulolytic protozoa in the
hindgut (Breznak, 1982; Slaytor, 1990). Studies have shown bacteria do not
digest enough cellulose to supply all the termite's energy needs (Slaytor, 1990).
But several studies have shown that bacteria fix nitrogen that may be critical to
the termite's survival (Breznak, 1975, 1982; French, Turner & Bradbury, 1976;
Protrikus & Breznak, 1977). This suggests that any cellulase digestion by bacteria
would be beneficial to termites, although perhaps not life sustaining.

1

A list of all abbreviations and acronyms can be found in Appendix A.
4

Industrial importance of cellulase. Cellulose occupies two fundamental
industrial areas. First, as a resource in the form of pulp, fiber, and chemical or
physical feedstocks. And second, as a waste product, after processing raw
materials that contained cellulose. Both forms greatly impact industry. Resource
cellulose funds the industry, while waste cellulose carries the cost of disposal.
Industry is constantly searching for methods of improving the quality of their
product. One area of research that shows promise in enhancing the value of
cellulose, or lowering the cost of disposal, is biotechnology (Coughlan, 1990;
Dhingra et al., 1993; Hartley et al., 1987; Kirk & Chang, 1990; Pommier et al.,
1990; Wood & Ingram, 1992).
Biotechnology involves the use of isolated enzymes or fermentation
conditions to alter natural or artificial substances. Cellulases, hemicellulases, and
ligninases are enzymes designed to digest the various components of plant
material. Currently cellulases and hemicellulases, mostly xylanases, are used to
modify the structure of cellulose-containing substances. Commercial ligninases
remain unavailable in quantity (Coughlan, 1990; Kirk & Chang, 1990). Even so,
modification of pulp with cellulase and hemicellulase enhances lignin removal
and lowers the amount of chlorine bleaching required to remove the remaining
lignin (Kirk & Chang, 1990). The Weyerhaeuser Company, one of the world's
largest pulp and paper manufacturers, currently uses several commerciallyavailable cellulases to improve pulping of wood chips. Piles of wood chips are
sprayed with a slurry of water and enzyme and allowed to sit for several hours
5

before processing. Partial digestion of the chips enhances the mechanical pulping
process, reducing effluents and cost (Brooks, 1994).
Additional uses of cellulases include treatment of chemical and animal
feedstocks. Uses of cellulosidic enzymes on straw can improve the digestibility of
animal feedstocks by improving the ratio of carbohydrates to the undigestible
fiber (Coughlan, 1990; Dhingra et al., 1993). Care needs to be exercised in the
treatment of silage though, because excessive treatment leads to collapse of the
cell walls and spoilage (Coughlan, 1990). Feedstocks used by the chemical
industry benefit from pretreatment with cellulosidic enzymes by making sugars
readily available for processing into ethanol or acetone while reducing the
mechanical strength of the feedstock (Dhingra et al., 1993; Wood & Ingram,
1992). However, the ready supply of a low-cost competitor for chemical
feedstock, petroleum, has limited the research and development of additional
enzymatic manipulation schemes for lignocellulosic materials (Hartley et al.,
1987; OTA, 1995; Saddler, 1993). Cellulose remains a moderate cost alternative
to petroleum-based chemical feedstocks, that someday may replace chemicals
derived from higher priced oil. In addition to resource cellulose, waste cellulose
is becoming a valuable source of raw materials. Enzymatic digestion of the waste
stream in many processes that involve cellulose improves the quality and reduces
the quantity of by-products, allowing easier utilization of valuable raw materials.
Often the waste stream of cellulose is a water-saturated mass, ideal for
microbial degradation but difficult to burn as fuel, a common disposal method.
6

Current industrial processes are aimed at developing enzymatic treatments of
waste cellulose to produce sugars (glucose), gaseous fuels (methane and
hydrogen), and liquid feedstocks (ethanol) (Coughlan, 1990; Dhingra et al., 1993;
Hartley et al., 1987; Saddler, 1993). Although not available in quantity, ligninase
shows promise in removing chlorinated compounds common in the bleaching
process waste stream in several pilot projects (Kirk & Chang, 1990).
Another waste stream where cellulases are utilized is the recycling
industry. For example, cellulases assist in deinking recycled paper by apparently
degrading the ink-containing outermost fibers and allowing easier removal of the
ink (Young, 1994; Zeyer et al., 1994). In addition to deinking, cellulases are used
to enhance the freeness, the rate of water drainage in pulp (Sarkar, Cosper &
Hartig, 1995), and upgrade the quality of recycled pulp fibers (Stork et al., 1995).
Most of these treatments of recycled fibers by cellulases are designed to lower the
energy input in the pulping process by providing more uniform, freer cellulose
fibers that integrate into the virgin fiber mix to form a consistent product. Even
with cellulase-driven enhancement of recycled fibers, limitations exist to the
extent cellulose fibers can be recycled. In the pulping process, fiber shortening
and degradation occur over time, limiting used fiber input to 35% by mass
(Virtanen and Nilsson, 1993). Cellulases are used to homogenize the fiber length
by degrading the shorter fibers preferentially over longer fibers.
Although purified enzymes allow more consistent control of the product,
living organism enzyme systems lower the overall cost of utilizing a resource
7

(Hartley et al., 1987; Saddler, 1993). Bacteria containing cellulolytic systems that
produce alcohol during sugar metabolism are very low cost producers of a
valuable resource. Bacterial fermentation of lignocellulosic materials can produce
ethanol at competitive rates with corn and sugar beets (Hartley et al., 1987; OTA,
1995; Saddler, 1993). The economic disadvantage in producing ethanol by
enzymatic fermentation includes the current restriction of monoculture input,
limiting sources to one type of material (OTA, 1995), and the low cost of
petroleum alternatives (Hartley et al., 1987; OTA, 1995; Saddler, 1993).

Research goals. This research focused on isolating symbiotic termite
microorganisms capable of digesting cellulose. Isolates were compared to known
cellulase producers and additional experiments were developed to further
characterize the cellulolytic abilities of each organism. Conclusions regarding the
industrial value of these isolates are discussed where appropriate.

8

Materials and Methods

Termites
Collections. Several dozen termites identified as Zootermopsis angusticollis, the
damp wood termite, were collected in September from a nest near Young's Cove,
Section 30, Range 2W Township 19N in Thurston County, State of Washington.
Identification of the termites was kindly provided by Dr. John Longino and Dr.
Robert Sluss of The Evergreen State College in Olympia, Washington. The
habitat surrounding the nest was second growth Pseudotsuga menziesii (Douglasfir), coastal fringe. The nest was located in an overturned Douglas-fir stump that
was partially buried. Specimens were collected from galleries that extended
below ground but still were surrounded by woody material of the stump. These
termites were kept in a 20 L container with Douglas-fir wood shavings for
approximately 2 weeks before dissection. No nutritional additions, except
sterilized deionized water, were provided during the 2 weeks.

