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Development of Termite Saliva as a
Potential Stabilizer for Gravel Road Layers
CROSSROADS CHALLENGE FUND, 2013
Prepared for:
Submitted By:
CROSSROADS
BLOCK 2, OFFICE C2, NYONYI GARDENS, KOLOLO KAMPALA, UGANDA.
SCHOOL OF ENGINEERING,
MAKERERE UNIVERSITY
August, 2014
CROSSROADS CHALLENGE FUND
PROJECT NO. CF4
Development of Termite Saliva as a Potential
Stabilizer for Gravel Road Layers
FINAL REPORT
By
The Research Group represented by Dr. Umaru Bagampadde as Principal Investigator and Mr. David Kaddu as Co-Investigator for the
School of Engineering, College of Engineering, Design, Art and Technology (CEDAT), Makerere University
“TERMABOND ROAD SYSTEM”:
“100mg of mucopolysacharides to oligosaccharides produced from 45 units of enzyme cellulase and treated with 0.3g of cellulose for 1 hour
and then treating the solution with water in a ratio of 1:100”
August, 2014
i
ACKNOWLEDGEMENTS
This work has been sponsored by the CrossRoads Challenge Fund (95%) and Makerere
University (5%). Numerous Government agencies have contributed directly and indirectly to the
success of this project like the Uganda National Roads Authority (UNRA) through their
upcountry stations, the Uganda Road Fund (URF) and the Ministry of Works and Transport
(MoWT). A lot of laboratory supervisory support has been exhibited by Mr. Fred Mukasa of the
Highways laboratory at Makerere University. The research team comprising of persons from
various research institutions and students of Makerere University has been coherent and
cooperative. Dr. Muhammad Ntale of the Department of Chemistry helped a lot in interpretation
of the XRD, AAS and FTIR results. Dr. Joseph Fuuna Hawumba of the Department of
Biochemistry, Makerere University greatly assisted in the Analysis of Termite samples,
optimization of enzymes and substrates and development of model compound. The Materials
Science laboratory at the Royal Institute of Technology, Stockholm, Sweden offered excellent
services in doing some fundamental tests on the soil materials.
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TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................... i
LIST OF FIGURES ......................................................................................................... iv
LIST OF TABLES ............................................................................................................. v
EXECUTIVE SUMMARY ................................................................................................... 7
CHAPTER 1: INTRODUCTION ...................................................................................... 13
1.1 BACKGROUND INFORMATION ................................................................................. 13
1.2 PROJECT SUMMARY ............................................................................................... 14
1.2.1 Study Need and Objectives ............................................................................... 14
1.2.2 Relevance of Study to the Road sub-sector......................................................... 15
1.2.3 Project Approach ............................................................................................. 16
1.2.4 Main Activities ................................................................................................. 16
1.2.5 Expected Outputs/Outcomes ............................................................................. 17
1.2.6 Benefits from the Study .................................................................................... 17
1.2.7 Contribution to CrossRoads Objectives ............................................................... 18
CHAPTER 2: THE INNOVATION .................................................................................... 19
2.1 THE INNOVATIVE TERMITE SALIVA TECHNOLOGY .................................................... 19
2.1.1 General Aspects on termite saliva technology ..................................................... 19
2.1.2 Soil Strengthening Mechanism of Termites ......................................................... 27
2.2 COMPARISON OF THE TECHNOLOGY WITH CLASSICAL STABILISATION TECHNIQUES 36
CHAPTER 3: METHODOLOGY ....................................................................................... 44
3.1 RESEARCH DESIGN ................................................................................................ 44
3.1.1 Study Extent/Coverage ..................................................................................... 44
3.1.2 Materials Used ................................................................................................. 45
3.1.3 Test Methods on Soils ....................................................................................... 48
3.1.4 Elemental analysis ........................................................................................... 48
3.1.5 Analysis of termite extracts ............................................................................... 49
3.1.6 Treatment of soil with extract ........................................................................... 50
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3.2 OPTIMISATION OF THE THEORIES OF SOIL MODIFICATION ..................................... 52
3.2.1 Treatment of soil with cellulase – Investigation of theory 1 .................................. 52
3.2.2 Treatment of soil with oligosaccharides – investigation of theory 2 ....................... 53
3.2.3 Preparation of the best oligosaccharide solution and investigation of theory 3 ....... 56
CHAPTER 4: PRESENTATION OF FINDINGS AND DISCUSSIONS ................................. 57
4.1 Results of mound soils and surrounding soils ............................................................ 57
4.1.1 Elemental analysis ........................................................................................... 57
4.1.2 pH as measured from the pH meter ................................................................... 61
4.1.3 Gradation of Soil Samples ................................................................................. 62
4.1.4 Maximum Dry Density ...................................................................................... 63
4.1.5 Atterberg’s Limits ............................................................................................. 65
4.2 Results from the test road section ............................................................................ 67
4.2.1 Gradation ........................................................................................................ 67
4.2.2 Atterberg’s Limits ............................................................................................. 67
4.2.3 Strength .......................................................................................................... 68
4.3 Analysis of extracts ................................................................................................. 68
4.3.1 Cellulase activity in the anthill soil, surrounding soil, termite head and abdomen
extracts 68
4.3.2 Isolation of mucopolysaccharides ...................................................................... 68
4.3.3 Treatment of in-situ soil with extract ................................................................. 69
4.4 Formulation and preparation of the Oligosaccharide .................................................. 70
4.4.1 Optimisation of time ......................................................................................... 70
4.4.2 Optimisation of enzyme units ............................................................................ 72
4.4.1 Optimisation of substrate .................................................................................. 74
4.5 Variation of the mucopolysaccharides ....................................................................... 76
4.6 Composition of model compound and treatment of in-situ soils .................................. 77
CHAPTER 5: CONCLUSIONS ........................................................................................ 82
CHAPTER 6: RECOMMENDATIONS .............................................................................. 85
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LIST OF FIGURES
Figure 2.1: Cellulolytic Systems in Lower Termites (Flagellate – Harboring) ............................ 32
Figure 2.2: Cellulolytic Systems in Higher Termites (Flagellate – free) .................................... 32
Figure 2.3: Endogenous digestive system in N. takasagoensis. .............................................. 34
Figure 3.1: Map of Uganda Showing Locations of sampling points .......................................... 45
Figure 3.2: Sampling points on an anthill ............................................................................. 46
Figure 3.3: Picking of samples from anthills ......................................................................... 47
Figure 3.4: Identification of live termites and separation of heads from abdomens .................. 47
Figure 3.5: Collection of soil extract, abdomen extract, thorax extract in a desiccator .............. 51
Figure 3.6: Conduction of consistency tests for soils treated with extract ............................... 51
Figure 3.7: Dissolution of weighed cellulase in a petridish with citrate buffer .......................... 54
Figure 4.1: Results from elemental analysis ......................................................................... 60
Figure 4.2: Analysis of pH of mound soils ............................................................................ 62
Figure 4.3: Gradation curves of mound soils ........................................................................ 63
Figure 4.4: Compaction test curves of mound soils ............................................................... 64
Figure 4.5: Maximum Dry Densities of mound soils ............................................................... 65
Figure 4.6: Liquid Limits of sampled material from mounds ................................................... 66
Figure 4.7: Plastic Limits of sampled material from mound soils ............................................. 66
Figure 4.8: Plasticity Indices of sampled material from mound soils ....................................... 67
Figure 4.9: Gradation curves of in-situ material .................................................................... 67
Figure 4.10: Values of in-situ soil CBR treated with dosage 1................................................... 70
Figure 4.11: CBR values of in-situ soil treated with dosage 2 oligosaccharides .......................... 71
Figure 4.12: Plasticity of in-situ soil treated with dosage 2 oligosaccharides .............................. 72
Figure 4.13: CBR values of in-situ soil treated with with the variation of enzyme units ............... 73
Figure 4.14: Plasticity of in-situ soil treated with the variation of enzyme units ……………………….73
Figure 4.15: CBR values of in-situ soil treated with with the variation of substrate .................... 74
Figure 4.16: Plasticity of in-situ soils with the variation of substrate ........................................ 75
Figure 4.17: CBR and Plasticity of in-situ soils with the variation of mucopolysaccharides ........... 76
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LIST OF TABLES
Table 2.1: Particle sizes mound soil and surround surface soil (Anusha, 2010) ......................... 23
Table 2.2: Composition of termite mound and surrounding soils ............................................. 23
Table 2.3: Tri-axial results on treated and untreated gravel (Mukasa, 2012) ............................ 27
Table 3.1: Details of sampling points for the soils of the anthills ............................................. 46
Table 3.2: Properties of enzyme cellulase procured ............................................................... 52
Table 4.1: Statstical Analysis of chemical results .................................................................... 60
Table 4.2: Plasticity of in-situ material ................................................................................... 67
Table 4.3: Strength of in-situ material ................................................................................... 68
Table 4.4: Yield and concentration of mucopolysaccharides ..................................................... 69
Table 4.5: Plasticity values for soil treated with extract from termite heads and abdomen ......... 69
Table 4.6: Values of CBR of in-situ soil treated with oligosaccharides (dosage 1) ...................... 70
Table 4.7: Values of CBR with soil treated with oligosaccharides (Dosage 2) ............................. 71
Table 4.8: Values of CBR of in-situ soil treated with oligosaccharides prepared with different
enzyme units....................................................................................................................... 72
Table 4. 9: CBR and Plasticty of Oligos +Mucopolysaccharides vs Oligos .................................. 77
Table 4. 10: CBR of soil treated with stabilised and unstabilised TermaBond ............................. 77
Table 4. 11: Direct costs in production of TERMABOND........................................................... 79
Table 4. 12: Other associated costs ....................................................................................... 80
Table 4. 13: Comparison of TERMABOND stabilized pavement road section and the conventional
road construction methods used. .......................................................................................... 81
vi
7
EXECUTIVE SUMMARY
E-1 This research employed an innovative approach of stabilizing in-situ / existing road
materials using termite saliva technology / concept and has been largely funded by the
CrossRoads Challenge Fund (CCF) and Makerere University. It arose out of the need to
develop a friendlier alternative stabilizer / soil bonding agent to be applied specifically to
the existing natural soil materials so as to overcome the costly and time intensive
haulage of new gravel from borrow pits and reduce the unit road construction cost and
preservation of the natural scenery.
E-2 The concept was developed from the termite building industry, the steps of mound
construction and the ingredients termite use to build their mounds commonly known as
anthills. World over, termites have been known to construct mounds in a bid to protect
the mother queen and the entire colony by picking small sized soil particles, mix it with
their saliva and pile it together. As a result of their communal nature, the compounded
effect in terms of the quantity of work executed is very well pronounced, in addition to
carrying out works over a twenty four hour period. The constructed structure when wet is
too weak. However on drying, it exhibits enhanced strength which is resistant to harsh
environmental conditions including rains.
E-3 The team therefore set out to establish the properties of the termite mounds and the
special ingredients making up the termite saliva in a bid to mimic the saliva by
constituting a model compound with properties similar to those of the saliva. This would
later be used to stabilize soils, construct pavement layers using this compound to provide
/ enhance the strength of the constituent layer materials. As opposed to classical
stabilizers, the termite saliva technology concept is noble and can result in utilization of
existing inferior material, environmental preservation/cost, extended service life,
reduced health and cleanliness concerns, and reduced potential for accidents. Similar to
the termite saliva, the product is non-toxic and has no negative impact to the
environment.
E-4 The research objectives were to: (i) chemically characterise the termite mound soils and
the surrounding soils; (ii) chemically analyse the termite saliva extracts from both the
abdomen and the head of live worker termites; (iii) determine the key engineering
properties of soils treated with the termite extracts from head and abdomen; (iv) develop
a model compound similar to termite saliva; (v) determine the engineering parameters of
8
E-5 The team anticipated the following benefits out of this study namely (i) Improved
strength of road soil material based on TERMITE stabilisation technology (ii) Increase the
knowledge base on use of TERMITE technology in road material improvement (iii) Avail
Research results on use of TERMITE technology to potential stakeholders for improving
material specifications or standards. If the technology is tested and found to be
successful, it might lead to longer maintenance-free periods, reduced material
demand, increased level of service and preservation of nature.
E-6 Termite mound soils were collected from different administrative regions of the Uganda
National Roads Authority along major national road corridors namely Northern region (Lira
district along Akia – Aloi – Olilim road); Western region (Mbarara and Masindi districts
along Mbarara – Bushenyi road); West Nile (Arua district near the UNRA Offices);
Southern region (Kasese district along Kasese – Kikorongo road); Karamoja (Kotido
district) and Central region (Luwero district along Nakseke – Ngoma road). Soil samples
were picked from the top of the mound, middle and bottom. Control samples were picked
from the 3m and 6m offsets from the anthill. The live work termite samples were
collected from Masulita in Wakiso district and from active termitarium near the School of
Food Technology, Makerere University. The study road was Lubowa - Ndejje road in
Wakiso district from which insitu soils were sampled and tested / treated with the model
termite saliva compound.
E-7 Both chemical and strength tests were carried out on these samples. The composition of
termite saliva was analyzed, the different components in the termite saliva that were
anticipated to enhance strength were obtained in synthetic form and optimized using soils
from Lubowa-Ndejje road. These were then reconstituted to form the model compound.
E-8 From the chemical characterization tests using the Atomic Absorption Spectrometer
(AAS), iron was found to be the most dominant for both the mound and surrounding soils
all values obtained were over 18,000ppm compared to other mineral elements such as
carbon, nitrogen and Potassium. Carbon and Nitrogen were also found to be present in
larger quantities in mound soils as compared to surrounding soils. Termites release fecal
material full of C and N which they use in construction of nests. The higher C and N
content in surrounding soils could be attributed to decomposing organic matter. Statistical
analysis revealed that termites are not selective in the soil materials they choose to work
with in regard to its chemical composition.
E-9 All soils were finer than the acceptable specification range for gravel wearing course and
9
subgrade layers. The PI for all the soils were between 10 and 25% Light compaction was
used to determine OMC and MDD and subsequently CBR was determined at three levels
of compaction (10, 25 and 55 blows).
E-10 Extracts from the surrounding soils, termite heads and abdomen were analyzed for
presence and activity of cellulase enzyme and mucopolysacharides. Cellulase activity in
the head was found to be 0.04 µmoles of glucose per minute per mg protein; Cellulase
activity in the abdomen was found to be 0.0648 µmoles of glucose per minute per mg
protein; Cellulase activity in the control soil sample was found to be 0.061 mg of glucose
per minute per g of soil and Cellulase activity in the anthill soil sample was found to be
0.08 mg of glucose per minute per g of anthill soil. The results confirmed the presence of
enzyme cellulase in the termite saliva. The specific cellulase activity in termite abdomens
was higher than that in worker heads by 62%. Cellulase activity in the control soil sample
was found to be 0.061 mg of glucose per minute per gram of soil. In comparison to the
activity in the mound soil, activity in control soil was 25% lower. It was therefore
concluded that termites increase enzyme cellulase concentration in mound soil and hence
the enzyme plays a role in the nest structural stability.
E-11 Mucopolysaccharides, the predicted gluing agents were isolated from the head and
abdomen of live termites and from mound soil. The highest concentration of the gluing
agent was found in the termite heads at a concentration of 8214 ppm, followed by
abdomen at 3250ppm and from the wet mound soil at 425ppm. The presence of
mucopolysacharides in the mound soil confirmed that they play a role in the structural
strength of the termitarium.
E-12 Consistency tests were carried out on insitu soils from Lubowa road treated with the
above mucopolysacharides. The treatment of soil with extract led to a reduction of both
PI and LS which is an indication of improved performance. PI reduced by 12% with
extract from fresh soil and by 39% with extract from the abdomen and finally by 46%
with extract from the head. The MoWT (2005) specification states that a material to be
used for sub-base layers of G30 materials should have a Maximum linear shrinkage value
of 8%. From the above results, it can be shown that head extracts provide linear
shrinkage values close to the maximum 8%.