Dissections. Dissections were carried out as described by Bignell, Oskarsson and
Anderson, (1980); Breznak and Pankratz, (1977); Hendee (1933); and Thayer
(1976). Termites were separated into two groups of fully developed workers and
soldiers. Approximately half the collected termites were used in the dissection
preparation. Prior to dissection each termite was exposed to a germicidal UV
lamp for 15 minutes with agitation of the open glass dish to ensure complete
9

exposure of the dorsal and ventral areas. Additionally, each termite was then
washed in a 70% ethanol solution. Termites were placed in four separate fivemember groups for dissection. One termite from each pool was randomly selected
and rolled over a tryptic soy broth (TSB) (Difco Co., Detroit, MI) agar plate to
check for external contamination. No growth occurred after overnight incubation
of the agar plates at 30EC. Each termite was decapitated using a scalpel under a
dissecting microscope and the digestive tract, including the crop, was pulled
through the opening. Each of these digestive tracts was carefully rinsed externally
with sterilized deionized water before being added to the pool.

Isolations. After dissection, each group of digestive tracts was either coarsely
chopped with a scalpel or homogenized with a sterile mortar and pestle in the
presence of alumina. Four samples (100 :l) of the liquid material from each pool
were then spread onto TSB plates and incubated at 30EC. Over several days each
unique colony type, based on surface morphology or color was sampled and
strains were isolated by repeated streaking on TSB plates. Twenty-four isolated
strains were stored at -70EC according to Maniatis, Fritsch and Sambrook, (1982).

10

Assays

Inocula. Samples were taken from -70EC glycerol stocks and grown overnight on
TSB agar plates at 30EC. Bacteria from these plates were either used directly to
inoculate experiments, or grown overnight in liquid TSB media in a shaking water
bath at 30EC and then used as inocula. Experiments which generated quantified
data were inoculated with measured volumes of overnight cultures. Experiments
that utilized cells but were not quantified, such as cell staining, were from cultures
grown overnight on TSB agar plates. A list of standard tests and media
preparations can be found in Appendix B.

Controls. Bacillus subtilis, wild type (B. subtilis, lab strain) and Escherichia coli,
K803 (E. coli, lab strain) were selected as positive and negative controls in all of
the following studies. Bacillus subtilis displays CMCase activity (Chan & Au,
1987; Lo et al., 1988) and limited cellulase activity (Chan & Au, 1987; Lo et al.,
1988). E. coli possess neither enzyme activity (Balows et al., 1992), but was used
to establish growing culture conditions, such as pH changes and depletion of
nutrients, without utilizing the cellulose additions of interest. E. coli cultures
were included in the experiment design to eliminate the possibility that changes in
cellulose properties, such as viscosity reductions or increases in reducing ends,
were due to changes in the media generated by the growth of cells and not the
enzyme systems being tested.
11

Screening. Bacterial species were screened for cellulolytic activity by
liquification of a 2.5% carboxymethylcellulose (CMC) solution (10 ml)
containing medium viscosity CMC (Sigma Co., St Louis, MO) in minimal salts
media (M9) as measured by visual inspection of the agitated test tubes containing
each culture of interest. Bacteria were also screened for growth on M9 agar plates
containing CMC or deionized water washed crystalline cellulose as the sole
carbon source.

Assays. Presence of CMCase activity was determined by measuring changes in
viscosity and reducing ends in media containing CMC (Chan & Au, 1987; Kricke
et al., 1994; Thayer, 1976, 1978; Wood & Kellogg, 1988a). Cellulase activity
was measured in test using both dyed and undyed crystalline cellulose in
procedures described by Weimer, Lopez-Guisa and French (1990) and Wood and
Kellogg (1988a). Three species that grew on cellulose or CMC-agar plates or
modified a liquid CMC medium by reducing viscosity were tested in a quantified
series of experiments for the ability to liquify CMC as measured by viscosity
change using a slightly modified Thomas-Stormer viscometer. The viscometer
was modified in the following manner. A Pasco Model 9260 photo-reflective
sensor was attached to the face of the analog dial and the output from the sensor
was connected to an Gerbrands Model G1270 electronic timer via a switching
circuit seen in Figure 3. When the analog clock hand passed the sensor it
triggered the timer to start. One full circuit of the analog
12

Figure 3. Schematic diagram of trigger circuit modification to Thomas-Stormer
viscometer.

dial was chosen because it was a convenient time span of 9 to 13 seconds,
allowing several repetitions of the experiment during a brief time period.
Additionally, one full circuit minimized the complexity of the electronic circuitry
required to trigger the timer by requiring only one pass of the analog clock hand.
Upon completing a full circuit, the hand again triggered the timer to stop, yielding
a time in seconds to four significant figures. This modification allowed repeatable
time measurements to within 0.1%, approximately an order of magnitude
improvement over hand timing with a stop watch.

13

Liquid media were inoculated with a 1000-fold dilution from a fresh
overnight culture into M9 medium containing 2.5% CMC and 0.25% casamino
acids (Difco Co., Detroit, MI), called CMCCA. Cultures were placed in a shaking
water bath at 30EC. Samples (6 ml) were removed from the flasks at regular time
intervals and subjected to centrifugation at 4,100g for 15 minutes. A sample (4
ml) of the supernatant was removed, taking care not to disturb the pellet, from
these centrifuged samples, diluted 20-fold into water and tested for change in
viscosity at 20EC. Results are given as percent change between samples and the
ratio of uninoculated CMCCA and water.
Cultures containing CMC were also assayed for reducing ends using the
3,5- dinitrosalicylic acid method as described by Baum and Bowen (1972). A
sample (100 :l) was removed from the centrifuged samples and added to 400 :l
3,5-dinitrosalicylic acid. The mixture was incubated 5 minutes in a water bath at
100EC. Sample absorbance was measured at 540 nm after dilution to a total
volume of 2 ml. Results are given as micromoles of reducing ends per ml, based
on a glucose dilution standard curve.
Crystalline cellulose assays were performed in two experiments. The first
experiment examined the release of dye from cellulose azure (Sigma Co., St
Louis, MO) according to methods described by Wood and Kellogg (1988a).
Liquid media were inoculated with a 1000-fold dilution from a fresh overnight
culture into M9 medium containing 0.5% cellulose azure and 0.25% casamino
acids, called DCCA. Samples (750 :l) were removed, subject to centrifugation in
14

Eppendorf tubes at 14,000g for 10 minutes. Supernatant (600 :l) was removed
taking care not to disturb the pellet and absorbance was measured against
uninoculated DCCA media without dyed cellulose blank at 568 nm using a
Hewlett Packard diode array spectrophotometer.
The second experiment, developed in this investigation, involved the
apparent loss of microfine crystals of cellulose. Liquid media were inoculated
with a 1000-fold dilution from a fresh overnight culture into M9 medium
containing 1.0% undyed crystalline cellulose, EMCOCEL50M, (Mendel Co.,
Patterson, NY) and 0.25% casamino acids, called UCCA. Cultures were placed in
a shaking water bath at 30EC. Samples (1 ml) were removed, subjected to
centrifugation in Eppendorf tubes at 14,000g for 10 minutes and then most of the
supernatant was carefully removed and discarded. One milliliter 95% ethanol was
added to each tube, followed by vigorous agitation to completely resuspend the
pellet. Samples were subjected to centrifugation at 14,000g for 10 minutes to
remove water-soluble proteins, and 600 :l supernatant was carefully removed.
Microfine crystals of cellulose remain suspended in solution for hours after
centrifugation. These crystals displayed greatest light scattering at 204 nm as
measured using a Hewlett Packard diode array spectrophotometer. Decrease in
absorption of light at 204 nm indicated a decrease in microfine crystals of
cellulose. For microscopic examination, a spot of each ethanol sample (0.5 :l)
was placed on a stud, air dried, and examined using a scanning electron
microscope.
15

Results and Discussion

Isolation of bacteria. Dissection of Zootermopsis angusticollis was
accomplished under a dissecting microscope as previously described. One
member of each pool of termites was checked for external contamination.
Because no external contamination was found, all isolates were considered to
originate in the stomachs of the dissected termites. Twenty-four different strains
of bacteria determined by colony type were isolated. Fungi were discarded.
Individual colonies were isolated on TSB plates and incubated overnight (30EC)
to ensure that each species was monotypical. Next, the isolated species were
characterized by a series of standard bacterial tests (Table 1), and screened for
cellulolytic enzymes using CMC liquification assays (Thayer, 1976, 1978; Wood
& Kellogg, 1988a) and agar plate growth utilizing CMC and crystalline cellulose
as the sole carbon source (Thayer, 1976, 1978; Wood & Kellogg, 1988a).

Cellulolytic screening.
Identification of candidate organism genus. Initial cellulolytic screenings were
conducted using plate growth on various crystalline cellulose and CMC sources.
Of the 24 bacterial species isolated, only 3 displayed cellulolytic activity. Three
isolates (HW25, HWM and HG5) which showed consistent cellulolytic ability
were used in further studies. These isolates were further characterized by various

16

Table 1. Bacterial Characterizations of Gut Isolatesa
G ram

C atalase

O xidase

M otility

Action

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+
-

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

LT
S
TS
TS
FS

CW 1
CW 3
CW 9
C W 12
C W 14
C W 16
HW 1
HW 3
HW 7
HW 9
HW 11
HW 13
HW 15
HW 18
HW 20
HW 22
HW 25
HW M
HG 1
HG 3
HG 5
HG 12
HG 14
HG 16

Abbreviations:
Action

LT
S
TS
FS

Local tw isting
Sw imming
Tw isting sw imming
Fast sw imming

C ell shape

R
C

R ods
C occi

C olony morphology

R
SG
CC

R ound
Stab grow th
C oncentric circles

Surface texture

S
FR
MR

Slick
Fine ripples
M edium ripples

FS
LT
S
S
FS
LT
S
LT
S
S
FS
LT
LT
LT
LT
LT

C ell
shape
R
R
R
R
R
R
C
R
R
C
R
R
R
C
R
R
R
R
R
R
R
R
R
C

Surface
texture
S
S
S
FR
S
S
S
S
S
S
S
MR
S
S
S
FR
MR
FR
S
S
MR
S
S
S

C olor
G
CB
CB
C
CB
OR
CB
C
C
G
C
C
C
G
CB
C
C
WG
CB
OB
CB
CB
G
W

Length
(: M )
2
1.5
2
2
1.5
0.5
0.5
1
2
1
1
1.5
1
0.5
3
2
2.5
2
1
2
2.5
1.5
0.5
0.5

W idth
C ell
(:M ) G rouping
0.5
SD
0.5
S
0.5
M CS
0.75
SLC
0.5
S
0.5
CL
0.5
CL
0.5
S
0.5
SSC
1
S
0.5
S
0.5
MC
0.5
SD
0.5
SC
0.75
SSC
0.75
SLC
0.75
SSC
0.5
SSC
0.5
DS
0.5
S
0.75
SD
0.5
SSC
0.25
S
0.5
SC L

C olor

G
CB
C
WG
OR
OB
W

G olden
C reamy beige
C ream
W hite gray
O range red
O range beige
W hite

G rouping
O rder indicates
most prevalent
grouping

S
SD
SSC
SLC
SC
SC L
DS
CL
M CS
MC

Singles
Singles & doubles
Singles & short chains(2-4)
Singles & long chains(2-10)
Short chains(2-4)
Singles & clusters(4-10)
D oubles & singles
C lusters
M edium chains(2-6) & singles
M edium chains(2-6)

+ Positive, - N egative

a

C olony
morphology
R
R
R
R
R
SG
CC
SG
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R

A brief description of each test listed in Tables 1, 2 and 3 can be found in Appendix B.

17

bacteriological tests (Tables 2 and 3). Tests showed that all 3 isolates were grampositive facultative anaerobic rods capable of growth on a wide variety of carbon
sources and under varying pH and temperature conditions. HW25, HWM and
HG5 all grew best under slightly acidic conditions, pH between 7.0 and 6.0, and at
42EC. However, all experiments conducted were run at 30EC and pH 7.0 to
enable comparisons between these experiments and those of Thayer (1976, 1978).
Resistance was low to a variety of antibiotics except for ampicillin, which could
be the result of low membrane permeability to the antibiotic. All isolates showed
endospore formation after treatment in 80EC water for 30 minutes. Spore staining
was used to determine the location and type of spore formation.
Table 2. Physical Characterizations of HW25, HWM and HG5

Gram
Catalase
Oxidase
Motility
Cell shape
Colony color
Length (:m)
Width (:m)
Spore tests