E-13 The soil modification theories were tested as follows: the action of oligosaccharides was
investigated by reacting cellulase with cellulose and optimising time, cellulose and cellulase
for the production of the most suitable solution for strength and plasticity improvement.
10
The optimisation of time was carried out by reacting 200 units of enzyme cellulase per ml
of buffer. The CBR value increased by 43% with enzyme substrate reaction stopped at 60
minutes. Therefore the best time for maximum production of oligosaccharides was
60minutes.
E-14 When the dosage was increased to 500 units of enzyme cellulase per ml of buffer, a
substantial increment in the value of CBR of soil treated with oligosaccharide solutions
prepared as dosage 2. The optimum time to produce the maximum concentration of
oligosaccharides was again recorded as 1hour. The CBR value increased from 8% to 38%
at 1hour of enzyme- substrate reaction. The plasticity Index of in-situ reduced when
treated with oligosaccharide solutions prepared at different time intervals implying
increased strength of soil. The highest reduction of plasticity was observed at 1hour of
enzyme- substrate reaction stoppage. Therefore the best time for highest yield of
oligosaccharides from cellulase-cellulose reaction was 1 hour.
E-15 The optimization of enzyme units was carried out by producing oligosaccharide solutions
through reaction of different units of enzyme cellulase with cellulose and stopping the
reaction at one hour. The solutions prepared were then treated with in-situ soil (ratio of
water: solution- 100:1) to formulate the dry CBR and plasticity values at 16 days curing.
There was substantial increment in the value of CBR of soil treated with oligosaccharide
solutions. This may be attributed to the increase in enzyme concentration and hence more
oligosaccharides for a given quantity of substrate. CBR value increased from 42% to 82%
at 45 units of enzyme per ml of buffer. The highest CBR was obtained at 45 units of
enzyme cellulase.
The plasticity Index of in-situ reduced when treated with oligosaccharide solutions
prepared with different enzyme units implying increased strength of soil. The highest
reduction of plasticity was observed at oligosaccharide solution prepared with 60 units of
enzyme cellulase (15.7%) Although the highest concentration of oligosaccharides for
maximum strength is a solution prepared with 45 units of enzyme cellulase, 60 units were
ideally chosen as the optimum enzyme concentration for the best yield of oligosaccharides.
E-16 The optimization of the substrate was conducted by treating 45 units and 60 units of
enzyme cellulase with cellulose. The CBR values gradually increased with variation of
substrate for both 45 units and 60 units. The highest CBR (63%) was obtained at 0.3 g of
substrate corresponding to 45 units of enzyme. Therefore the maximum production of
oligosaccharides for maximum increase in soil strength is prepared by treating 45 units of
11
enzyme cellulase with 0.3g of cellulose for 1 hour. The CBR curve of 60 units was slightly
lower than that of 45 units with the maximum CBR (61%) at 0.35g of cellulose.
E-17 The plasticity index of soil treated with oligosaccharides prepared with 45 units of enzyme
cellulase reduced with increased variation of substrate while that of soil treated with
oligosaccharide solution prepared with 60 units of enzyme cellulase increased. The lowest
reduction in plasticity was obtained with 0.3g of cellulose. Plasticity values correspond with
CBR values. Therefore, in order to produce the best concentration of oligosaccharides for
the best strength and plasticity characteristics of in-situ lateritic soils, 45 units of enzyme
cellulase are treated with 0.3g of cellulose for 1 hour from which solution is treated with
water in a ratio of 1:100.
E-18 The best supernatant of oligosaccharides was supplemented with mucopolysacharides in
different quantities in reference to theory 3 of soil modification by termites. 10ml of the
resultant mixture was dissolved in 1000ml of water and treated with in-situ soil samples to
determine dry CBR and plasticity readings at 4 days curing. The mucopolysacharides led to
an increase in soil CBR treated with oligosaccharides from 63% to 75% with the addition
of 100mg of mucopolysacharides. The CBR value then reduced with further addition of
mucopolysacharides. This was due to the formation of complex bonds between
mucopolysacharides and oligosaccharides from cellulose digestion due to their interaction.
The mucopolysacharides reduce plasticity of the soil treated with oligosaccharides. In
conclusion, 100mg of mucopolysacharides were chosen to be the ideal quantity for the
best effect of strength improvement.
E-19 The constitution of model compound was accomplished by addition of 100mg of
mucopolysacharides to oligosaccharides produced from 45 units of enzyme cellulase
treated with 0.3g of cellulose for 1 hour from and then treating the solution with water in
a ratio of 1:100. Two compounds were produced. Compound one was produced by mere
addition of mucopolysacharides to the solution of oligosaccharides and the mixture was
left to stand overnight before treatment with the soils. Compound two was produced and
immediately a stabilizer was added to inhibit any possible reaction between the
oligosaccharides and mucopolysacharides. The two samples were used to treat in-situ
soils from Lubowa road and both plasticity and strength values were recorded. Lower CBR
values (50%) were exhibited by un-stabilized compound compared to the stabilized
12
compounds (50%) possibly because of the formation of complexes between
oligosaccharides and mucopolysacharides.
E-20 It is possible to achieve higher strength values using this compound should the
concentration of oligosaccharides be increased. The stabilization to some degree dilutes
the concentration of the oligosaccharides and affects the complex formation mechanism.
A higher concentration of oligosaccharides would therefore check the above.
E-21 It is major recommendation from this study that the results from this study be verified by
field applications by putting up test sections in some of the areas with problematic soils
around Uganda. Comparisons from the above studies can be used to correlate the above
findings. In addition, a firm base should be protected from the adverse effects of
weather by installation of a sealant to stop ingression of water into the deeper layers that
would reduce its structural integrity. The type of surfacing to best serve the above
purpose without necessarily increasing the cost will be determined and assessed from the
field trials.
E-22 The TERMABOND road construction system is a cheaper option compared to the single,
double and asphalt concrete surfacing systems, with figures standing at UGX
356,114,500/= for a TERMABOND system, UGX 420,000,000/=, 580,000,000/= and
1,690,000,000/= for single surface dressing, double surface dressing and asphalt
concrete surfacing options. It is more expensive than the gravel system however the life
cycle cost analysis shall render the latter option more expensive. At a large scale, a total
saving of UGX 64,000,000/= is made on a TERMABOND system compared to the single
surface dressing. Interventions to waive taxes and reduction in the importation and
shipping costs shall further lower the construction costs of the TERMABOND road system.
E-23 It is suggested further to this study, that the above test sections should be constructed
using the TERMABOND road system and results compared with the conventional /
traditional road construction systems. The road sections should be setup in the same
areas where the different roads should experience same traffic and environmental
conditions. Thereafter, monitoring of the above sections should commence with and last
for at least 1 year. The shelf life of the stabilizer, TERMABOND also needs to thoroughly
be investigated to determine the expiry and strategies to preserve it longer improvised.
13
CHAPTER 1: INTRODUCTION
1.1 BACKGROUND INFORMATION
The project was conceived with the view of harnessing nature by mimicking the use of termite
saliva in strengthening soil. Since in situ road soil is normally dumped and new material imported,
it was thought that application of a model material that mimics termite saliva action on the in situ
material will go a long in improving gravel roads performance more cheaply and in a more
environmentally friendly manner. Specifically, the team anticipated improving strength of road soil
material based on TERMITE stabilization technology, avail information on use of Termite Saliva
technology in improving material design and performance specifications/standards, create
expertise on use of termite saliva in road material improvement, and reduced unit cost of gravel
roads mechanized routine maintenance.
As a stabilization / bonding agent, the mimicked model compound is to aid in strength
enhancement of in-situ soils which are otherwise condemned and ferried away from site in
preference of more suitable materials. This raises the unit cost of road construction.
This study was initiated and has been largely funded through the “innovation grant” by the
CrossRoads Challenge Fund (CCF). CrossRoads is an acronym for “Creating Opportunities for
Sustainable Spending on Roads”. The major CrossRoads programme goal is to ensure that
Uganda gets better roads and higher value for money when building, maintaining and repairing
these roads.
The Programme has two main objectives and these are;
to help improve the quality of Uganda’s road network; and
to help improve the efficiency of the Government of Uganda’s spending on roads
The programme has provided a CCF to a tune of GBP 1 million (approx. UGX 3.8 billion) to fund
industry entrepreneurs and research establishments to develop innovative solutions to roads
issues. The aim is to provide consultants and contractors with more cost effective ways of
designing and implementing roads projects and thereby achieving better value for money in the
road sub-sector.
14
1.2 PROJECT SUMMARY
1.2.1 Study Need and Objectives
Various soil stabilizing agents are available on the local markets including lime and cement. Other
chemical stabilizers are imported into the country once there is a general need. Where project
resources are scarce, mechanical stabilization involving the use of sands, quarry dust etc has been
proposed and used. However, once used, the conventional materials (lime, cement, quarry dust,
sands etc) have resulted into very un-durable roads as evidenced by the fast deterioration with
time. In addition, the advanced enzymic chemical stabilizers are very expensive since they require
importation. With the dwindling stocks of road construction material deposits, innovations into
improvement of existing ground materials to reduce construction time and costs will come in
handy. One of these techniques is to mimic the natural environment, the termite world in
particular.
Termite anthills and mound soils are commonly found along road construction projects in many
parts of Uganda. They are mainly made up of clayey materials which are highly plastic and very
weak in terms strength (CBR and UCS). On their encounter with such weak and un-durable
materials, construction engineers remove them and deposit them far away from the project site
and then import new and suitable materials. However, this has associated problems such as fast
deterioration with time, environmental nuisances from the disposed materials and also from the
destruction of the scenery at the borrow pits. The whole process is very expensive. It is this same
soil that termites inject their saliva into to form hard, strong, durable and weather resistant
anthills.
Anthill mound soils are characterized by high strength properties and the mechanism of termite
saliva stabilization of soil is of interest to the highway community as this could be used to stabilize
road base and sub-base layers. This study has established the contents of termite saliva (physical
and chemical properties) which they inject into the soil and then modelled a compound which can
be locally manufactured in our industries in large amounts. This would go a long way in reducing
road construction costs incurred during the importation of stabilizers (such as cement) while at
the same time harnessing nature.
15
To contribute to providing solutions towards this need, this research project was undertaken to
develop a model compound to stabilize the existing soil for better performance of roads with
importing materials from elsewhere by making use termite saliva technology.
Specifically, the research was aimed at achieving the following objectives;
i. To chemically characterize selected termite mound soils from different parts of the
country.
ii. To determine engineering and mechanical properties of termite mound soils.
iii. To treat in-situ soils with the extract from head and abdomen of the termites.
iv. To test effectiveness of a chemical model compound similar to termite saliva in improving
soil strength.
Termites build their antihills using saliva which they inject into the soil to enhance strength. The
end result is a hardened soil mass which does not deteriorate even under very extreme conditions
of weather. This is to critically examine the anthill building agents and develop products similar to
termite saliva, stabilize the soils with these products and report comparative assessments about
their interrelationships with the soils.
1.2.2 Relevance of Study to the Road sub-sector
Conventional stabilizers are expensive and have showed premature failures in roads. Innovative
and cheaper methods of improving existing ground materials to reduce construction time and cost
are important. This research was undertaken to mimic the natural construction methods used by
termites to enhance soil strength. A Model compound similar in chemical setup like termite saliva
has been produced and can be used to stabilize road materials.
There is a wide variation of termite mounds all over the country and these have been considered
as hazards to human activities in addition to destroying the beautiful scenery. These are also
commonly found along road corridors. Maintenance crews have always destroyed them by
spraying of chemicals to kill the building agents – termites, which may end up introducing
dangerous chemicals in the soil media. In other cases, anthills are destroyed by manually digging
them out to avoid any further destruction to the road. This is done as part of the clearing and
grubbing operation under bill item 31.01 and in accordance with section 3000 of the General
16
Specifications for Road and Bridge Works (2005), which is time wasting. It is also very costly to
remove the hard mound soils regardless of the method used. Little or no study has been done to
assess the performance of this mound soil as pavement / subgrade materials. The researchers in
this study, were interested in not only assessing the suitability of mound soils as pavement /
subgrade materials but also to explore the major constituents of the building agents. This in turn
would give rise to the model compound that can be manufactured directly or indirectly, treat the
soil with the model compound and study the performance of the soil – compound mixture. If
found successful, this would lead to reduction in costs arising out of importation / borrowing of
materials for pavement layer construction as this would utilize the existing in – situ soils.
1.2.3 Project Approach
In this study, formulation of a model compound as a potential stabilization material for road base
and sub-base using termite saliva technology was done. The research was laboratory based
covering soils from anthills from different regions of Uganda including Luweero, Mbarara, Lira,
Kotido, Masindi, Arua and kasese. Elemental analysis and physical properties analysis was carried
out on termite mound soils and offsets of 3m and 8m away from the anthill in each of the region
in order to compare and contrast the results. Termite saliva was extracted and analyzed, treated
in situ soils with the extract and results compared with untreated soils, a model compound was
formulated, test with naturally existing soils strength properties improvement and a comparison
made with specification requirements to check whether the strategy is suitable for use.
1.2.4 Main Activities
The main activities undertaken involved literature review to establish the current state of the art;
material sampling from the different selected parts of the country; materials conditioning followed
by laboratory experiments (including characterization, mechanical and durability tests), chemical
analysis of the termite saliva extract and termite mound soil, treatment of the saliva extract with
the in situ soils, optimization of the different parameters and formulation of the model compound
with similar properties as termite saliva, treatment of in situ soils with the model compound,
stabilization of road soils and analysis of the data from the laboratory testing programme to draw
conclusions. The factors investigated included chemical composition both termite saliva and
sampled soils, for the different termites in Uganda (Macrotermes Subhyalinus (MS) and
Macrotermes Bellicosus (MB)), compaction effort (10, 25 and 55 blows), and curing time 3 days.
17
1.2.5 Expected Outputs/Outcomes
This study has disseminated results on the model compound application which when applied
properly on the road can result into
i. Improved strength of road soil material based on termite stabilisation technology.
ii. Research results on use of termite technology availed to potential stakeholders for
improving material specifications/standards.
iii. Increased knowledge base on use of termite technology in road material improvement.
iv. Reduced unit cost of road material stabilisation.
1.2.6 Benefits from the Study
The study has given results showing that for most parts of the country, in-situ road materials
can be utilized instead of being cart to waste. If implemented, the findings can be beneficial to
several stakeholders as follows:
1. Government of Uganda (GoU), whether through force account or contracting, will spend
less on sourcing, transporting and spreading material during rehabilitation of gravel
roads. This saves time and money, and hence leads to reduced construction unit cost.
2. The contractors can avoid the burden of looking for borrow materials (laterites) which
are not easily found in some areas of Uganda. Besides the land tenure system gives a
lot of freedom to private owners to determine prices for these materials.
3. The local artisans can be protected from unfavorable and unhealthy environmental
effects like the dust nuisance for roadside settlements/businesses, open borrow pit
areas which breed disease vectors, and others.
4. The road users will enjoy reduced travel times, reduced vehicle operating costs,
comfortable rides, less damage to their vehicles, less fuel consumption and others.
All these benefits will help Government in realizing the goals set out in its programme to
improve the road sub-sector of transportation. This is aimed at increasing the percentage of
the road network in a fair to good condition. Some of these programmes include the 10-
year Road Sector Development Programme (2001/02 – 2010/11), 15-year Uganda National
Transport Master Plan (2008 – 2023), the 5-year National Development Plan (2010/11 –
2014/15), and others.
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1.2.7 Contribution to CrossRoads Objectives
Since the principal themes of crossroads program is to support research into road materials and
methods, the major outcomes from this study will help in improvement of road material design
specifications, harnessing nature, reduce road construction and maintenance unit costs and
provide a foundation for construction of dust and mud free durable roads.
No road materials related research has been carried out yet on simulating termite saliva as
potential stabilizers of road construction materials. All studies have been based previously on lime,
cement and amine based stabilizers. All these are either mineral or liquid chemical stabilizers. This
research is distinct because it relates to a stabilizer that is natural and less damaging to the
environment.
19
CHAPTER 2: THE INNOVATION
2.1 THE INNOVATIVE TERMITE SALIVA TECHNOLOGY
2.1.1 General Aspects on termite saliva technology
Stabilization refers to the treatment of soil or gravel in order to improve its stability and bearing
capacity. Stabilization can be traced as far back as 2000 years ago when the Romans first used a
form of lime treatment for their tracks that carried heavy transport wagons. This process is
therefore as old as the road construction industry and will continue to be a relevant one for as
long as good quality and cheap road aggregates remain scarce. A review of previous works shows
that stabilization research efforts have been centred more on traditional than their non-traditional
counterpart chemical stabilizers. Some termites are dated as old as 400 years, so basing on this
fact, this study has come to analyze the unique technology the termites use to construct their
nests
Termites
Termites, often misnamed white ants, are small to medium-sized, soft-bodied insects which range
in color from dull white to light and dark brown, and belong to the insect order lsoptera. Termites
also exhibit polymorphism, and a well-defined grouping of individuals into different functional
castes, viz. larvae, workers, pseudergates, soldiers, nymphs and reproductives. Of these, the first
four are non-winged and incapable of reproduction. Pseudergates retain their ability to
differentiate and nymphs develop to alated reproductives.
Termites are social insects belonging to order Isoptera considered to be of the cockroach order
Blattodea whose diversity and classification are described in Eggleton (2011) and Eggleton,
(2011). With over 2600 known specifies (Eggleton, 2011), termites are classified into seven
families namely (Lo and Eggleton, 2011):
i) Mastotermitidae: This is the most primitive family with only one wood-feeding termite
species recorded in Australia.
ii) Kalotermitidae: These consume materials from dry wood
iii) Termopsidae: these nest in and feed on wet dead logs
iv) Hodotermitidae: they are commonly referred to as grass-harvesters
v) Rhinotermitidae: this is a wood consuming family commonly located in the temperate
zones.
20
vi) Serritermitidae: these have few species known and only found in SouthAmerica. They
could therefore be classified within the Rhinotermitidae family
vii) Termitidae: Is the largest family, consisting of approximately 2000 species and accounting
for almost 75% of all known termites.
There are over seven sub-families of the family Termitidae and these comprise of namely
Macrotermitinae, Sphaerotermitinae, Foraminitermitinae, Apicotermitinae, Termitinae,
Syntermitinae, and Nasutitermitinae (Eggleton, 2011; Lo and Eggleton, 2011). Termitidae have
diverse feeding preferences, the majority feeding on soil. A few subfamilies such as Termitinae,
Nasutitermitinae, and Syntermitinae contain wood- or litter-feeding termites. Termites of the sub-
family Macrotermitinae cultivate basidiomycete fungi (Termitomyces sp.) in their nests, and are
thus commonly known as fungus-growing termites.
Based on the presence or absence of flagellated protistan symbionts in the hindgut of termites,
they are conventionally grouped into lower and higher termites. The first six families, all of which
harbor protistan symbionts in the hindgut, are referred to as “lower termites”. The remaining
family, Termitidae, which lack protistan symbionts in the hindgut, are traditionally referred to as
“higher termites” (Lo and Eggleton, 2011).
Termites in Uganda
There are two dominant species of termites in Uganda namely Macrotermes Subhyalinus (MS) and
Macrotermes Bellicosus (MB). The latter is commonly found in central Uganda while the former in
western Uganda. Mounds of both macrotermes bellicosus and macrotermes subhyalinus contain
less sand than the subsoil when the subsoil is sandy, but only macrotermes subhyalinus mounds
contain less clay when the surrounding soil has high clay content.
Nests
Since termites have a soft cuticle and are easily desiccated, they live in nests that are warm,
damp, dark, and sealed from the outside environment. These nests are constructed by workers or
old nymphs. The high relative humidity in the interior of the nest (90 to 99 percent) probably is
maintained in part by water production resulting from metabolic processes of individual termites.
Nest construction
Some termites build nests in wood, some in trees and posts, and some below the ground.
Although MS and MB begin nests below ground, they form large mounds or towers of soil. The
21
nest is constructed of sand and clay. The workers burrow into the subsoil bringing back a sand
grain and a "mouthful" of clay which becomes moistened with saliva. The sand particle is stuck in
position in the nest and cemented with the mortar of clay and saliva. Building activities are most
intense at the beginning of the wet season.
Studies showed that an individual termite would pick up a soil pellet of approximate area 10-6 m2,
crush and masticate it by the buccal appendices and the mandibles and in the process saliva is
added. During the study, groups of termite workers of different sizes were observed around a
queen and found that individual termites pick up a piece of soil near the queen, transport the
pellet to the site of deposition (at a distance of about 2 to 5 cm from the queen) where they
deposit and cement the pellet. This leads to the construction of pillars or columns that are
lengthened until they reach a height of 0.5 to 0.8 cm, when workers start to build lamellae which
are extended and connected to one another to form a roof over the queen.
Some termite classes utilize faeces in the construction of mound material. These faeces are used
to cement and stick in position sand grains in the mound.
Nest structural stability
In soils, stability is usually a function of the organic (largely polysaccharide and glycoprotein)
content of the micro and macro aggregates and of the availability multivalent cations. The
following are sources of structural strength in termite mounds according to literature.
Termites use their saliva to break down cellulose to polysaccharides and finally to glucose.
The increase of polysaccharide within soil matrix is associated with increase in soil stability.
It is therefore possible that the broken down polysaccharide is a primary strength additive
to mound soils.
The presence of enzyme activity in the saliva including Endo Beta 1,4 Glucanase among
others are additional contributors to termitarium strength. Endo-beta 1,4 glucanase digests
cellulose to beta glucans which have proved to be effective in soil stability according to a
study in Korea. In this study, a purified biopolymer (β-1,3/1,6-glucan) was used as an
engineered soil additive to ordinary residual soil. Micro (i.e., small strain) geotechnical
properties of β-1,3/1,6-glucan-treated Korean residual soil were measured via non
destructive laboratory tests. The results showed that shear modulus increased as the β-
22
1,3/1,6-glucan content in soil increases. Meanwhile, β-1,3/1,6-glucan treatment had no
effect on the compression stiffness of soil.
Although soil organic matter (SOM) is usually considered as a cement ensuring the soil
structural stability of mound soil, studies that SOM has a negligible role and that clay can
be considered as a key component to understand the structural stability of macrotermes
mound soil. Additionally, reports indicate that there is a significant increase of clay content
in termite mound soil compared to organic matter.
Withstanding this, some scholars have argued that complex carbohydrates exist in the
termite mouth in form of mucopolysacharides. These are believed to enhance the gluing
properties of the soil hence making it harder.
Nest soil texture
From studies carried out on wet sieve analysis and hydrometer analysis for soil texture profiles, termite
mounds exhibit higher clay and silt fractions than surrounding soils. Table 2.1 provides the particle sizes
of mound soil and surrounding surface soils.
Nest chemical composition
The XRF analysis indicated that the main oxides present in sample pair 1 were SiO2, Al2O3 and Fe2O3.
About 75% to 80 % of sample pair 2 was contributed by SiO2. From the XRF studies, it was seen that
the amount of Al2O3 was higher in both mound soils compared to nearby soils. Therefore it may be
possible to suggest that the higher percentage of Al2O3 may have contributed to the cementation of
mound soils (Anusha et al., 2010). XRD analysis indicated that the termite mounds consisted
dominantly of clay minerals with quartz and mica; whereas the corresponding soils without termites
contained more iron rich clay minerals as shown in Table 2-2.
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Table 2.1: Particle sizes mound soil and surround surface soil (Anusha, 2010)
Where;
TM 1 and TM 2 are Termite mound soils collected during summer.
SS 1 and SS 2 are surrounding (control) soils that were collected during summer.
TM 3, TM 4 and TM 5 are Termite mound soils that were collected during the rainy season.
SS 3, SS 4, and SS 5 are surrounding (control) soils that were collected during rainy season.
Table 2.2: Composition of termite mound and surrounding soils
Composition % Anusha et al., (2010)
TM(1) SS(1) TM(2) SS(2)
SiO2 37.4 36.3 75.2 83.2
Al2O3 24.7 21.6 17.5 10.3
K2O 0.7 0.64 0.54 0.49
Fe2O3 13.1 15.53 4.07 3.10
TiO3 1.6 1.35 1.21 1.25
CaO 0.5 0.53 0.51 0.61
MgO 0.9 0.71 0.43 0.38
Composition Sample Pair 1
Sample Pair 2
Sample Pair 3
Sample Pair 4
Sample Pair 5
TM 1 SS 1 TM 2 SS 2 TM 3 SS 3 TM 4 SS 4 TM 5 SS 5
Gravel (%) 0 11.8 0 0.5 0 0.3 0 0.3 0 15.9
Coarse sand (%) 5.6 23.2 0.0 2.3 0.3 2.9 0.05 0.9 0.4 5.9
Medium sand (%)
10.8 14.6 29.8 37.4 14.0 28.1 33.6 34.2 21.0 18.7
Fine sand (%) 7.7 10.6 29.7 32.3 20.8 24.7 33.9 50.2 33.5 27.3
Silt (%) 51.5 30.3 22.7 19.3 38.9 29.9 24.7 8.2 36.6 26.1
Clay (%) 24.3 9.6 17.8 8.3 25.9 14.1 7.8 6.2 8.5 6.1
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Enzyme based stabilization
Enzymes are natural, nontoxic, non-flammable, non-corrosive liquid formulation fermented from
vegetable extracts that improve the engineering properties of the soil. Great work has been done
on the use of enzymes as stabilizing agents.
Review of previous works shows that trials of enzyme based stabilisers in different countries as a
soil stabilising agent have been done. For instance World Bank study in Paraguay, recorded
consistent road improvements and better performance from the roads stabilised with the enzyme
based stabilisers compared to untreated roads. The conclusions of the study were drawn based on
data gathered on a large-scale study from multiple sites using commercial enzymes and
documentation of road performance for up to 33 months. Stabilization with enzymes has been
used in several countries including India and Malaysia. Good performance of these roads despite
the heavy traffic and the high rainfall has been found. Besides an increase in the strength and
durability of the roads, a reduction in project cost has also been achieved.
In Mendocino County, California Department of Transportation has conducted several tests of a
compaction additive based on enzymes. This natural product helped the road base to set very
tightly, reducing dust and improving chip-seal applications. With air quality and water quality
agencies requiring dust reduction, enzymes are a potentially effective new product, cheaper than
asphalt.
Enzyme Action to Soil
Generally, enzyme agents work well over a wide range of climates and environments and work
particularly well on material containing a high percentage of clay or in iron rich soils according to
literature. They are ineffective on material containing a low percentage of fines or where loose
surface gravel is present. Additionally, enzyme dust suppressants must be “intimately mixed” and
compacted at optimum moisture content. Quality control during construction is difficult and is
likely a major contributing source of the range of variation in experimental results to date. At this
time enzyme solutions are generally considered promising, but are still in the experimental stage
of development.
25
Mechanism
When exposed to air, the bacteria multiply rapidly producing large organic molecules that are
absorbed into the soil or clay particle lattice structure. This reaction first causes a slight swelling,
followed by a tightening or compacting effect. The reaction, in effect, mirrors the natural process
of forming shale but increases the process from millions of years to a number of days or even
hours. The procedural development is as follows;
1) Negative charge on the surface of clay particles attracts positive (hydrogen) end of water
molecule.
2) Water molecules are arranged in a definite pattern-adsorbed layer
3) Enzyme catalyzes the reaction between the clay and the organic cations and accelerates
the cation exchange process to reduce the adsorbed layer thickness.
4) Enzyme replaces adsorbed water with organic cations, thus neutralizing the negative
charge on a clay particle.
5) The organic cations also reduce the thickness of the electrical double layer. This allows
enzyme treated soils to be compacted more tightly together
Enzyme promotes the development of cementatious compounds as shown by the general reaction below:
H 2O + clay Calcium Silicate Hydrates
Enzyme cellulase as a soil stabiliser
Studies on the cellulase enzyme effect on engineering properties of black cotton soil and lateritic
soils treared indicated that;
Lateritic soil treated with 1 % cellulase produced higher values of strength at 3 days curing
compared to untreated soil. There was however no significant difference in the strength of
soil treated with 1.5% cellulase.
The strength of black cotton soil became 1.22 times higher when treated with 1.5 %
cellulase at 3 days curing. There wasn’t any significant difference in strength with soil
treated with 2 % cellulase at 3 days curing.
Enzyme
26
Polymer based stabilisation
Polymers are large molecules composed of many similar smaller molecules linked together. The
individual smaller molecules are called monomers. When small organic molecules are joined
together, giant molecules are produced. These giant molecules are known as macromolecules.
The mound soil is cemented together with chewed up partially digested wood, saliva and faeces
to produce a more durable building material. These reworked soils are more stable than the
surrounding ground mass. This therefore implies that oligosaccharides (polymers) of cellulase
digestion are key contributors to termiterium strength.
How polymers work
Many road gravels have adequate strength to resist traffic stresses when they are dry but
dramatically lose this strength with the increases in moisture contents that often occur in service.
When wet, the clay fines within the gravel become ‘greasy’ and lubricate the larger stones. This
allows them to slide relative to each other to produce rutting when subjected to wheel loads.
Strength loss can be particularly pronounced for gravels that have smooth, rounded coarse stones
and highly plastic fines.
Polymers act to preserve the ‘adequate’ dry strength of water-susceptible gravels by a dual
process of ‘external’ and ‘internal’ water proofing. This involves creating a hydrophobic soil matrix
between the larger stones, which reduces permeability and so limits water ingress. Also, because
the polymer is so strongly attached to clay particles, it displaces water from the clay. Thus
softening and lubricating effect of any moisture that does enter the pavement is much reduced.
This enables soil particles to agglomerate. (Mihai et al., 2005)
Con-Aid, a polymer based stabilizer was treated with gravel to generate the tri-axial test results as
shown in Table 2-3. It can be seen that polymers improve soil properties such as shear strength
and hence reduce the plasticity index. Soaked CBR testing of treated and untreated gravels was
the method most commonly used to indicate the degree of improvement in shear strength
achieved for particular gravels. The results for soaked CBR of the polymer treated samples were
significantly higher than the control samples.
Soaked CBR testing of treated and untreated gravels was the method most commonly used to
indicate the degree of improvement in shear strength achieved for particular gravels. The results
for soaked CBR of the polymer treated samples were significantly higher than the control samples.
27
In so doing, the polymer treated base courses remain drier in service, they exhibit reduced plastic
deformality and so the rate at which wheel path deformations accumulate is reduced (Mukasa,
2012).
Table 2.3: Tri-axial results on treated and untreated gravel (Mukasa, 2012)
Test Specimen Moisture
Content
(%)
Cohesion
(kPa)
Friction Angle
(Degrees)
Dry Density
g/cm3
Dry untreated gravel 1.6 450 39 2.18
Wet untreated gravel 8.2 0 22 2.11
Wet polymer treated
gravel
8.9 125 37 2.02
2.1.2 Soil Strengthening Mechanism of Termites
Termites have been known to construct termite mounds using soils which is a result of
decomposition of dead plants and animals. The dry matter of plants or plant biomass is generally
referred to as Lignocellulose. This is the most abundant biomass on earth commonly composed
of cellulose, hemicelluloses and lignin. It is through the digestion of lignocellulose that biomass is
converted into biofuels, hence recognized as a potential sustainable source of biofuels and
biomaterials production (Himmel et al., 2007; Ragauskas et al., 2006). The study of termites,
which have been known to decompose liginocellulose have identified numerous cellulose
hydrolysis enzymes (Kudo, 2009; Ohkuma, 2003; Yamada et al., 2005).