HW25
+
+
+
Rods
Cream
2.5
0.75
+

HWM
HG5
+
+
+
+
+
+
Rods
Rods
White gray Creamy beige
2.0
2.5
0.50
0.75
+
+

+ Positive, - Negative

18

Table 3. Growth Response of HW25, HWM and HG5 to Varied Conditions

Carbon source
Glucose
Lactose
Maltose
Glycerol
CMC
Fructose
Mannitol
Sucrose
Na Acetate
Galactose
MacConkey
EMB
Purple Broth
Mannitol salt
$ hemolysis
TSI Slant
Butt
Gas
Hydrogen Sulfide
Anaerobic growth
Antibiotic resistance
Ampicillin
Streptomycin
Chloramphenicol
Tetracycline
TSB plates
pH conditions
pH5.0
pH6.0
pH7.0
pH8.0
Temperature
30EC
37EC
42EC

HW25

HWM

HG5

+
+
+
+
+
+
+
+
+
+
+
+
A
A
+

+
+
+
+
+
+
+
+
+
+
+
+
+
K
A
+

+
+
+
+
+
+
+
+
+
+
+
K
A
+

+
-

+
-

+
+
+
-

+
+
+
+

+
+
+
+

+
+
+

+
+
+

+
+
+

+
+
+

+ Growth, - No Growth, A Acid, K Alkaline

19

These test results suggest the isolates are in the genus Bacillus. According
to Balows et al. (1992) Bacillus bacteria degrade cellulose or modified cellulose.
HW25, HWM and HG5 fail to meet criteria for all genera of cellulose degraders
listed in The Prokaryotes (Balows et al., 1992), except Bacillus. In addition,
Breznak and Pankratz (1982) and Thayer (1976, 1978) report the presence of
Bacillus bacteria in the gut of lower termites. Therefore the assumption was made
that HW25, HWM and HG5 are in the Bacillus genus. No attempt was made to
characterize these bacteria to species level.

Growth rates. As shown in Figure 4, B. subtilis, E. coli, HW25 and HWM all
grow at approximately the same rate in liquid media at the stated conditions. HG5
lags slightly behind the other four bacterial strains but eventually grows to near
the maximum density of approximately 1010 cells per ml. Plate counts of cells
were hampered by the tendency of HW25, HWM and HG5 to chain in lengths of 2
to 5 cells. Cell counts were conducted using a hemacytometer once cultures
reached stationary phase.

Quantitative assays. After initial screens for cellulolytic enzymes were
successfully completed, more quantitative tests were developed using CMC and
crystalline cellulose. All tests were carried out over several weeks using inocula
from fresh overnight cultures grown on TSB media at 30EC. These test
procedures were used to minimize differences in inoculum volumes and
20

Figure 4. Growth curves for TSB cultures in 5 ml Corex centrifuge tubes in a
shaking water bath at 30EC. ! E. coli, ? B. subtilis, > HW25, Ë HWM,
× HG5
culture startup conditions. Most tests were conducted in triplicate, or were
performed several times over a period of 2 years. Results note when multiple
sampling was used.
21

Carboxymethylcellulose assays. CMCCA media (50 ml) were inoculated with
fresh overnight culture (50 :l) and incubated at 30EC in a shaking water bath.
Samples (6 ml) were taken from triplicate flasks, subject to centrifugation, and
then viscosity of the supernatant was measured using a modified Thomas
viscometer. Concentration of reducing ends in the supernatant was determined by
3,5-dinitrosalicylic acid assays.
Reduction in media viscosity was measured as described in materials and
methods. Viscometer readings (in seconds) were converted to percent change to
facilitate comparisons with other experiments (Thayer, 1976, 1978). Percent
change in viscosity was calculated from the average value of all the measurements
for the uninoculated CMCCA over the length of the experiment period versus an
average value for water. These average values were 11.91 seconds ± 0.074
seconds (P = 0.05) for the uninoculated CMCCA and 9.93 seconds ± 0.047
seconds (P = 0.05) for water. The average difference between the uninoculated
CMCCA and water was 1.98 seconds.
The bacteria (B. subtilis, HW25, HWM and HG5) displayed a decrease in
viscosity of the liquid medium and an increase in reducing ends over time (Figure
5 and 6) that is consistent with digestion of CMC chain in a random manner.
Digestion of CMC from the ends of the cellulose chain would not result in an
observable increase in reducing ends and would slowly reduce viscosity. Random
chain breakage would increase reducing ends and show rapid viscosity reduction
(Thayer, 1976, 1978; Wood & Kellogg, 1988a). The E. coli culture displayed
22

Figure 5. Change in viscosity in liquid culture over time. Liquid media (50 ml)
containing 2.5% CMC and 0.25% casamino acids in M9 salts was inoculated with
fresh overnight culture (50 :l) and grown at 30EC in a shaking water bath.
Samples were taken from triplicate flasks, subjected to centrifugation at 4,100g
for 15 minutes and viscosity of the supernatant was tested using a Thomas
viscometer. P CMCCA, ! E. coli, ? B. subtilis, > HW25, Ë HWM, × HG5; bars
represent ± one standard deviation.
23

Figure 6. Change in reducing ends in liquid culture over time. Liquid media (50
ml) containing 2.5% CMC and 0.25% casamino acids in M9 salts was inoculated
with fresh overnight culture (50 :l) and grown at 30EC in a shaking water bath.
Samples (100 :l) were taken from triplicate flasks, subjected to centrifugation at
4,100g for 15 minutes and the supernatant was tested for reducing ends using 3,5dinitrosalicylic acid assays. P CMCCA, ! E. coli, ? B. subtilis, > HW25,
Ë HWM, × HG5; bars represent ± one standard deviation.
24

slightly lower viscosity and no significant change in reducing ends over the length
of the experiment. This decrease in viscosity could result from slow digestion of
the terminal glucose ends by $-glucosidase or some other undetermined enzyme
system.
There were slight variations in values for the triplicate samples (Figure 5
and 6). Uninoculated CMCCA media viscosity was measured to determine the
consistency of the viscometer readings, based on variations in sample volume,
dilution factors and temperature readings. All viscosity measurements of identical
samples varied less than 0.1% from the mean. Measurements were done in
triplicate at each test point. Sample volumes were measured at 70, 75, 80 and 85
ml. One tenth of one percent variation in time measurements for sample volumes
centered at 80 ml fell between 78.5 ml and over 90 ml. Dilution factors were
measured at 1, 2, 3, 4 and 5 ml. One tenth of one percent variation in time
measurements for dilution factors centered at 4 ml fell between 3.98 ml and 4.02
ml. Temperatures were measured at 19.0, 19.5, 20.0, 20.5 and 21.0 EC. One
tenth of one percent variation in time measurements for temperature centered at
20.0EC fell between 19.9EC and 20.1EC. Because sample volume, dilution factor
and temperature changes were held to under 0.1% and the modified viscometer
gave repeatable measurements within 0.1%, variation of readings was due to
conditions within the growing culture and not due to fluctuations in instrument
readouts. Indeed, inocula induced changes in viscosity and reducing ends were
consistent for all tests run over a period of 2 years.
25