The termite intestinal tract
The intestinal tracts of termites are axially structured microenvironments with differences in
metabolic activities and microbial community structures (Köhler et al., 2012). The termite gut
generally consists of the foregut, midgut, and hindgut. The foregut is an esophageal tract of
ectodermal origin with an enlarged anterior segment (“the crop”) and a posterior segment (“the
gizzard”) that plays a role in mechanical grinding of ingested wood fragments. The midgut, which
in insects is chiefly a secretion site for digestive enzymes and nutrient absorption, is columnar and
28
uniform with an endodermal origin. The midgut is located posterior to the foregut. The Malpighian
tubules are usually attached to the end of the midgut to excrete nitrogen wastes to the gut
lumen.
The hindgut of ectodermal origin, is the largest organ. The hindgut can be further subdivided into
P1, P2, P3, P4, and P5 segments (Watanabe and Tokuda, 2010). Among these segments, P3 is
typically enlarged to harbor numerous microorganisms. The gut microbial community contains all
three domains of organisms: Archaea, Bacteria, and Eukaryotes (protists). Relatively little
microbiota is found in the foregut and midgut, whereas abundant microbiota is found in the
hindgut (Hongoh, 2011; Köhler et al., 2012). The hindgut compartments of higher termites are
more developed and complex than those of lower termites. Except for Macrotermitinae,
Sphaerotermitinae, and Foraminitermitinae, higher termites have developed a “mixed segment”
(half of the gutwall consists of midgut tissue; the remaining is hindgut tissue) between the midgut
and the hindgut, but the precise function of the mixed segment has not been elucidated. In
addition to the intestinal tract, the salivary glands also significantly contribute to the digestive
physiology of termites. Detailed descriptions of termite gut structures are documented elsewhere
(see Bignell, 1994, 2011; Lo and Eggleton, 2011 and references therein).
Cellulose and cellulase
Lignocellulose consists of cellulose (20–50%), hemicellulose (15–35%), and lignin (18–35%).
Cellulose, a linear polysaccharide consisting of β-1,4-linked D-glucopyranosyl units, is the major
component of plant material (20–40%) and the most abundant biomass on earth (Tomme et al.,
1995). Cellulases, found in the gut of lower and higher termites, are produced by organisms that
catalyze the cellulolysis (or hydrolysis) of cellulose, and three classes have been identified in
cellulolysis. Endo-β-1,4-glucanases (EC 3.2.1.4) hydrolyze cellulose chains in a non-processive
(random) manner, whereas exoglucanases such as cellodextrinases (EC 3.2.1.74) or
cellobiohydrolases (EC 3.2.1.91) depolymerize cellulose chains from their reducing or non-
reducing ends in a processive or ordered manner. β-Glucosidases (EC 3.2.1.21) cleave cello-
oligosaccharides (especially cellobiose) to liberate glucose.
Glucanases are enzymes that break down a glucan, a polysaccharide made of several glucose sub-
units. As they perform hydrolysis of the glucosidic bond, they are hydrolases.They are of different
types summarized as hereunder;
i) α-glucanases: These are composed of
α-1,4-glucanase, an enzyme that breaks down α-1,4-glucans
29
α-1,6-glucanase, an enzyme that breaks down α-1,6-glucans
Pullulanase, a specific kind of glucanase that degrades pullulan
ii) β-glucanases; these are composed of:
β-1,3-glucanase, an enzyme that breaks down β-1,3-glucans such as callose or
curdlan
β-1,6 glucanase, an enzyme that breaks down β-1,6-glucans
Cellulase, an enzyme that perform the hydrolysis of 1,4-beta-D-glucosidic linkages
in cellulose, lichenin and cereal β-D-glucans.
Xyloglucan-specific endo-beta-1,4-glucanaseXyloglucan-specific exo-beta-1,4-
glucanase
Cellulolytic systems in lower termites
Distribution patterns of cellulolytic enzymes in the gut of termites have been studied extensively,
but relevant studies on soil-feeding termites are limited. The distribution patterns and expression
of cellulolytic enzymes in the termite gut varied by termite caste and developmental stages (Fujita
et al., 2008; Shimada and Maekawa, 2010). Thus, this review focuses on mature worker-caste
termites that feed on lignocellulosic materials and have significant digestive roles among the
castes.
Generally, lower termites possess strong hydrolytic activity (45–85% of total gut activity) against
carboxymethylcellulose (CMC) (representing endo-β-1,4-glucanase [EG] activity) in the salivary
glands (Tokuda et al., 2004), whereas these termites possess stronger cellulolytic activity (40–
88%) in the hindgut than in the salivary glands when microcrystalline cellulose is the substrate
(primarily representing cellobiohydrolase [CBH] activity) (Tokuda et al., 2005). Regarding β-
glucosidase (BG), lower termites have stronger enzymatic activities both in the salivary glands and
the hindgut than higher termites (Slaytor, 2000; Tokuda et al., 2002). The relevant genes
encoding these enzymes have been identified (Lo et al., 2011; Watanabe and Tokuda, 2010).
Based on peptide sequence similarities, glycoside hydrolases are classified into more than 100
families. Members of the same family frequently have different substrate specificities but share
structural similarities that, in turn, reflect their evolutionary origins (Henrissat and Bairoch, 1993).
According to this classification, all endogenous EGs are affiliated with the glycoside hydrolase
family (GHF) 9 (Leonardo et al.,2011; Tartar et al., 2009; Watanabe and Tokuda, 2010; Zhang et
al., 2012b), while all endogenous BGs belong to GHF1, except for one putative endogenous GHF3
BG which has been identified from the salivary gland EST library of Hodotermopsis sjostedti
30
(Termopsidae) (Yuki et al., 2008). Detailed cellulase gene structures of termites are reviewed by
Lo et al. (2011).
Compared to host cellulases, symbiotic protistan communities in lower termites produce more
complex cellulolytic enzymes. Metatranscriptomic sequencing techniques have provided
comprehensive information about cellulolytic enzymes from the protistan symbionts of five genera
of termites, Mastotermes darwiniensis (Mastotermitidae), Neotermes koshunensis
(Kalotermitidae), H. sjostedti, Reticulitermes speratus (Rhinotermitidae), Reticulitermes flavipes,
and Coptotermes formosanus (Rhinotermitidae) in addition to the closely-related wood roaches
Cryptocercus punctulatus (Scharf and Tartar, 2008; Sethi et al, 2013; Tartar et al., 2009; Todaka
et al., 2007, 2010a; Xie et al., 2012). In the protistan cellulolytic system, GHF5 EGs, GHF7 EGs,
and GHF7 CBHs appear to function as a core enzyme set that might be conserved across protistan
generations during the history of symbiosis between termites and intestinal protists. On the other
hand, the GHF45 EGs might supplement these core cellulases; this family is absent from N.
koshunensis and forms multiple clades in the phylogenetic tree (Todaka et al., 2010a). The
symbiotic protists primarily express GHF3 BGs, which are predicted to perform similar glucose
release functions as endogenous GHF1 enzymes (Scharf and Tartar, 2008; Tartar et al., 2009;
Todaka et al., 2007; Xie et al., 2012).
Lower termites produce both EGs and BGs primarily in the salivary glands. These endogenous
cellulases probably hydrolyze amorphous regions of cellulose during its passage through the
midgut. A combination of endogenous EG and BG can release glucose from filter paper (Zhang et
al., 2010) and pine lignocelluloses (Scharf et al., 2011). An essential contribution of an
endogenous EG to termite survival and fitness has been verified with RNAi investigations (Zhou et
al., 2008b). Partially degraded cellulosic fragments being moved to the hindgut are endocytosed
by the symbiotic protists. Because no (or only a trace of) endogenous cellulases enter the hindgut
(Fujita et al., 2010; Nakashima et al., 2002; Watanabe et al., 2006), cellulolysis in food vacuoles is
conceivably accomplished synergism of multiple cellulolytic enzymes that originated from protists.
Because ingested cellulosic materials are masticated and fragmented (less than 20 μm in the
foregut; Fujita et al., 2010), this micro-fragmentation of food could have increased surface area,
facilitating access of cellulolytic enzymes and stimulating endocytosis by symbiotic protists. In
addition, the presence of CBHs in the protistan cellulolytic system apparently confers efficient
digestibility of crystalline cellulose in the hindgut. This dual cellulose digestion system has been
31
proposed as a model of efficient cellulose hydrolysis in lower termites (Nakashima et al., 2002;
Tokuda et al., 2007; Watanabe and Tokuda, 2010).
Lower termites secrete endogenous endo-β-1,4-glucanases (EGs) and β-glucosidases (BGs) in the
salivary glands. These enzymes are probably mixed with wood particles ingested by the termites
and may act primarily against amorphous cellulose. Chewed wood particles are partially degraded
during passage through the midgut. Remaining wood particles enter the hindgut, where they are
digested in the food vacuoles of symbiotic flagellates with protistan EGs, BGs, and
cellobiohydrolases (CBHs) that are highly active against crystalline cellulose. These protists also
produce hemicellulolytic enzymes. Such cellulolytic systems of wood-feeding higher termites have
been chiefly studied in Nasutitermitinae, which secrete endogenous BGs both in the salivary
glands and the midgut, whereas endogenous EGs are secreted only in the midgut. These enzymes
also act primarily against amorphous cellulose, but may have an undescribed mechanism that
enhances digestibility of crystalline cellulose (Tokuda et al., 2012). The hindgut harbors symbiotic
bacteria and archaea.
Among them, Treponema and Fibrobacteres are primarily involved in cellulolysis with EGs and
BGs. Like these cellulases, cellobiose- and cellodextrin-phosphorylases (GHF94) appear to be
involved in cellulolysis, but there is no evidence that CBHs are present in the hindguts of higher
termites. Nevertheless, the hindgut precipitates contain activity against crystalline cellulose
(Tokuda and Watanabe, 2007). Many different hemicellulolytic enzymes are also produced by
these bacterial symbionts.
32
Figure 2.1: Cellulolytic Systems in Lower Termites (Flagellate – Harboring): Source:
(Ni and Tokuda, 2013)
Figure 2.2: Cellulolytic Systems in Higher Termites (Flagellate – free); Source: (Ni and
Tokuda, 2013)
Cellulolytic systems in higher termites
Distribution of cellulase activities in higher termites is more variable. The reason for this is
somewhat obscure, but ecological events may have affected cellulolytic systems during the
evolution of higher termites (Fig. 1.2). In most wood-feeding termites of the subfamily
Nasutitermitinae, EG activities are confined to the midgut, but Nasutitermes lujae share an almost
equal amount of EG activity both in the midgut and the hindgut (Slaytor, 2000). In addition, some
species of Termitinae have most EG activity in the hindgut, which is presumably attributable to
the symbiotic amoeba (Slaytor, 2000) whose nature has not been clarified yet. Similar to the
distributions of EG activities, distribution of BG activities is also variable. Some termites, including
Nasutitermes spp., have BG activity primarily in the salivary glands and the midgut (Slaytor, 2000;
Tokuda et al., 1997). Other termites possess up to 99.5% BG activity in the hindgut (Slaytor,
2000). Distribution of cellulase activities in the fungus growing termites (subfamily
Macrotermitinae) is more complicated (Rouland-Lefèvre, 2000) partially due to varying
dependence on the symbiotic fungi among species as a nutritional carbon source (Hyodo et al.,
2003). Contributions of the hindgut microorganisms to cellulose digestion in higher termites have
long been neglected, but wood fiber associated cellulase activities were reported in the hindgut of
Nasutitermes takasagoensis and Nasutitermes walkeri (Tokuda and Watanabe, 2007). These
cellulase activities were significantly decreased if antibiotics were administrated to termites. These
bacterial cellulolytic activities in the hindgut accounted for ~50% compared to cellulase activity in
33
the midgut if microcrystalline cellulose was the substrate (Tokuda and Watanabe, 2007). Thus,
there is strong evidence that the endogenous cellulolytic system in the midgut of higher termites
is more important than that of lower termites. As with lower termites, endogenous EGs in higher
termites are affiliated with GHF9 and are secreted from the midgut, except for the fungus-growing
termite Odontotermes formosanus, in which EG genes are still expressed in the salivary glands
(Tokuda et al., 2004). Endogenous GHF1 BGs of N. takasagoensis were expressed and secreted in
the midgut and the salivary glands (Tokuda et al., 2009, 2012), whereas the fungus-growing
termite Macrotermes barneyi expressed an endogenous GHF1 BG gene primarily in the midgut
(Wu et al., 2012).
As mentioned above, the endogenous cellulolytic system of woodfeeding higher termites is
thought to contribute to cellulose digestion more significantly than lower termites. Figure. 2
illustrates the endogenous cellulolytic system of Nasutitermes as a representative of wood-feeding
higher termites based on Tokuda et al. (2012).Masticated cellulosic fragments would be first
attacked by salivary BGs that do not enter the midgut, but the actual role of salivary BGs is
ambiguous. A salivary BG tends to more preferentially hydrolyze β-1,3-glucans (found in fungal
cell walls) than β-1,4 linkages generally recognized in cellulose chains (Uchima et al., in press). A
recent study on labial gland secretion of termites indicated the presence of p-arbutin that is
hydrolyzed by BG to produce hydroquinone, a precursor of p-benzoquinone, an irritating defensive
secretion (Sillam-Dussès et al., 2012). In addition, a possible function of BG as a proteinaceous
pheromone and reproductive suppressor has also been suggested (Korb et al., 2009; Matsuura et
al., 2009). It is probable that salivary BGs in higher termites play roles other than cellulolysis.
Ingested cellulosic particles are fragmented into less than 150 μm in the foregut. The midgut
epithelium secretes both EGs and BGs thatmake essential contributions to cellulose digestion.
Luminal concentrations of these enzymes are extremely high (~1500 U/ml for EG and ~80 U/ml
for BG), which may be important for primarily hydrolyzing amorphous regions of cellulose.
Although a purified or recombinant EG of this termite shows trace activity against microcrystalline
cellulose (Hirayama et al., 2010; Tokuda et al., 1997), the midgut crude enzyme can hydrolyze
highly crystalline cellulose to some extent (Tokuda et al.,2012). However, a detailed mechanism
of this phenomenon has yet to be elucidated. Short cello-oligosaccharides released by these
enzymes would be degraded in the ectoperitrophic space where BGs accumulate and glucose
products would be absorbed across the midgut wall. As shown in Fig. 1, remaining cellulosic
fragments enter themixed segment and then the hindgut, but as mentioned before, the role of
themixed segment in cellulose digestion is unclear. Cellulosic fragments would be further
34
hydrolyzed by prokaryotic cellulases in the hindgut. However, the detailed mechanism underlying
the cellulolytic system requires further study. In addition, it is likely that otherwood-feeding higher
termites, especially those belonging to Termitinae, have different cellulolytic systems than
Nasutitermes (Slaytor, 2000). Hence, further explorations of xylophagous species are required to
fully understand cellulolytic systems in higher termites. In fungus-growing termites, clear
conclusions are not available regarding the mechanism of cellulose digestion. For example, in the
case of O. formosanus, intestinal cellulase activity may be insufficient to produce the necessary
amount of glucose required for respiration (Tokuda et al., 2005) and the symbiotic fungus is likely
a sole carbon source based on the stable carbon isotope ratio (Hyodo et al., 2003). In spite of
these studies, its cellulolytic mechanism has been investigated recently, suggesting an “acquired
enzyme hypothesis”— that lignocellulolytic enzymes acquired fromthe symbiotic fungi act
cooperatively with endogenous digestive enzymes in termites (Deng et al., 2008a,b).
Figure 2.3: Endogenous digestive system in N. takasagoensis. : PM, peritrophic
membrane
Wood fragments ingested by the termites may first mix with the salivary BGs. The wood particles
in the foregut are broken down to 150 μm in diameter and enter the midgut. The midgut tissue
secretes both EGs and BGs into the luminal space. Based on the midgut luminal volume (approx.