B. subtilis, HW25 and HWM demonstrated similar results when grown in
CMCCA. HG5 increased reducing ends and lowered viscosity more slowly than
the other isolates. However after 14 days incubation viscosities of the supernatant
were similar and reducing ends in HG5 samples were only slightly lower than the
other isolates. This delay could be a slower growth rate or lower efficiency in
cellulase activity. There were approximately 30% fewer cells in the HG5 cultures
than in the other cultures, although consistent values were not obtained due to the
viscous nature of the media and the chaining of the cells. In addition HG5 grew
more slowly on the nutrient-rich nonselective media TSB (Figure 4). If HG5
cultures were indeed 30% less concentrated than those of other bacterial cultures,
then the rate of hydrolysis of the CMC was approximately the same by HG5 as by
B. subtilis, HW25 and HWM.

Crystalline cellulose assays. Cellulolytic activity was measured in liquid media
containing either cellulose azure, a dyed crystalline cellulose, or undyed
crystalline cellulose. Release of the dye indicated cellulolytic activity in the
cellulose azure. Loss of microfine crystals in the undyed cellulose test was
assumed to be due to cellulolytic activity (Weimer, Lopez-Guisa & French, 1990).
Dyed cellulose assays are standard tests for measuring crystalline cellulose
digestion. The undyed cellulose assay was developed in this report to eliminate
the possibility that the dye in the dyed cellulose assay inhibited the digestion of
crystalline cellulose.
26

Dye release from cellulose azure was measured by an increase in
absorbance (568 nm) in the supernatant indicating cellulolytic activity (Wood and
Kellogg, 1988a). Figure 7 shows the result of a 14 day experiment that was run
concurrently with the CMC assays in Figures 5 and 6. HW25 cultures produced a
marked release of dye into the supernatant. HWM cultures produced a small, but
significant release of dye by day 14. B. subtilis and HG5 cultures showed slightly
higher levels over uninoculated DCCA and E. coli samples. These results
indicated that HW25 and HWM have complete cellulolytic enzyme systems, as
defined as the ability to digest crystalline cellulose. B. subtilis and HG5 have a
limited impact on the crystals of cellulose. Literature citations support this
limited cellulase activity in B. subtilis (Balows et al., 1992; Chan & Au, 1987;
Wood & Kellogg, 1988a).
Release of dye from the cellulose could be muted if the cells were able to
digest the dye molecule. Bacterial growth experiments on dyes used in staining
the crystalline cellulose were conducted on agar plates without additional
nutrients and showed that none of the bacteria were able to significantly digest the
dye. These controls also indicated that none of the test species had significant
ligninolytic abilities. Dyes used to color cellulose closely resemble the lignin
precursor molecule and are used in assays for ligninases (Wood & Kellogg,
1988b).

27

Figure 7. Digestion of crystalline cellulose as measured by release of dye.
Triplicate samples of liquid media (5 ml) containing 1% cellulose azure and
0.25% casamino acids in M9 media were inoculated with fresh overnight culture
(5 :l) and grown at 30EC in a shaking water bath. Samples were diluted two-fold,
subjected to centrifugation at 14,000g for 10 minutes and absorbance was
measured using a diode array at 568 nm. Results are given for undiluted samples
of 1 ml. P DCCA, ! E. coli, ? B. subtilis, > HW25, Ë HWM, × HG5; bars
represent ± one standard deviation.
28

Another crystalline cellulose assay utilized measurements of microfine
undyed cellulose crystal loss. This loss of fibers was measured as described in
materials and methods. These assays were conducted in addition to the dyed
cellulose assays to eliminate the possibility that crystalline cellulose digestion was
inhibited by the dye molecules. Microfine crystals of cellulose, less than 2 :m in
length, remained suspended in the ethanol even after centrifugation. These tests
were performed on cultures grown in UCCA at t = 14 days. When compared to
the uninoculated UCCA HWM cultures showed a 25% decrease in absorption,
and E. coli, B. subtilis, HW25 and HG5 showed a 10% decrease (results not
shown). A spot of each sample (0.5 :l) was examined using a scanning electron
microscope. The HWM spot was almost completely devoid of crystals. The
uninoculated UCCA spot contained several dozen crystals approximately 1-2 :m
in length. E. coli, B. subtilis, HW25 and HG5 spots had slightly lower numbers of
crystals than the uninoculated UCCA but all had at least one to two dozen
crystals.

Summary of results.
Both crystal cellulose and CMC assays indicated that B. subtilis, HW25,
HWM and HG5 contained enzyme systems capable of degrading cellulose.
HW25 and HWM could digest both crystalline cellulose and CMC. B. subtilis
and HG5 could digest only CMC with any detectable efficiency. Tests designed
to measure inhibition of by-products were performed, but were generally
29

unsuccessful due to the long duration of the experiments. Additions of glucose
and cellobiose, by-products from cellulose degradation, to growing samples
caused some inhibition in cellulose degradation initially (results not shown). But
inhibition of cellulose degradation disappeared after a day and maintaining
constant levels of inhibitors was difficult. Once a reasonable level of 0.25% for
casamino acids was established early in the investigation of these isolates no
further tests on supplemental nutrient optimization were conducted. Levels of
nutrient additives were considered optimal if cell density reached 108 cells/ml
within 24 hours, but cellulose degradation was not inhibited beyond the first day.
The undyed crystalline cellulose assay, the dyed cellulose assay, and the
CMC assay displayed slightly different results from the isolates. All three isolates
digested CMC, reducing viscosity and increasing reducing ends in the media. The
dyed cellulose assay showed that HW25 contains an enzyme capable of releasing
dye molecules from the crystalline cellulose. Finally, the undyed cellulose test
demonstrated that HWM removed microfine crystals of cellulose from solution.
The undyed cellulose assay was unique to this study. Although Weimer, LopezGuisa and French (1990) claimed microfine crystal loss was the result of
digestion, they failed to develop an assay. The undyed cellulose assay provides a
good alternative to dyed cellulose tests, where the presence of dye might inhibit
digestion of the crystalline cellulose. HWM displayed cellulase activity on the
undyed assay, but only marginal activity on the dyed assay. HW25 displayed
significant dye release, but the culture showed only minimal levels of microfine
30

crystal digestion. This undyed cellulose assay demonstrated different results than
the standard dyed assay used by many investigators.