75 nl), the concentrations of EG and BG activities are approximately 1500 units/ml and 80
units/ml, respectively (one unit is the amount of enzyme that produces 1 μmol of reducing sugar
35
or glucose/min). It is presumed that amorphous regions of cellulose exposed on the surface of the
wood particles are digested primarily by these enzymes, and released glucose might be absorbed
across the midgut tissue. Finally, the partially digested wood particles are moved to the hindgut
via the mixed segment. The role that the mixed segment plays in digestion has yet to be clarified.
Termites can live on crystalline cellulose and secrete cellulase in regions which are either devoid
of microorganisms or contain very small numbers of microorganisms. Slaytor (1992) based on this
evidence to conclude that cellulase which is secreted in the salivary glands, the foregut and mid
gut of all termites is endogenous. The cellulases found are very similar: they consist of multiple
endo-beta-1,4- glucanase and beta-1,4-gluscosidase components. He established no evidence of
presence of exo-beta-1,4-glucanase or even its necessity since all the cellulases produce sufficient
glucose from crystalline cellulose.
Slaytor (1992) confirmed that the predominant sites where cellulase activity is found in termites
are salivary glands, the foregut and the midgut. These are sites for secretion of other hydrolytic
enzymes generally considered endogenous such as amylase, maltase, invertase and hemicellulose.
There is hardly any carbohydrate activity present in the hindgut which is usually associated with
the highest number of microorganisms. The only exceptions occur in the lower termites which
additionally have cellulase activity in the hindgut associated with cellulytic Protozoa. There lacks
evidence of presence of bacterial cellulases in the higher termites.
Cellulose Degrading Systems
Endogenous cellulase of termite origin (endo-_-1,4- glucanase and _-glucosidase) which are
excreted from the salivary glands or the gut have been identified and characterized in both higher
and lower termites. Molecular analysis reveals these endogenous enzymes are members of
glycosyl hydrolase family 9 (GHF9). In higher termites the endogenous cellulolytic activity meets
the metabolic requirement. In lower termites, substantial cellulolytic activity is found in the
hindgut. Thus the ingested cellulose can be partially degraded by the endoglucanase of termite
origin, then unhydrolyzed cellulose travels to the hind gut, where it can be endocytosed and is
fermented by the symbiotic protists in lower termites. Termites grind and crunch the ingested
material, which may enhance digestion by increasing the exposed surface area. Two main types
of cellulases exist: endo- beta-1,4- glucanases (EGs) and cellobiohydrolases (CBHs) (EC numbers
3.2.1.4 and 3.2.1.91, respectively; see Enzyme Nomenclature of the NC-IUBMB,. EGs hydrolyse
36
the interior parts of the cellulose chain (Subodh et al., 2012), while CBHs attack the reducing ends
of the cellulose polymer. A wide variety of bacteria and yeasts including cellulolytic and
hemicellulolytic ones have been isolated from the termite gut (Prillinger et al, 1996; Schater et al,
1996; Wenzel et al, 2002). Furthermore, the termite gut is expected to be the source of novel
microorganisms with wide ranging industrial applications (Tokuda et al; 2004).
The term ‘cellulase’ traditionally includes two types of enzymes, exoglucanase (primarily
cellobiohydrolase [EC 3.2.1.91] and occasionally cellodextranase [EC 3.2.1.74]) that hydrolyses
cellulose from its non-reducing or reducing ends, and endo-beta-1,4-glucanase [EC 3.2.1.4] that
hydrolyses along the glucan chain of cellulose fibres randomly. Cellooligosaccharides produced by
these enzymes are further hydrolysed to glucose by beta-glucosidase (Tokuda et al; 2005).
Carboxymethylcellulose, which measures endo- beta -1,4- glucanase activity, is one of the most
popular artificial substrates for measuring cellulase activity because of its high solubility in water.
Thus, carboxymethylcellulose has been preferentially used in most studies of cellulose digestion in
termites and other insects (Tokuda et al; 2005).
2.2 COMPARISON OF THE TECHNOLOGY WITH CLASSICAL STABILISATION
TECHNIQUES
The use of TERMITE saliva technology to treat and strengthen in-situ road material is a new
concept in the stabilization field. It entails transforming a poor material into a better and improved
material for construction of gravel roads. Classical additives include lime, cement, enzymes like
terrazyme, etc. Limited success has been recorded in their use for stabilizing in-situ marginal
materials. When this technology is embraced in Uganda can result in;
Extended period over which gravel roads will be above the threshold of an acceptable level
of service before need for major rehabilitation.
Environmental preservation and cost saving since limited borrow pit opening, purchase,
transportation, spreading as well as poor spoil material deposition would be done.
Transition from the classical practice of using borrow and transported material to the use
of a seemingly inferior in-place material that would otherwise be cart to spoil.
It will reduce the cost of upgrading of gravel roads to either higher class or paved
standard.
Reduced dustiness from loose fine material on road surfaces.
37
Reduced potential for loss of control vehicle accidents due to loose gravel (skidding) and
impaired visibility due to excessive dust.
Since termite mounds are found with in every region in Uganda, it is concrete evidence that the
technology can be applied in any part of the country.
Major findings related to Engineering Properties
This review has alluded to fundamental aspects of termite mound building. The building behavior
of termite workers is a step by step process and motivated by the need to protect the queen while
providing shelter and protection for each other. The process of picking up a soil pellet,
transporting it to the site of deposition and cementing the pellets together seems to be a logical
process following a critical path of controlled activities. That Termites start by constructing pillars
(or columns) that are lengthened until they are approximately 0.5-0.8cm in height and then build
lamellae which are extended and connected to one another to form a roof over the queen while
Inter-pillar spaces are also filled with pellets to produce walls. This is a typical construction
procedure similar to that developed and being utilized by man.
In a study carried out by Millogo et al., (2011), it was discovered that the termite mound is
composed of 46% sand, 44% silt and 10% clay, was of medium plasticity (Santos, 1989), and
could not be adequately shaped by extrusion. For mounds shaping, it is asserted that, termites
resort to their saliva (Hesse, 1955) and gather different shaped pellets (5–1000 μm in size),
consisting of organic matter, minerals or both (Eschenbrenner, 1986 ). These micro-aggregates
represent the basic ‘building blocks’ of the mound. The measured magnitudes of compressive and
bending strengths of nest building samples were 5.1±0.3 and 1.3 MPa, respectively, and were
found to be as strong as cement-stabilized crude bricks (Azeredo et al., 2007; Venkatarama and
Gupta, 2005). The linear shrinkage of nest soil test-pieces was found to be <5% as a result of the
presence of a significant amount of fillers (principally quartz), and possibly due to the absence of
swelling clay minerals.
Anusha et al., (2010) studied the surrounding soil sample (Ssoil) and the termite mound (Mound soil)
and the following were noted:
Natural Water Content
Two soil sample pairs were collected during summer season and a set of three sample pairs during
rainy season. The first two samples had an increase in water content for the mound soil possibly due to
38
the: (i) Presence of organic content, (ii) increase in clay content, (iii) higher percentage of
chemical/enzymes in the mound soil and (iv) presence of finer soil particles.
Organic Content
Organic content was determined using the hydrogen peroxide method. It was observed that the
percentage of organic content for mound soils and the nearby soil is higher when compared to other
normal soils. Comparing the mound soil and the nearby soil, the reduction in organic content of mound
soil may be due to the decomposition of organic matter by the action of termites.
Liquid Limit
The liquid limit results showed that mound soil had values greater than that of Ssoil in all the cases.
This increase in liquid limit for the mound soil may ascribed to: (i) presence of organic content, (ii)
increase in clay content, (iii) higher percentage of chemical /enzymes in the mound soil and (iv)
presence of finer soil particles. And the increase in water content of the surrounding soil in the other
three samples is due to the impermeability of mound soil compared to the surrounding soil.
Plastic Limit
The plastic limit test was conducted according to ASTM (D424–59). The plastic limit values show that
because the clay content of the mound soil is more than the surrounding surface soil; its plastic limit
value is also higher.
Plasticity Index
Considering the plasticity index values, the mound soils have higher plasticity indices than the Ssoil,
which indicate that they have higher clay fractions, hence an increase in cohesion. The other
reasons for the increase in the plasticity values of the mound soils are the higher percentage of
chemical/enzymes of termites, organic content and the clay content.
Specific Gravity
The specific gravity values of all soil samples are less compared to normal soils found in the
corresponding region. Generally reduction of specific gravity is mainly due to the presence of high
organic content and lesser amount of iron content.
39
pH
To measure the pH of the soil, 10g sample mixed with 100ml of distilled water standing 24 hours. pH
of the soil was determined using a digital pH meter. All the samples were acidic, mound soil being less
acidic compared to the controls.
Particle size Analysis
Grain size distribution for the soil texture profile samples of the mound and surrounding materials was
determined using wet sieve analysis and the results are given in Table 1.
Table 1: Grain size distribution results for the samples that were tested
Where; TM 1 and TM 2 are Termite mound soils collected during summer.
Ssoil 1 and Ssoil 2 are surrounding (control) soils collected during summer.
TM 3, TM 4 and TM 5 are Termite mound soils collected during the rainy season.
Ssoil 3, Ssoil 4, and Ssoil 5 are surrounding (control) soils collected rainy season.
Chemical Analysis
The XRF analysis indicated that the main oxides present in sample pair 1 were SiO2, Al2O3 and Fe2O3.
About 75 to 80 % of sample pair 2 is contributed by SiO2. From the XRF studies it can be seen that the
amount of Al2O3 is higher in both mound soils compared to nearby soils. Hence it may be possible to
suggest that the higher percentage of Al2O3 may contribute to the cementation of mound soils. Table 2
shows the chemical composition of termite mounds (TM) and Surrounding soil (Ssoil). The XRD analysis
indicated that the termite mounds consisted dominantly of clay minerals with quartz and mica; whereas
Composition Sample Pair
1
Sample Pair
2
Sample Pair
3
Sample Pair
4
Sample
Pair 5
TM 1 Ssoil 1 TM 2 Ssoil 2 TM 3 Ssoil
3
TM 4 Ssoil 4 TM 5 Ssoil
5
Gravel (%) 0 11.8 0 0.5 0 0.3 0 0.3 0 15.9
Coarse sand (%)
5.6 23.2 0.0 2.3 0.3 2.9 0.05 0.9 0.4 5.9
Medium sand (%)
10.8 14.6 29.8 37.4 14.0 28.1 33.6 34.2 21.0 18.7
Fine sand (%) 7.7 10.6 29.7 32.3 20.8 24.7 33.9 50.2 33.5 27.3
Silt (%) 51.5 30.3 22.7 19.3 38.9 29.9 24.7 8.2 36.6 26.1
Clay (%) 24.3 9.6 17.8 8.3 25.9 14.1 7.8 6.2 8.5 6.1
40
the corresponding soils without termites contained more iron rich clay minerals. The analysis indicated
that there is no significant difference in clay minerals which is kaolinite in all the samples.
Table 2: Chemical composition and elemental analysis of the samples
ND – Not Detected
Discussion and Implications of the Review
From the review of literature, the following were noted:
Compositio
n
Sample
Pair 1
Sample Pair
2
Trace Elements
(µg/g)
Sample Pair
1
Sample Pair
2
TM 1 Ssoil
1
TM 2 Ssoil
2
TM 1 Ssoil 1 TM 2 Ssoil
2
SiO2% 37.4
1
36.2
6
75.24 83.20 V 243 286 78 73
Al2O3 (%) 24.7
4
21.5
9
17.52 10.25 Cr 232 396 45 35
TiO2 (%) 1.62 1.35 1.21 1.25 Co 19 14 7 5
MnO (%) 0.10 0.08 0.01 0.02 Ni 166 94 16 ND
Fe2O3 (%) 13.1
3
15.5
3
4.07 3.10 Cu 59 31 17 13
CaO (%) 0.47 0.53 0.51 0.61 Zn 91 72 55 53
MgO (%) 0.93 0.71 0.43 0.38 Rb 44 28 30 20
Na2O (%) 0.09 0.12 0.19 0.38 Sr 38 48 54 42
K2O (%) 0.73 0.64 0.54 0.49 Zr 953 966 948 965
P2O5 (%) 0.22 0.24 0.10 0.14 Nb 24 24 24 24
LOI (%) 20.3
3
22.7
2
Nil Nil Ba 228 193 273 142
Total (%) 99.7
7
99.7
7
99.8
1
99.8
2
La 59 48 59 43
Ce 124 93 116 92
Pd 32 33 32 33
41
1. The termite mound material consisted of conventional building components namely: mortar
(kaolinite), aggregate (quartz) and plaster (organic matter).
2. Magnitudes of the measured physical properties of the termite mounds were similar to those of
manufactured earth blocks.
3. For kaolinite and organic matter availability, it appears that termites resorted to the
modification of the structure of microcline and produced organo-metal complexes.
4. The mound resistance to rainfall was apparently linked to the good physical characteristics, wall
coating and absence of swelling clay minerals.
5. The liquid limit of mound soil was generally higher than the surrounding soils.
6. The mound soil showed an increase in the plasticity index value. This indicates that the termite
mounds have more cohesion than surrounding soils due high fines content.
7. The specific gravity values obtained were in the range of 2.1 to 2.42, indicating the presence of
organic content in the soils. This was confirmed by conducting organic content test and showed
that organic content is in the range of 5 - 9%.
8. From the sieve analysis results, it can be concluded that for the building up of mound, the
termites are using more percentage of medium sand compared to fine sand and more
percentage of silt content compared to clay content. That is proper gradation of soil is
considered by the termites for the construction of termite mound, in order to make the mound
stable.
9. The main difference in percentage between the chemicals present in mound and nearby soils is
for Al2O3 and Fe2O3 and these chemicals might have given the cementation/cohesive property
to the mound soils.
10. The strength of the cellulase treated lateritic soil is 1.19 times more for soil treated with 1 %
cellulase for 3 days curing compared to untreated soil and does not give significant difference in
strength with soil treated with 1.5% cellulase for 3 days. Percentage increase in strength is
19.16%.
11. The strength of the cellulase treated black cotton soil is 1.22 times more for soil treated with
1.5 % cellulase for 3 days curing compared to untreated soil and does not give significant
difference in strength with soil treated with 2 % cellulase for 3 days. Percentage increase in
strength is 21.37 %.
42
Way forward from the above review
A detailed literature search and review has been done from which the research team has decided
to forge a way forward as follows:
i) Termites feed on cellulose as their main source of food. It is therefore important that they
adopt to the above by possessing structures that can help in breaking down the complex
cellulose into smaller digestive foods in this case glucose. Termites therefore have glands in
the foregut, middle gut and hind gut that help them to accomplish the digestion
requirements. The foregut produces numerous secretions which consist of various types of
enzymes, the most dominant being cellulase, that masticates and fragments cellulose
assisted by the gizzard and foregut before it is passed on to the midgut for further
breakdown. They also use the same salivary secretions to build the mounds while their nests
are constructed using their fecal materials. The salivary secretions have the head as their
origin. These are mainly composed of cellulase as the major enzyme. Another sub
component of cellulase responsible for the breakdown of cellulose is the endo-beta-1,4-
glucanase, which is only found in the foregut and the midgut. This is also produced by the
salivary glands.
ii) During the process of building mounds, termite workers, in an effort to protect the queen,
secrete saliva which contains among other things, cellulase and end-beta-1,4-glucanase and
mix it with the dry soil. When the soil is moist, a termite molds it into a small pellet which it
carries in its mouth up to the stage of deposition. The process continues until the worker
termites are sure that the mother queen has been given adequate protection. On drying, a
hard solid mass forms around the queen that stands for as long as the anthill remains
active. It is now clear that the hardness of the soil is as a result of salivary secretions. The
soils to be worked must contain cellulose for the salivary secretions to break down. It was
therefore important to ascertain the presence of cellulose in the termite mound soils and
also the surrounding soils.
iii) What role do the general inorganic elements play in the hardness of the soil? To answer this
question, the team set out to collect termite mound soils from across the country along the
national grid routes namely: Luweero, Lira, Kotido, Arua, Masindi, Mbarara and Kasese
regions. Termites have been known to build anthills almost everywhere, so there was need
to ascertain whether there is some special mineral elements that termites look for to
accomplish their tasks. Mineral elements were thus identified in each of the above mound
soils and their contribution in terms of significance was statistically determined.
iv) What are the engineering properties of termite mound soils and how do they compare with
those of the surrounding soils? Termite mound soils and surrounding soils were also
collected and taken to the laboratory for investigation, to determine the key engineering
properties i.e. gradation, compactability, plasticity and strength.
v) Extracts from the termites. Worker termites were collected and divided into the head and
abdomen from which cellulase and total carbohydrates were assayed. The procedure for
assaying for cellulase and total carbohydrates is explained in the later sections. However, we
43
were able to collect extracts containing cellulase enzyme and total carbohydrates from both
the head and abdomen which were used to treat the soil. An investigation into the presence
of cellulose in the soils was also done. Indeed there was presence of cellulose in both the
mound soil and the surrounding soils. However, cellulase enzyme was more concentrated in
the mound soils than the surrounding soils.
vi) Treatment of soil with soil extracts; the extracts were reconstituted into liquid form using
distilled water. The plasticity index tests were carried out on road soils and results indicated
a reduction in the PI for treated soils compared with the conventional soils.
44
CHAPTER 3: METHODOLOGY
3.1 RESEARCH DESIGN
The research was sequential flow of activities as shown in figure 3.1. This study mainly involved
collection of termite mound soil, in-situ soil and live termites to formulate the model compound.
Mechanical characterization tests and chemical analysis tests that were carried out are also
explained in this chapter.
3.1.1 Study Extent/Coverage
This study was aimed at developing an improved material formulation embracing termite saliva
technology to be applied to Ugandan soils on gravel roads in an economical and sustainable way.
Preliminary field visits to different districts of Uganda were made to identify active mounds and
obtain the necessary permission before sampling. (See Figure 3-1)
Table 3-1 shows the areas that were finally selected for sampling of materials for use in the study.
The sampling regions in the whole country were determined based on the fact that the research
desired to establish the variation in termite mounds nature from all regions in the country given
the diversity in geological properties of soils in Uganda. The termite mounds were thus sampled
from Arua, Lira, Masindi, Mbarara, Luwero, Kasese and Kotido.
Study termite mound technology
Collect soil/mound
Samples
Characterize Mound Soil
samples
Study Impact of
Termite action
Characterize Composition
of termite Head and Gut Extracts
Formulate model stabilizer
that mimics termite stabilizer
Study soils stabilized with
the model stabilizer
Make inferences
45
Figure 3.1: Map of Uganda Showing Locations of sampling points
3.1.2 Materials Used
Dry Soil sampling
Materials were collected from different layers of the anthills namely Top (0-0.5m), Middle (0.5-
1.0m) and Bottom (1.0-1.2m) as can be seen in Figure 3-2. Control samples were also taken from
distances offset from the centerline of the mound at 3.0m and 6.0m. At each of these offset
distances, samples were picked from the top (0.2-0.5m) and Bottom (0.5-1.0m). Samples were
picked for two major reasons namely chemical characterization and determination of engineering
parameters. Samples for chemical analysis were sealed in water tight bags to avoid loss of
moisture while those for engineering parameters were packed in sisal bags, loaded on trucks and
delivered to Highways laboratory in the Department of Civil and Environmental Engineering,
College of Engineering, Design, Art and Technology for subsequent testing.
Material source
46
Figure 3.2: Sampling points on an anthill
Table 3.1: Details of sampling points for the soils of the anthills
Anthill 3m Offsets 6m Offsets
Top Middle Bottom Top Bottom Top Bottom
A B C D E F G
(0-0.5m) (0.5-1.0m) (1.0-1.2m) (0.2-0.5m) (0.5-1.0m). (0.2-0.5m) (0.5-1.0m)
Location
Position of the sample
Anthill Top
(0 – 0.5m)
Anthill Middle
(0.5 – 1.0m)
Anthill Bottom
(1.0 – 1.2m)
Offset 3m
(0.2 – 0.5m)
Offset 3m
(0.5 – 1.0m)
Offset 6m
(0.2 – 0.5m)
Offset 6m
(0.5 – 1.0m)
Kotido
Lira
Arua x
Masindi x
Luwero
Kasese
Mbarara x
47
Figure 3.3: Picking of samples from anthills
Wet soil sampling
Wet mound soil was sampled after an overnight re-construction by worker termites. It was
sampled directly from the anthill and stored in air tight bags and delivered to the laboratory for
subsequent testing. Control soil samples were picked 10m away from the anthill.
Sampling of termite samples
Worker termites were manually picked from anthills Collected from Masulita, Wakiso and
Makerere, Upper FST Block and kept in air entraining tins. Upon delivery to the laboratory,
termites were divided into head and abdomen to facilitate collection and comparison of extracts
from the abdomen, head and the wet mond soils.
Figure 3.4: Identification of live termites and separation of heads from abdomens
(a) Picking samples from top of anthill
(b) Picking samples from 6m offset from anthill
48
3.1.3 Test Methods on Soils
The following tests were done at the Highways Laboratory, Makerere University in accordance
with ASTM and BS standards on soils obtained from the roadside:
i. Grading/ Sieve analysis ASTM D422-63
ii. Atterberg’s Limits ASTM D4318
iii. Proctor tests/ Compaction tests BS 1377 – 4
iv. California Bearing Ratio ASTM D1883 -99
v. Natural Moisture Content tests ASTM D2216-71
In this study, the light manual Proctor compaction test method was employed in determining
the maximum dry density and optimum moisture content. This was because the soils used
generally had very insignificant gravel fraction as was established from grading results
presented later. In this compaction, a 50mm diameter rammer of 2.5kg falling through a height of
300mm was applied with 25 blows on three layers in a 1-liter compaction mould. Subsequently,
the CBR at the respective optimum moisture content was then determined using the 2-liter
cylindrical BS mould. Variations were made of the factors that are mentioned earlier when
determining the CBR.
3.1.4 Elemental analysis
Chemical tests were carried out on samples collected from different part both from within and
around the anthill using the Atomic Absorption Spectroscopy (AAS) to determine mineralogical
composition of mound samples and samples from the surrounding soils. In the AAS procedure,
chemical composition data is collected by analyzing powdered form of the soil samples for
different elements like potassium, sodium, iron, magnesium, calcium and manganese. The
amounts (wt %) of the elements are obtained in terms of oxides although oxides may not
necessarily naturally occur in the soil. Contents of potassium, sodium, iron, magnesium, calcium
and manganese are determined by digesting 200 mg of soil samples, ground to minus 100 mesh
sieve, with 10 mL of hydrofluoric acid (conc. 40%) reagent mixed with 3 mL of perchloric acid
(conc. 70%). The mixture of the acids and the sample is heated in a 50 mL beaker for one hour
on a hot plate having a surface temperature of 200°C and then allowed to cool at room
temperature for five minutes. The resulting solution is diluted with distilled water, filtered and
analyzed using an atomic absorption spectrophotometer.
49
3.1.5 Analysis of termite extracts
The extraction and analysis of the above extracts was based on the following pre-determined
assumptions / theories gathered from literature. It was argued that the hardness of the soil was
as a result of the following:
i. The presence of cellulase enzyme in the termite saliva which breaks down cellulose
present in the soil into smaller monosaccharides.
ii. There exists complex carbohydrates in the termite mouth in form of mucopolysaccharides
that introduce and enhance the gluing properties of the soil to make harder.
iii. The hardness of the soil could be a result of the breakdown of the cellulose by the
cellulose enzyme into shorter chain polymers. This is called incomplete digestion since it
does not result into simpler monosaccharides.
With all the above in mind, it was important to analyze for the presence of cellulase (cellulase
assay) in the termite head and abdomen and also in freshly built termite mound soil. This was
followed by the extraction and analysis of complex carbohydrates in form of mucopolysaccharides
to determine both their presence and concentration.
Cellulase assay
In order to demonstrate the presence of cellulase in both the abdomens and heads of worker-
termites, the worker-termites were obtained and sectioned into the head and abdomen until at
least 2 g of each section was obtained. Each of the portions were ground using a mortar and
pestle and the contents of each portions mixed with 0.05 M citrate buffer in a ratio of 1 g of
termite to 2 ml of buffer and the mixture stirred using a glass rod. After 20 min the supernatants
of the two portions were collected after centrifuging (MSE…. England) for 15 minutes at 3000
rpm. Cellulase activity was assayed using a modified filter paper method developed by Mandels
et al. (1976). Typically, Whatman No.1 filter paper strip, 1.0 x 6.0 cm supplied as a substrate, was
cut into smaller pieces, was fully immersed in 1.6 mL of citrate buffer (0.05 M, pH 4.8) and the
reaction was started by adding 0.4 mL of enzyme extract after equilibration at 50oC. Digestion of
the filter paper was done for 1 hour after which the reaction was stopped by boiling in the water
bath for 5 min. The amount of reducing sugar was estimated spectrophotometrically at the
absorbance at 540 nm after reacting the liberated reducing sugar with 3.0 ml of
3,5-Dinitrosalicylic acid reagent. The amount of reducing sugar liberated was estimated from the
50
glucose standard curve and protein was estimated using the Biuret method. Specific cellulase
activity was defined as the amount of reducing sugar liberated per min per mg protein.
Assay for mucopolysaccharides
In order to isolate total mucopolysaccharides from both the abdomen and heads of worker-
termites, 14-g and 8-g samples of the frozen worker-termite abdomens and heads, respectively,
were ground in a motor and the total lipids extracted using the method of Bligh and Dyer, (1959).
Briefly, the ground samples were homogenized for 2-5 minutes in of 52mL and 24mL, respectively
of a mixture of chloroform and methanol (ratio of 1:2). To the resultant homogenate was then
added 14 mL and 8 mL chloroform and after blending for 30 seconds, 14 mL and 8 mL,
respectively, distilled water was added and blending continued for another 1-2 minutes. The
homogenate was filtered through Whatman No. 1 filter paper. After complete separation and
clarification, the volumes of the chloroform and the aqueous layers are visible. The chloroform
layer was discarded and the aqueous layer retained. The residues of the abdomen and heads
were removed from the filter funnels, re-suspended in water and re-extracted for another 30 min
of continuous stirring. Thereafter, the supernatants were collected by centrifuging at 6000 rpm
and the supernatants de-proteinized using equal volumes of chloroform. After spinning at 600-
rpm, the aqueous layer was removed and pooled with the one collected earlier. Thereafter
mucopolysaccharides were precipitated by two volumes of ethanol saturated with sodium acetate.
The precipitate was collected as described earlier, dissolved in sodium acetate buffer and dialyzed
exhaustively (72 hours) against distilled water and then freeze-dried and weighed.
3.1.6 Treatment of soil with extract
In order to formulate a chemical compound with properties similar to termite saliva, it was
important to initially prove the capability of the actual termite saliva. The supernatant obtained
after treating termite heads with buffer was taken to the soil laboratory for soil examination. The
supernatant was dissolved in 100ml of distilled water, stirred for 1 minute and left to stand at
room temperature for six hours as shown in Figure 3.5. The general observation was that this
mixture was more sticky compared to the plan distilled water.
51
Figure 3.5: Collection of soil extract, abdomen extract, thorax extract in a desiccator
Soil sample passing the sieve No. 40 from the test road section was treated with this mixture by
conducting the standard consistency test to determine both plasticity index and linear shrinkage
tests as shown in Figure 3-6. In order to mimic the actual duration taken for the freshly
constructed mound soil to gain strength, the mixture was first left to stand for about 24 hours
before being tested. The procedure above was repeated for the extracted samples from the
abdomen. A control test was also conducted to facilitate result analysis.
Figure 3.6: Conduction of consistency tests for soils treated with extract from the
freshly built termite mound
52
3.2 OPTIMISATION OF THE THEORIES OF SOIL MODIFICATION
3.2.1 Treatment of soil with cellulase – Investigation of theory 1
Cellulase enzyme was procured with properties described in Table 3.2 and treated with in-situ
soils from Lubowa-Ndejje road at chainage 0+300.
Table 3.2: Properties of enzyme cellulase procured
Mass (g) 102.4
pH 5.7
Units 100,000
Colour White powder
Corrosivity Non-corrosive
Temperature for store 2.0 °C – 8.0 °C
Soils from Chainage 200 – Lubowa Ndejje road were treated with crude cellulase enzyme from the
local market and both Plasticity and strength values obtained are herewith presented in Table 3-3.
Table 3.3: Plasticity and CBR of in-situ soils treated with cellulase
Dosage (%) PI (%) CBR (%)
0.0 23.1 25.1
0.5 16.4 28.4
1.0 12.6 36.0
1.5 11.2 36.2
From the above results, it can be shown that strength increases with percentage of cellulase up to
1%, No considerable increase with 1.5%. (Substrate depleted, glucose formed). The above
results were also compared with the MoWT (2005) specifications as shown hereunder;
53
Strength Requirements (CBR):
Gravel Wearing Course: GW = 25%
Subbase Layers: G30 = 30% after 4 days soaking.
Cellulase generally leads to increased strength with maximum strength obtained at a dosage pf
1.5%.
Plasticity Requirements (PI%):
Fill layers: G15: Max. 25
Subbase layers: G45: = Max. 14
Subbase layers: G30: = Max. 16
The addition of cellulase enzyme generally leads to increased strength and reduced plasticity of
especially materials to be used as fills and gravel wearing courses.
3.2.2 Treatment of soil with oligosaccharides – investigation of theory 2
In general, oligosaccharides were prepared by treating cellulase with cellulose with the
optimization of the following classified parameters:
i. Time of reaction
ii. Quantity of cellulase utilised
iii. The amount of substrate i.e cellulose
The best oligosaccharide solution was prepared as a combination of the optimized parameters.
3.2.2.1 Optimisation of Time
In order to determine the optimum quantity of oligosaccharides, different units of enzyme were
treated with cellulose and the enzymatic reaction stopped at separate time intervals. Soil CBR was
determined in accordance with BS 1924, Part 2: 1990 by treating soil with the supernatants from
the oligosaccharide dosages (ratio of water: dosage prepared 1:100) prepared as follows.
i. 0.1g of cellulose was weighed and added to 12 glass test tubes labelled 0minutes,
30minutes, 60 minutes, 1.5hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours and 4 hours.
ii. 9.9ml of citrate buffer (PH 5.7) was pipetted and added to each of the test tubes and
mixed with the white solid for 1 minute.
54
iii. The mixtures was then incubated at 37ºC for 10 minutes in a shaking incubator.
iv. Enzyme concentration (500 units per ml of buffer) was prepared by adding 20ml of citrate
buffer to 10.24g of cellulase in a petridish. The resultant solution was relatively yellow as
shown in the Figure 3.7.
Figure 3.7: Dissolution of weighed cellulase in a petridish with citrate buffer
v. 0.1ml of the prepared solution was added to each of the incubated cellulase buffer
mixtures to make 10ml of cellulase- cellulose buffer mixture. The time of start of reaction
was noted and the test tubes were placed in a mechanical shaker at 170rpm and 37°C.
vi. The reaction per test tube was stopped by placing the glass test tube in a boiling water
bath at half hour time intervals as labelled then centrifuged at 4000 rpm for 5 minutes
from which the supernatant was decanted.
vii. The supernatants decanted were taken to the highways laboratory for CBR and Plasticity
tests after adding 10ml of supernatant to 1000ml of water in a glass cylinder and shaken
vigorously to obtain a uniform mix.