31

Conclusions

Isolates. Of the 24 different strains isolated from the gut of Zootermopsis
angusticollis, only three showed consistent cellulolytic activity. These isolates
were selected for further study. B. subtilis and E. coli were selected to serve as
positive and negative controls. Tests on HW25 and HWM indicated they
contained both crystalline cellulose and CMC digesting enzyme systems. HG5
showed only CMCase activity. All three isolates grew under a variety of
moderate conditions that lend themselves to many industrial processes.
Among the three isolates, HW25 and HWM displayed complete cellulase
systems capable of digesting crystalline cellulose. In particular, HW25 released
significant amounts of dye from the crystalline cellulose after 6 days of growth.
None of the other isolates showed significantly elevated dye levels in the
supernatant until day 10 and they never released as much dye as HW25, even after
14 days of incubation. HW25 grows as rapidly as B. subtilis and to a slightly
higher titer. It grew under most conditions tested. This adaptability, cellulase
production, and rapid growth, make HW25 the best candidate for further studies.

Assays. A number of reliable assays for measuring cellulose degradation exist,
especially those of in Wood and Kellogg (1988a). The CMC assay used in these
experiments was essentially that of Thayer (1978) and Wood and Kellogg
(1988a), with slight modifications necessitated by the available equipment. The
32

major modification was the addition of the electronic trigger mechanism on the
mechanical viscometer. This modification allowed reproducible measurements on
samples over long periods of time in the experiments. The assay using dyed
crystalline cellulose was adapted from that of Wood and Kellogg (1988a) to
enable comparisons with other assays of cellulolytic activity. Adaptation of the
assay consisted of changing the medium to match those used in CMC and undyed
cellulose assays. The undyed cellulose assay was developed specifically for this
project. Development of this assay stemmed from the observation that absorption
in the UV range decreased when samples incubated for 14 days. This change in
absorption was due to the loss of microfine crystals of cellulose. Loss of crystals
was confirmed by electron microscopy. This undyed cellulose assay provided
another crystalline cellulose test that showed slight differences between HWM
and HW25, both of which contained cellulases capable of digesting crystalline
cellulose. All three assays, CMC, dyed cellulose and undyed cellulose gave
slightly different information, each unique to the specific nature of the assay.
CMC assays were simple and rapid, useful to screening unknown isolates for
CMCase activity. The dyed and undyed crystalline cellulose assays were useful in
determining cellulase activity capable of digesting crystalline cellulose.
Dyed cellulose assays are used by many investigators to measure
cellulolytic activity. The assay is inexpensive, easy to perform with a minimum of
standard laboratory equipment and yields measurable results. However, the dye
molecule used in the prepared cellulose may inhibit enzymatic digestion of the
33

crystals through competitive inhibition or lowered binding efficiency. The undyed
cellulose assay developed in this investigation has many of the same advantages
as the dyed assays. Low cost and ease of performance are the most appealing
aspects of this assay. The undyed cellulose has the additional advantage of
eliminating the potential inhibiting effects of the dye molecule. The
disadvantages of this assay are the need for a scanning electron microscope to
confirm that microfine crystals were digested and the difficulty in quantifying the
results. The signal decrease in the assay was significant, but difficult to measure
without additional treatments that increased the potential for false readings.

Comparisons to other cellulase producers. Since all the assays involved cell
cultures, comparison with purified cellulolytic proteins were hampered. B.
subtilis was chosen as a positive control because it possesses strong CMCase
activity and shows slight crystalline cellulase activity. All three isolates showed
similar CMCase activity to that of B. subtilis. HW25 and HWM surpassed the
activity of B. subtilis in digesting crystalline cellulose. Commercially available
enzymes were used to show that the assays were working; however rapid
digestion of cellulose by purified enzymes was difficult to compare to the slow
process in the growing cell cultures in which by-products of cellulose digestion
were metabolized.
Another bacterial cellulase producer, Bacillus stearothermophilus ( B.
stearothermophilus) shows great promise in industrial application, because of its
34

rapid growth rate, wide range of temperature tolerance, and production of ethanol
from lignocellulosic materials (Hartley, 1987). Isolates examined in this project
should be tested for useful fermentation products and temperature tolerance.
HW25 and HWM already show an ability to digest cellulose and grow rapidly on
simple media. Mutation and selection of HW25 and HWM might produce a strain
that produces ethanol from sugars released in the metabolism of cellulose.
Combined with a high temperature tolerance and rapid growth, these strains could
provide a viable alternative to B. stearothermophilus with yet unknown attractive
properties such as higher efficiency in cellulose digestion or better batch growth
response.
Additional tests on the isolates should measure their ability to fix nitrogen,
metabolize unusual carbon sources, or grow at extreme conditions of temperature
and pH. These tests could reveal important abilities helpful in industrial processes
to provide a low cost, but valuable conversion of raw materials into refined goods.

Industrial value. Although almost all commercially available cellulases used in
industry are purified from fungal sources, new and novel uses for cellulases are
stimulating the search for enzymes capable of specific activity under special
conditions. Purified enzymes are preferred over cell cultures in industrial settings
because of the ease of controlling the reaction in a purified enzyme system
(Brooks, 1994). If purified enzymes were available from the isolates described in

35

this report, extensive testing for optimal and extreme conditions would be
warranted.
Protein sequencing of the purified enzymes would allow a better
understanding of the protein's function, structural homologies to other proteins,
and potentially, any inherent positive or negative traits. Using current
biotechnology skills, genes could be mutated to produce proteins suited for
specific applications. One or several of the proteins in these isolates could
provide a novel solution to binding specificity or catalysis of cellulose, or other
carbon sources. These isolates contain genes that code for cellulases.
Transferring the cellulase genes into other hosts could increase the cellulase
activity of a current cellulase producer through synergism. Alternatively gene
transfer could add cellulase ability to a host that ferments glucose into a variety of
useful products.
These isolates exhibited CMCase and crystalline cellulases capable of
digesting cellulose under simple conditions. Although the cellulase systems were
not tested for unusual activities such as temperature or pH tolerance, they may
serve a specific function in the ever-changing landscape of biotechnology.
Additional studies of interest would include testing a broader range of bacteria
found in Zootermopsis angusticollis, and the isolation and purification of the
enzyme systems found. With a better understanding of the capabilities of the
isolated bacteria, industrial applications could be developed that are suitable for
the isolates. Purification of enzymes from the isolates would allow more
36

meaningful comparisons with currently available cellulases. This understanding
of the enzyme systems in the isolates would indicate where each of the enzymes
would best serve industry as a digester of cellulose.