3.2.2.2 Optimisation of Enzyme Units
i. 0.1g of cellulase was added to 12 glass test tubes labelled dually as 20 units, 40 units, 60
units, 80 units, 100 units and 120 units.
ii. 9.0 ml of citrate buffer was added to each of the labelled test tubes and the mixture
shaken for 30 seconds.
55
iii. The test tubes were then incubated for 10 minutes in a shaking incubator at 37ºC and 140
rpm.
iv. 40µl, 80µl, 120µl, 160µl, 200µl and 240µl of enzyme buffer mixture as prepared earlier
were added to a mini test tubes. The test tubes were topped to 1ml with citrate buffer.
The resultant yellow solutions were added dually to the incubated testubes and the time
for the start of reaction noted. These were placed in the shaking incubator and allowed to
stand for 1hour.
v. The enzyme substrate reaction was stopped after one hour by placing the test tubes in
boiling water bath for 10 minutes and then centrifuged at 4000 rpm for 5 minutes from
which the supernatant was decanted.
vi. The supernatants decanted were taken to the highways laboratory for CBR and Plasticity
tests after adding 10ml of supernatant to 1000ml of water in a glass cylinder and shaken
vigorously to obtain a uniform mix.
3.2.2.3 Optimisation of Substrate
i. 0.1g, 0.2g, 0.3g, 0.4g and 0.5g of cellulose was weighed and added to labelled glass test
tubes in pairs.
ii. 9.0 ml of citrate buffer was added to each of the labelled test tubes and the mixture
shaken for 30 seconds.
iii. The test tubes were then incubated in a shaking incubator at 37ºC and 170rpm for 10
minutes.
iv. 90µl and 120µl of enzyme-buffer mixture as prepared in dosage 2 were each added to 5
plastic mini test tubes and topped up to 1ml by adding 910µl and 880µl respectively.
v. The resultant yellow solutions were added independently to the incubated glass test tubes
and labelled as added and the time for the start of reaction noted.
vi. The glass test tubes were then incubated for 1 hour in a shaking incubator at 37ºC and
140rpm thereafter the reaction between enzyme and substrate was stopped by placing
test tubes in a boiling water bath for 10 minutes and then centrifuged at 4000 rpm for 5
minutes from which the supernatant was decanted.
56
vii. The supernatants decanted were taken to the highways laboratory for CBR and Plasticity
tests with 10ml of supernatant added to 1000ml of water in a glass cylinder and shaken
vigorously to obtain a uniform mix.
3.2.3 Preparation of the best oligosaccharide solution and investigation of theory 3
Having optimized the different parameters, the best oligosaccharide solution was prepared by
reacting 45 units of enzyme cellulase with 0.3g of cellulose for 1 hour. 50mg, 100mg, 150mg,
200mg and 250mg of mucopolysaccharides were then added to the oligosaccharide solution prior
to testing for engineering parameters i.e CBR and Plasticity.
3.2.3.1 Stabilization of the oligosaccharide and mucopolysaccharides
The final solution containing mucopolysaccharides and oligosaccharides was stabilized by addition
of 25% glycerol as follows
i. 4.2g of cellulose was weighed and added to a glass beaker.
ii. 120ml of citrate buffer was then added to the glass beaker and the mixture shaken for 30
seconds and incubated in a shaking incubator at 37ºC and 170rpm for 10 minutes.
iii. 1260µl of enzyme-buffer mixture as prepared in dosage 2 was added to the incubated
glass beakers and the time for the start of reaction noted. The beaker was then topped up
with citrate buffer to amount to 140ml.
iv. The glass beaker was then incubated for 1 hour in a shaking incubator at 37ºC and
140rpm thereafter the reaction between enzyme and substrate was stopped by placing it
in a boiling water bath for 10 minutes. The resultant mix was centrifuged at 4000 rpm for
5 minutes from which the supernatant was decanted.
v. 70ml of citrate buffer was mixed with 70ml of glycerol and the resultant solution added to
the supernatant to amount to 25% of glycerol.
vi. 1400mg of mucopolysaccharides were then added to the glycerol oligosaccharide mixture.
vii. The solution was then taken to the highways laboratory for CBR and Plasticity tests with
the addition of 10ml to 1000ml of water in a glass cylinder in accordance to the standards.
57
CHAPTER 4: PRESENTATION OF FINDINGS AND DISCUSSIONS
4.1 RESULTS OF MOUND SOILS AND SURROUNDING SOILS
4.1.1 Elemental analysis
Chemical tests were carried out on samples collected from different part both from within and
around the anthill using the Atomic Absorption Spectroscopy (AAS) to determine mineralogical
composition of mound samples and samples from the surrounding soils.
The data collected on the soils that were stabilized were analyzed both objectively and
statistically. Statistical analysis was done using the Analysis of Variance (ANOVA) technique.
The AAS analysis indicated that the main oxides present in are Fe2O, CaO, MgO and Sodium,
Potassium, and Sulphur present in trace quantities. The XRD analysis indicated that the termite
mounds and surrounding soils consisted dominantly of clay minerals with Iron, Magnesium and
Calcium with no significant difference in clay minerals which is kaolinite in all the samples.
The following nomenclature was used to represent the different positions in the mound and
surrounding soils from which samples were picked.
1 = Top of mound
2 = Middle of mound
3 = Bottom of mound
4 = 3m offset from mound – Top
5 = 3m offset from mound – Bottom
6 = 6m offset from mound – Top
7 = 6m offset from mound – Bottom
Location = Different Regions
Lateral = Positions within and offset
mound
It can be observed that Iron was the most abundant in all the termite mound samples collected
including the surrounding soils. For clarity, plot (a) shows the variation of mineral elements
across the termite mound and surrounding soils while the plot (b) indicates the same but with no
iron.
58
(a) (b)
(a) (b)
(a) (b)
59
(a) (b)
(a) (b)
(a) (b)
60
Figure 4.1: Results from elemental analysis
Statstical Analysis of chemical results
Generally, chemical composition laterally, was not significant, location was significant for some
elements e.g. C and N as per the table below;
Table 4.1: Statstical Analysis of chemical results
Location = Different Regions; Lateral = Positions within and offset the mound
DEPENDENT VARIABLE: IRON
Source Type III Sum of Squares
df Mean Square F Sig.
Location 2681.429 4 670.357 7.544 0.000
Lateral 151.143 6 25.190 0.283 0.939
Error 2132.571 24 88.857
Corrected Total 4965.143 34
a. R Squared = 0.570 (Adjusted R Squared = 0.392)
(a) (b)
61
4.1.2 pH as measured from the pH meter
i. ii.
iii. iii.
v. vi.
62
vii.
Figure 4.2: Analysis of pH of mound soils
The results of pH as shown in Figure 4-2 indicate that pH does not differ between the different
sample collection points across the regions.
4.1.3 Gradation of Soil Samples
Gradation is used to determine distribution of the different grain sizes in both mound soil and
surrounding soils. Dry sieve analysis was done on the samples that had been initially air dried to
ensure that there was no agglomeration of soil particles. The results of sieve analysis are
summarized in Table C1 in ANNEX C of this report. The results are plotted on the classical semi
logarithmic graph as seen in Figure 4.3.
63
Figure 4.3: Gradation curves of mound soils
According to USCS, soil positions 1, 2, 3 were classified as CL i.e lean clay for anthills. Mound soils
possess higher percentage of fines than surrounding soils. Within the mound, the top of the
anthill exhibited the highest percentage of fines. Soil becomes coarser textured with distance from
the termite mounds. The significantly higher content of fines in termite mound soils compared
with surrounding soils confirmed findings by Lepage et al., (1989) that termites select fine soils
for mound construction.
4.1.4 Maximum Dry Density
Generally, termite mound soils required less compactive effort as compared to the surrounding
soils as illustrated in Figure 4-4 and 4-5.
64
Figure 4.4: Compaction test curves of mound soils
65
Figure 4.5: Maximum Dry Densities of mound soils
4.1.5 Atterberg’s Limits
Liquid Limit
Figure 4-6 shows the various liquid limit values in percentage for mound soils and surrounding
soils obtained using the cone penetrometer method. It can be observed that the liquid limit values
for mound soil is generally greater than that of surrounding soil for most of the sample areas. The
increased liquid limit values may be attributed to presence of organic matter, increase in the clay
content and presence of finer materials that are a result of the disintegration processes of the
termites as they build their mounds. The termite mound soils also have reduced water content
compared to surrounding soils partly due to the impermeability of mound soils compared to
surrounding soils.
66
Figure 4.6: Liquid Limits of sampled material from mounds
Plastic Limit
The plastic limit test was conducted according to ASTM (D424 –59). Comparing the plastic limit
values in Figure 4-7, as the clay content of the mound soil is more than the surrounding surface
soil, its plastic limit value is also higher.
Figure 4.7: Plastic Limits of sampled material from mound soils
Plastic Index
Considering the plasticity index values in Figure 4-8, the mound soils are more stable since the
value is more for mound soil than the control soil. Higher values of plasticity index and low
moisture content indicate that there is an increase in cohesion which makes the mound stable.
67
Figure 4.8: Plasticity Indices of sampled material from mound soils
4.2 RESULTS FROM THE TEST ROAD SECTION
4.2.1 Gradation
Figure 4.9: Gradation curves of in-situ material
4.2.2 Atterberg’s Limits
Table 4.2: Plasticity of in-situ material
CHAINAGE 0+00 0+300 0+900
LL (%) 54.2 44.5 53.3
PL (%) 26.4 21.4 24.4
PI (%) 27.8 23.1 28.9
Plasticity of in-situ material as described in Table 4-2 is relatively high. This may be attributed to
the high percentage of fines as illustrated in the gradation curves from Figure 4-9.
68
4.2.3 Strength
Table 4.3: Strength of in-situ material
CHAINAGE 0+00 0+300 0+900
CBR (%) 8 21 8
MDD (Kg/m3) 1392 1806 1539
OMC (%) 14.2 8.7 15
4.3 ANALYSIS OF EXTRACTS
4.3.1 Cellulase activity in the anthill soil, surrounding soil, termite head and abdomen
extracts
Cellulase activity in live worker heads and abdomen was determined from the Biochemistry
laboratory in Makerere University. Details of the results are shown below;
i. Cellulase activity in the head: = 0.04 µmoles of glucose per minute per mg protein
ii. Cellulase activity in the abdomen= 0.0648 µmoles of glucose per minute per mg protein.
iii. Cellulase activity in the control soil sample: = 0.061 mg of glucose per minute per g of soil
iv. Cellulase activity in the anthill soil sample = 0.08 mg of glucose per minute per g of anthill
soil
The results confirmed the presence of enzyme cellulase in the termite saliva. The specific cellulase
activity in termite abdomens was higher than that in worker heads by 62%.
Cellulase activity in the control soil sample was found to be 0.061 mg of glucose per minute per
gram of soil. In comparison to the activity in the mound soil, activity in control soil was 25%
lower. We therefore drew conclusion that termites increase enzyme cellulase concentration in
mound soil and hence the enzyme plays a role in the nest structural stability.
4.3.2 Isolation of mucopolysaccharides
Mucopolysaccharides were isolated from the head and abdomen of live termites and from mound
soil. The highest concentration of the gluing agent was found in the termite heads as shown in
Table 4.4. The presence of mucopolysaccharides in the mound soil confirmed that they play a role
in the structural strength of the termitarium.
69
Table 4.4: Yield and concentration of mucopolysaccharides
Sample Yield (g) Concentration (mg % w/w) Concentration (ppm)
Abdomen 0.026 0.325 3250
Head 0.115 0.8214 8214
Soil 0.017 0.00245 425
4.3.3 Treatment of in-situ soil with extract
Atterberg limits namely Liquid Limit, Plastic Limit, Plasticity Index and Linear Shrinkage values
were obtained experimentally with treatment of soil from Lubowa Ndejje road at chainage 0+900
with the extract of the termite heads, abdomen and freshly constructed mound soil and presented
in Table 4.5.
Table 4.5: Plasticity values for soil treated with extract from termite heads and
abdomen
Soil Sample LL, % PL, % PI, % LS %
Neat Soil 53.3 24.4 28.9 13.5
Soil + Extract from Wet soil 57.9 32.1 25.8 12.1
Soil + Extract from Abdomen 53.6 32.8 20.8 10.1
Soil + Extract from Head 54.7 34.9 19.8 9.3
It can be seen that treatment of soil with extract led to a reduction of both PI and LS which is an
indication of improved performance. PI reduced by 12% with extract from fresh soil and by 39%
with extract from the abdomen and finally by 46% with extract from the head. The MoWT (2005)
specification states that a material to be used for sub-base layers of G30 materials should have a
Maximum linear shrinkage value of 8%. From the above results, it can be shown that head
extracts provide linear shrinkage values close to the maximum 8%.
70
4.4 FORMULATION AND PREPARATION OF THE OLIGOSACCHARIDE
In accordance with theory 1 of soil modification, oligosaccharides were generally prepared by
reacting cellulase with cellulose and optimising time, cellulose and cellulase for the production of
the most suitable solution for strength and plasticity improvement.
4.4.1 Optimisation of time
Dosage 1 (200 units of enzyme cellulase per ml of buffer)
The enzyme-substrate reaction was stopped at different time intervals. The solutions were treated
with in-situ soil to formulate the CBR readings at 4 days curing in Table 4.6.
Table 4.6: Values of CBR of in-situ soil treated with oligosaccharides (dosage 1)
Time (mins) Neat / Control 0 30 45 60 75 90 105 120
CBR % 5 6 8 8 11 10 10 10 8
Figure 4.10: Values of in-situ soil CBR treated with dosage 1
CBR rose by 43% with enzyme substrate reaction stopped at 60 minutes as shown in Figure 4-10.
It then reduced with the increased time frame of reaction. From the above table, we drew
conclusion that the best time for maximum production of oligosaccharides was 60minutes.
71
Dosage 2 (500 units of enzyme cellulase per ml of buffer)
The enzyme-substrate reaction was stopped at different time intervals. The solutions prepared
were then treated with in-situ soil to formulate the CBR readings in Figure 4-11 and Table 4-7.
Table 4.7: Values of CBR with soil treated with oligosaccharides (Dosage 2)
Time
(Hours) 0 0.5 1 1.5 2 2.5 3 3.5 4
CBR % 8 23 38 33 33 24 20 24 29
Figure 4.11: CBR values of in-situ soil treated with dosage 2 oligosaccharides
Unlike dosage 1, there was substantial increment in the value of CBR of soil treated with
oligosaccharide solutions prepared as dosage 2. This may be attributed to the increase in enzyme
concentration and hence more oligosaccharides for a given quantity of substrate. Like in Dosage
1, the optimum time to produce the maximum concentration of oligosaccharides was 1hour as
shown in Figure 4-1 and Figure 4-10. CBR value increased from 8% to 38% at 1hour of enzyme-
substrate reaction. CBR value then reduced gradually for the subsequent time intervals to 20% at
3hours of enzyme substrate reaction. The ratio of oligosaccharides to reducing sugars increased
up to 1 hour of enzyme substrate reaction as seen by the increase in CBR. With time, the ratio of
72
oligosaccharides to reducing sugars reduced and this led to a decrease in CBR value as illustrated
in Figure 4-11.
Figure 4.12: Plasticity of in-situ soil treated with dosage 2 oligosaccharides
The plasticity Index of in-situ reduced when treated with oligosaccharide solutions prepared at
different time intervals implying increased strength of soil. The highest reduction of plasticity was
observed at 1hour of enzyme- substrate reaction stoppage. We drew conclusion that the best
time for highest yield of oligosaccharides from cellulase-cellulose reaction was 1 hour. (See Figure
4-12)
4.4.2 Optimisation of enzyme units
Oligosaccharide solutions were prepared by reacting different units of enzyme cellulase with
cellulose and the reaction stopped at one hour. The solutions prepared were then treated with in-
situ soil (ratio of water: solution- 100:1) to formulate the dry CBR and plasticity values at 16 days
curing.