37

References

Balows, Albert, William J. Hauser, Kenneth L. Herrmann, Henry D. Isenberg and
H. Jean Shadomy (Eds.). (1991). Manual of Clinical Microbiology. (5th
ed.). American Society for Microbiology, Washington, D.C..
Balows, Albert, Hans G. Truper, Martin Dworkin, Wim Harder and Karl-Heinz
Schleifer (Eds.). (1992). The Prokaryotes: a handbook on the biology of
bacteria: ecophysiology, isolation, identification, applications. (Vols. 14). (2nd ed.). Springer Verlag, New York.
Banks, N., and T. E. Snyder. (1920). A revision of the Nearctic termites (Banks)
with notes on biology and geographic distribution (Snyder). U.S. National
Museum Bulletin. 108, 1-228.
Baum, Stuart J. and William R. Bowen. (1972). Exercises in Organic and
Biological Chemistry. The MacMillan Company, New York. 52, 211.
Bignell, D.E., H. Oskarsson and J.M. Anderson. (1980). Distribution and
Abundance of Bacteria in the Gut of a Soil-feeding Termite Procubitermes
aburiensis (Termitidae, Termitinae). Journal of General Microbiology.
117, 393-403.
Bisaria, Virendra S. and Saroj Mishra. (1989). Regulatory Aspects of Cellulase
Biosynthesis and Secretion. Critical Reviews in Biotechnology 9, 61103.
Breznak, John A. (1975). Symbiotic Relationships between Termites and Their
Intestinal Microbiota. Society for Experimental Biology Symposium. 29,
559-580.
Breznak, John A and H. Stuart Pankratz. (1977). In Situ Morphology of the Gut
Microbiota of Wood-Eating Termites [Reticulitermes flavipes (Kollar) and
Coptotermes formosanus Shiraki]. Applied and Environmental
Microbiology. 33, 406-426.
Breznak, John A. (1982). Intestinal Microbiota of Termites and Other
Xylophagous Insects. Annual Review in Microbiology. 36, 323-343.
Brooks, Dr. Bob. (1994). Senior Scientist for Specialty Pulp, The Weyerhaeuser
Corporation, Tacoma, WA. Telephone interview. July 7, 1994.
38

Chan, Kwong-Yu and K.S. Au. (1987). Studies on cellulase production by a
Bacillus subtilis. Antonie van Leeuwenhoek. 53, 125-136.
Claus, G. William. (1989). Understanding Microbes A Laboratory Textbook for
Microbiology. W.H. Freeman and Company, New York.
Coughlan, M.P. (Ed.) (1990). Enzyme Systems for Lignocellulose Degradation.
Elsevier Applied Science, London.
Dhingra, H.K., Preeti Shivhare, K.S. Panesar, P.R. Bohidar, V.K. Mohindru and
R. Pant. (1993). Application of biotechnology for organic solid waste
utilization. Indian Pulp & Paper Trade Association. 5, 111-123.
Hartley, B.S., F.R.S., P.M.A. Broda and P.J. Senior. (Eds.). (1987). Technology
in the 1990s: Utilization of Lignocellulosic Wastes. Philosophical
Transactions of the Royal Society of London. 321, 403-568.
Eutick, M.L., R.W. O'Brien and M. Slaytor. (1978). Bacteria from the Gut of
Australian Termites. Applied and Environmental Microbiology. 35, 823828.
French, J.R.J., G.L. Turner, and J.F. Bradbury. (1976). Nitrogen Fixation by
Bacteria from the Hind-gut of Termites. Journal of General
Microbiology. 95, 202-206.
Hendee, Esther C. (1933). The Association of the Termites, Kalotermes minor,
Reticulitermes hesperus, and Zootermopsis angusticollis with Fungi.
University of California Publications in Zoology. 39, 111-134.
Kirk, T. Kent and Hou-Min Chang. (Eds.) (1990). Biotechnology in Pulp and
Paper Manufacture. Butterworth-Heinemann, Boston.
Klyosov, Anatole A. (1990). Trends in Biochemistry and Enzymology of
Cellulose Degradation. Biochemistry. 29, 10577-10585.
Kricke, Jorn, Ajit Varma, David Miller and Frank Mayer. (1994).
Carboxymethyl Cellulase from Bacillus sp.: Isolation, Macromolecular
Organization, and Cellular Location. Journal of General Applied
Microbiology. 40, 53-62.

39

Lo, Amy C., Ron M. MacKay, Vern L. Seligy and Gordon E. Willick. (1988).
Bacillus subtilis $-1,4-Endoglucanase Products from Intact and Truncated
Genes are Secreted into the Extracellular Medium by Escherichia coli.
Applied and Environmental Microbiology. 54, 2287-2292.
Maniatis, T., E.F. Fritsch and J. Sambrook. (1982). Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
New York.
Office of Technology Assessment (OTA). (1995). Renewing Our Energy Future,
OTA-ETI-614. Washington, DC: U.S. Government Printing Office.
Pommier, Jean-Claude, Gerard Goma, Jean-Luc Fuentes and Christian Rousset.
(1990). Using enzymes to improve the process and product quality in the
recycled paper industry Part 2: Industrial applications. Tappi Journal.
73, 197-202.
Potrikus, C.J. and John A. Breznak. (1977). Nitrogen-Fixing Enterobacter
agglomerans Isolated from Guts of Wood-Eating Termites. Applied and
Environmental Microbiology. 33, 392-399.
Ross, Peter, Raphael Mayer, and Moshe Benziman. (1991). Cellulose
Biosynthesis and Function in Bacteria. Microbiological Reviews. 55, 3558.
Saddler, J. N.. (Ed.) (1993). Bioconversion of Forest and Agricultural Plant
Residues. CAB International, Wallingford, U.K..
Sarkanen, Simo, Ramon A. Razal, Thomas Piccariello, Etsuo Yamamoto, and
Norman G. Lew. (1991). Lignin Peroxidase: Toward a Clarification of Its
Role in Vivo. The Journal of Biological Chemistry. 266, 3636-3643.
Sarkar, Jawed M., David R. Cosper, and Edward J. Hartig. (1995). Applying
Enzymes and Polymers to enhance the Freeness of Recycled Fiber. Tappi
Journal. Feb. 1995. 89-95.
Slaytor, Michael. (1992). Cellulose Digestion in Termites and Cockroaches:
What roles do Symbionts Play?. Comparative Biochemistry and
Physiology. 104B, 775-784.
Solomons, T. W. Graham. (1988). Organic Chemistry. (4th ed.). John Wiley &
Sons, New York.

40

Stork, G., Helena Pereira, Thomas M. Wood, Eva Maria Düsterhöft, Annette Toft,
and Juergen Puls. (1995). Upgrading Recycled Pulps Using Enzymatic
Treatment. Tappi Journal. Feb. 1995. 79-88.
Tewari, Yadu B. (1990). Thermodynamics of Industrially-Important, EnzymeCatalyzed Reactions. Applied Biochemistry and Biotechnology. 23, 187203.
Thayer, D.W. (1978). Carboxymethylcellulase Produced by Facultative Bacteria
from the Hind-gut of the Termite Reticulitermes hesperus. Journal of
General Microbiology. 106, 13-18.
Thayer, D.W. (1976). Facultative Wood-digesting Bacteria from the Hind-gut of
the Termite Reticulitermes hesperus. Journal of General Microbiology.
95, 287-296.
Thorne, Barbara L., Michael I. Haverty, Marion Page and William L. Nutting.
(1993). Distribution and Biogeography of the North American Termite
Genus Zootermopsis (Isoptera: Termopsidae). Annals of the
Entomological Society of America. 86, 532-544.
Virtanen, Yrjö and Sten Nilsson. (1993). Environmental Impacts of Waste Paper
Recycling. Earthscan Publications Ltd., London.
Weimer, P.J., J.M. Lopez-Guisa and A.D. French. (1990). Effect of Cellulose
Fine Structure on Kinetics of Its Digestion by Mixed Ruminal
Microorganisms In Vitro. Applied and Environmental Microbiology. 56,
2421-2429.
Wood, Brent E., and L.O. Ingram. (1992). Ethanol Production from Cellobiose,
Amorphous Cellulose, and Crystalline Cellulose by Recombinant
Klebsiella oxytoca Containing Chromosomally Integrated Zymomonas
mobilis Genes for Ethanol Production and Plasmids Expressing
Thermostable Cellulase Genes from Clostridium thermocellum. Applied
and Environmental Microbiology. 58, 2103-2110.
Wood, Willis A. and Scott T. Kellogg. (Eds.) (1988a). Methods in Enzymology:
Biomass Part A Cellulose and Hemicellulose. Vol 160. Academic Press,
Inc, New York.
Wood, Willis A. and Scott T. Kellogg. (Eds.) (1988b). Methods in Enzymology:
Biomass Part B Lignin, Pectin, and Chitin. Vol 161. Academic Press,
Inc, New York.
41

Young, Jim. (Ed.) (1994). Enzone Bleaching, Enzynk Deinking Advance to
Pilot Plant Trials. Pulp and Paper. Nov. 1994. 81-82.
Zeyer, Christian, Thomas W. Joyce, John A. Heitmann, and James W. Rucker.
(1994). Factors Influencing Enzyme Deinking of Recycled Fiber. Tappi
Journal. Oct. 1994. 169-177.

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Appendix A
List of Abbreviations and Acronyms
Abbreviated Term

Full Term

B. stearothermophilus
B. subtilis
Cx-type
CMCase
CMC
CMCCA

Bacillus stearothermophilus
Bacillus subtilis
Noncrystalline cellulose
Carboxymethylcellulase
Carboxymethylcellulose
M9 medium, 2.5% CMC and 0.25%
casamino acids
M9 medium, 0.5% cellulose azure and
0.25% casamino acids
Eosin Methylene blue
Escherichia coli
Minimal Salts Media
Tryptic Soy Broth
Triple Sugar Iron
M9 medium, 1.0% undyed crystalline
cellulose and 0.25% casamino acids

DCCA
EMB
E. coli
M9
TSB
TSI
UCCA

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Appendix B
List of Microbiological Assays
Gram Stain:

Catalase:

Oxidase:

Motility:

Cell Length & Width:

Spore stain:

Carbon source:

MacConkey:

EMB:

Purple broth:

Mannitol salt:

Standard Gram stain utilizing log-phase cells.
Gram stain tests for cell membrane structure
(Balows et al., 1991, p. 1306).
Standard catalase assay utilizing cells grown
overnight. Catalase assay tests for catalase enzyme
presence (Balows et al., 1991, p. 1290).
Standard oxidase assay utilizing cells grown for 3
days. Oxidase assay tests for oxidase enzyme
presence (Balows et al., 1991, p. 1298).
Hanging drop microscopic examination of logphase cells. Hanging drop examination tests for
motility in bacteria.
Determined by light microscopy of Gram stain cells
using oil immersion magnification 1000X (Balows
et al., 1991, p. 704).
Standard spore stain (Wirtz-Conklin) utilizing heattreated cells. Spore stain tests for spores within the
cell (Balows et al., 1991, p. 1311).
Agar and M9 salts, with appropriate carbon source
at 1% by mass added, streaked with cells grown
overnight. (Claus, 1989, p. 195-198).
MacConkey agar streaked with cells grown
overnight. MacConkey is a selective medium that
selects for lactose fermentation (Balows et al.,
1991, p. 1208).
Eosin methylene-blue agar streaked with cells
grown overnight. EMB agar is a selective medium
that usually inhibits growth of gram-positive
bacteria (Claus, 1989, p. 480).
Purple broth agar with 2% galactose added streaked
with cells grown overnight. Purple broth with
galactose is a selective medium that selects for
galactose fermentation (Balows et al., 1991, p.
1270).
Mannitol salt agar streaked with cells grown
overnight. Mannitol salt agar is a selective medium
that usually inhibits growth of most bacteria, except
staphylococci (Balows et al., 1991, p. 217).

44

$ hemolysis:

TSI slant:

Antibiotic resistance:

pH conditions:

Temperature conditions:

Sheep's blood agar streaked with cells grown
overnight. Blood agar is a selective medium that
tests for growth on red blood cells. $ hemolysis is
the complete lysis of the erythrocytes (Claus, 1989,
p. 422).
Triple-sugar-iron slants inoculated with cells grown
overnight. TSI slants test for sugar fermentation,
gas production and anaerobic growth. Acid
production shows fermentation of sugar. Alkaline
production shows no sugar fermentation. Bubbles
display gas production . Black color shows H2S
production (Claus, 1989, p. 286-288).
Tryptic soy broth agar streaked with cells grown
overnight onto plates with antibiotic added. Levels
of antibiotic: ampicillin (100 :g/ml), streptomycin
(30 :g/ml), chloramphenicol (20 :g/ml),
tetracycline (15 :g/ml) (Claus, 1989, p. 397-399).
Tryptic soy broth agar streaked with cells grown
overnight. Phosphate buffers were used to adjust
the pH of the plates before pouring. Additional pH
measurements were conducted on the agar plates
after several days of incubation at 30EC. The pH
levels remained stable in the TSB agar plates.
Tryptic soy broth agar streaked with cells grown
overnight. Agar plates incubated at stated
temperatures.

45

Media

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