Table 4.8: Values of CBR of in-situ soil treated with oligosaccharides prepared with
different enzyme units
Units 0 20 40 60 80 100 120
CBR value 43 69 84 77 49 65 77
73
Figure 4.13: CBR values of in-situ soil treated with oligosaccharides prepared with
different enzyme units
There was substantial increment in the value of CBR of soil treated with oligosaccharide solutions.
This may be attributed to the increase in enzyme concentration and hence more oligosaccharides
for a given quantity of substrate. CBR value increased from 42% to 82% at 45 units of enzyme
per ml of buffer. CBR value then reduced gradually to 45% at 80 units of enzyme cellulase. From
the curve, the highest CBR was obtained at 45 units of enzyme cellulase. The ratio of
oligosaccharides to reducing sugars increased up to 45 units as seen by the increase in CBR. With
time, the ratio of oligosaccharides to reducing sugars reduced and this led to a decrease in CBR
value as illustrated in Figure 4-13. The gradual increase of the curve thereafter may have been
due to the introduction of enzyme activity of the second enzyme component of enzyme cellulase.
Figure 4.14: Plasticity of in-situ soil treated with oligosaccharides prepared with
different enzyme units
74
The plasticity Index of in-situ reduced when treated with oligosaccharide solutions prepared with
different enzyme units implying increased strength of soil (See Figure 4-14). The highest
reduction of plasticity was observed at oligosaccharide solution prepared with 60 units of enzyme
cellulase (15.7%) Although the highest concentration of oligosaccharides for maximum strength is
a solution prepared with 45 units of enzyme cellulase, 60 units were ideally chosen as the
optimum enzyme concentration for the best yield of oligosaccharides.
4.4.1 Optimisation of substrate
Substrate was optimised by treating 45 units and 60 units of enzyme cellulase with cellulose. The
CBR values gradually increased with variation of substrate for both 45 units and 60 units. The
highest CBR (63%) was obtained at 0.3 g of substrate corresponding to 45 units of enzyme. We
drew conclusion that the maximum production of oligosaccharides for maximum increase in soil
strength is prepared by treating 45 units of enzyme cellulase with 0.3g of cellulose for 1 hour. The
CBR curve of 60 units was slightly lower than that of 45 units with the maximum CBR (61%) at
0.35g of cellulose.
Figure 4.15: CBR values of in-situ soil treated with oligosaccharides prepared with
different substrate concentrations
It can be concluded that the higher the number of units, the greater the amount of substrate for
maximum production of oligosaccharides.
75
Figure 4.16: Plasticity of in-situ soils treated with oligosaccharides prepared with
different substrate concentrations
The plasticity index of soil treated with oligosaccharides prepared with 45 units of enzyme
cellulase reduced with increased variation of substrate while that of soil treated with
oligosaccharide solution prepared with 60 units of enzyme cellulase increased. The lowest
reduction in plasticity was obtained with 0.3g of cellulose. Plasticity values correspond with CBR
values. Therefore, we drew conclusion that in order to produce the best concentration of
oligosaccharides for the best strength and plasticity characteristics of in-situ lateritic soils, 45 units
of enzyme cellulase are treated with 0.3g of cellulose for 1 hour from which solution is treated
with water in a ratio of 1:100.
The oligosaccharides produced from treatment of 45 units of cellulase with cellulose interlink more
with addition of cellulose. The concentrations of oligosaccharides increase and hence less
plasticity results. On the other hand, increase of substrate concentration with 60 units of cellulase
results in higher plasticity. The greater the ratio of enzyme: cellulose, the greater the activity of
the enzyme and hence the faster release of the second component of cellulose. This results in the
formation of weaker interlinks between oligosaccharides.
76
4.5 VARIATION OF THE MUCOPOLYSACCHARIDES
The best supernatant of oligosaccharides was supplemented with mucopolysaccharides in
different quantities in reference to theory 3 of soil modification by termites. 10ml of the resultant
mixture was dissolved in 1000ml of water and treated with in-situ soil samples to determine dry
CBR and plasticity readings at 4 days curing. Results are shown in Figure 4-17 below.
Figure 4.17: CBR and Plasticity of in-situ soils treated with different concentrations of
mucopolysaccharides
The mucopolysaccharides led to an increase in soil CBR treated with oligosaccharides from 63% to
75% with the addition of 100mg of mucopolysaccharides. The CBR value then reduced with
further addition of mucopolysaccharides. This was due to the formation of complex bonds
between mucopolysaccharides and oligosaccharides from cellulose digestion due to their
interaction. The mucopolysaccharides reduce plasticity of the soil treated with oligosaccharides.
77
Table 4. 9: CBR and Plasticty of Oligos +Mucopolysaccharides vs Oligos
OLIGOS + MUCOS OLIGOS
CBR 63 (100mg) 75
PLASTICITY INDEX 19.6 (250mg), 22.2 (100mg) 21.3
In conclusion, 100mg of mucopolysaccharides were chosen to be the ideal quantity for the best
effect of strength improvement. However, regarding plasticity, we recommend that the termite
saliva be modified with lime.
4.6 COMPOSITION OF MODEL COMPOUND AND TREATMENT OF IN-SITU SOILS
As mentioned above, the constitution of model compound was made by addition of 100mg of
mucopolysacharides to oligosaccharides produced from 45 units of enzyme cellulase and treated
with 0.3g of cellulose for 1 hour and then treating the solution with water in a ratio of 1:100. Two
compounds were produced. Compound one was produced by mere addition of
mucopolysacharides to the solution of oligosaccharides and the mixture was left to stand
overnight before treatment with the soils. Compound two was produced and immediately a
stabilizer was added to inhibit any possible reaction between the oligosaccharides and
mucopolysacharides. The two samples were used to treat in-situ soils from Lubowa road and both
plasticity and strength values obtained are as indicated in the Table 4.10 below.
Table 4. 10: CBR of soil treated with stabilised and unstabilised TermaBond
CBR %
SAMPLE 1 SAMPLE 2 AVERAGE
STABILISED COMPOUND 55 44 50
UNSTABILISED COMPOUND 17 16 17
Lower CBR values were exhibited by un-stabilized compound compared to the stabilized
compound possibly because of the formation of complexes between oligosaccharides and
mucopolysacharides.
4.7 TRADE NAME OF THE MODEL COMPOUND
From the above results, it is clearly evidenced that the role of the model compound is to bond the
loose soil particles together to form a hard / strong medium. This is achieved through interlink-
78
aging of short chain polymers, namely, oligosaccharides, upon addition of more substrates. The
formation of such linkages reduces the surface area of the soil particles resulting into reduction in
plasticity index. Addition of the mucopolysaccharides on the other hand leads to an increase in the
soil strength in terms of CBR due to the formation of more complex bonds between the
mucopolysaccharides and oligosaccharides from cellulose digestion due to their interaction. The
mucopolysaccharides reduce plasticity of the soil treated with oligosaccharides. The whole above
process is majorly a bonding mechanism of soil similar to the way termites use their saliva
secreted by salivary glands to bond soil. The model compound therefore derives its trade name
“TERMABOND” from the above process.
4.8 COST IMPLICATIONS
It was not possible to conduct a detailed life cycle cost analysis of this technology since the
optimum dosage of TERMABOND for different soils has not been determined. However, the costs
below have been derived from the conventional methods of road pavement layer construction in
order to establish the costs incurred in stabilization and treatment of 1km of in – situ soils. A
standard road width of 7m with a thickness of 150mm resulting total volume of 1050m3, as the
bulk of in-situ soils to be treated with a field optimum moisture content of 14%.
Assumptions
i) In the 1:100 solution of TERMABOND, the density of the concentrate is approximately
equal to the density of water.
ii) The treatment of in situ soils shall be done at the optimum moisture content in the field.
The bulk mass of soil to be treated is thus = kgx 000,100,220001050
The dry weight of soil to be soil = kgM c
b 105,842,114.1
000,100,2
1
The concentrate is a combination of 100mg of mucopolysacharides, 45 units of cellulase enzyme
treated with 0.3g of cellulose substrate. The individual costs for each of the ingredients are as
shown below.
79
The above results into 600ml mixture which is used to stabilize 5kg of soil. The mass of soil to be
treated in a 1km stretch is 1,842,105kg.
Therefore the amount of mixture required = mls421,3685
105,842,1
Therefore the total cost for the stabilizer production = 895,82$600
135421,368 x
Table 4. 11: Direct costs in production of TERMABOND
DIRECT COSTS IN PRODUCTION OF TERMABOND Approximate Costs
Cost of Mucopolysacharides: 100
5000
650$x
$13 $13
Cost of Cellulase Enzyme (I tin = 100,000units): 1500$
000,100
45x
$0.7 $1.0
Cost of cellulose required for this mixture1 3.0
1000
50$x
$0.02 $0.1
Sub – Total 1.0 $13.7 $14.1
Add Shipping and Transportation costs $100 $100
Sub – Total 1.1 $113.7 $114
Add 18% VAT $20.5 $21
Sub – Total 1.2 $134.2 $135
1Cost of Cellulose was estimated at $50 per kg
Other Expenditures
With the above cost, it is possible to achieve a firm base as a result of treatment of in-situ soils
with TERMABOND. However, having a firm base which is not protected from weather will result
into slipperiness in addition to exposing the surface to progressive failures. It is therefore
80
proposed to provide a sealant to prevent entry of water into the pavement. However, to increase
skid resistance of traffic (both motorized and non – motorized), we propose to spray a layer of
aggregates on top. This will result into a paved road with a surface dressed like pavement with a
firm base. The surface is however non – structural providing protection against water ingression,
increasing the skid resistance and enhancing the riding conform of users. The table below
highlights the major expenses incurred in attaining the above.
Table 4. 12: Other associated costs
Item Description Unit Qty Rate Amount
Hire of Equipment
1 Grader Days 4 1,500,000 6,000,000
2 Roller Compactor Days 4 1,000,000 4,000,000
3 Water Bowzer Days 4 500,000 2,000,000
4 Spray Pump / Bitumen Spray Days 2 1,500,000 3,000,000
5 Chip Spreader Days 2 1,000,000 2,000,000
Materials
-
1 Aggregates Tons 250 85,000 21,250,000
2 Fuel Liters 5,000 3,400 17,000,000
3 Pen Grade Bitumen 80/100 200L Drums 18 1,500,000 27,000,000
Other Costs
-
Allowance for Drainage Works LS 1 20,000,000 20,000,000
Allowance for Labor Expenses LS 1 20,000,000 20,000,000
-
Sub – Total 1.0
122,250,000
Add Contingency 15%
18,337,500
Sub – Total 1.1
140,587,500
SUMMARY COSTS FOR STABILISATION AND SEALING OF 1KM
ITEM / DESCRIPTION AMOUNT
Production of TERMABOND stabilizer 82,895 2,600 215,527,000
Equipment and other Costs
140,587,500
GRAND TOTAL
356,114,500
Therefore, to have a 1km stretch of road fully constructed, complete with drainage and sealed, a
total of UGX 356,114,500 /=(US$14,838) is required. However it can be noted that most of the
costs are directly related to importation. Therefore, it is important that the manufacture of the
above ingredients is done locally.
81
Table 4.13 below gives a comparison of TERMABOND stabilized pavement road section and the
conventional road construction methods used.
Initial construction costs per kilometer for different types of road pavements
Table 4. 13: comparison of TERMABOND stabilized pavement road section and the conventional road construction methods used.
TYPE OF INTERVENTION AMOUNT IN US$ AMOUNT (UGSHS)
TERMABOND Road 1136,967 356,114,500
Gravel Road to undergo periodic Maintenance every 3 years 15,385 40,000,000
Single Surface Dressing 161,539 420,000,000
Double Surface Dressing 223,077 580,000,000
Asphalt Concrete Surfacing 650,000 1,690,000,000
It can be seen that the TERMABOND road system is a cheaper option compared to the single,
double and asphalt concrete surfacing systems. It is more expensive than the gravel system
however the life cycle cost analysis shall render the latter option more expensive. At a large scale,
a total saving of UGX 75,000,000/= is made on a TERMABOND system compared to the single
surface dressing.
82
CHAPTER 5: CONCLUSIONS
From the above studies, the following conclusions can be made:
iii) Results of the Atomic Absorption Spectrometer (AAS) indicate that iron is the most
dominant mineral element available in both the mound and surrounding soils. Carbon and
Nitrogen were also found to be present in larger quantities in mound soils as compared to
surrounding soils. Termites release fecal material full of C and N which they use in
construction of nests. The higher C and N content in surrounding soils could be attributed
to decomposing organic matter. Statistical analysis revealed that termites are not selective
in the soil materials they choose to work with in regard to its chemical composition.
iv) All soils were finer than the acceptable specification range for gravel wearing course and
subgrade layers. Probably this is the reasons as to why they are not desired in
construction.
v) Extracts from the surrounding soils, termite heads and abdomen were analyzed for
presence and activity of cellulase enzyme and mucopolysacharides. Termites increase
enzyme cellulase concentration in mound soil and hence the enzyme plays a role in the nest
structural stability.
Cellulase activity in the head was found to be 0.04 µmoles of glucose per minute per mg
protein; Cellulase activity in the abdomen was found to be 0.0648 µmoles of glucose per
minute per mg protein; Cellulase activity in the control soil sample was found to be 0.061
mg of glucose per minute per g of soil and Cellulase activity in the anthill soil sample was
found to be 0.08 mg of glucose per minute per g of anthill soil. The results confirmed the
presence of enzyme cellulase in the termite saliva. The specific cellulase activity in termite
abdomens was higher than that in worker heads by 62%. Cellulase activity in the control
soil sample was found to be 0.061 mg of glucose per minute per gram of soil. In
comparison to the activity in the mound soil, activity in control soil was 25% lower. E-11
vi) Mucopolysaccharides, the predicted gluing agents were isolated from the head and
abdomen of live termites and from mound soil. The highest concentration of the gluing
agent was found in the termite heads at a concentration of 8214 ppm, followed by
abdomen at 3250ppm and from the wet mound soil at 425ppm. The presence of
mucopolysacharides in the mound soil confirmed that they play a role in the structural
strength of the termitarium.
83
vii) Consistency tests were carried out on insitu soils from Lubowa road treated with the above
mucopolysacharides. The treatment of soil with extract led to a reduction of both PI and LS
which is an indication of improved performance. PI reduced by 12% with extract from fresh
soil and by 39% with extract from the abdomen and finally by 46% with extract from the
head. The MoWT (2005) specification states that a material to be used for sub-base layers
of G30 materials should have a Maximum linear shrinkage value of 8%. From the above
results, it can be shown that head extracts provide linear shrinkage values close to the
maximum 8%.
viii) From the tests on predicted soil modification theories, the action of oligosaccharides was
investigated by reacting cellulase with cellulose and optimising time, cellulose and cellulase
for the production of the most suitable solution for strength and plasticity improvement.
The optimisation of time was carried out by reacting 200 units of enzyme cellulase per ml of
buffer. The CBR value increased by 43% with enzyme substrate reaction stopped at 60
minutes. Therefore the best time for maximum production of oligosaccharides was
60minutes.
ix) Increase of enzyme dosage to 500 units of enzyme cellulase per ml of buffer resulted into a
substantial increment in the value of CBR of soil treated with oligosaccharide solutions
prepared as dosage 2. The optimum time to produce the maximum concentration of
oligosaccharides was again recorded as 1hour. The CBR value increased from 8% to 38%
at 1hour of enzyme- substrate reaction. The plasticity Index of in-situ reduced when treated
with oligosaccharide solutions prepared at different time intervals implying increased
strength of soil. The highest reduction of plasticity was observed at 1hour of enzyme-
substrate reaction stoppage. Therefore the best time for highest yield of oligosaccharides
from cellulase-cellulose reaction was 1 hour.
x) Although the highest concentration of oligosaccharides for maximum strength is a solution
prepared with 45 units of enzyme cellulase, 60 units were ideally chosen as the optimum
enzyme concentration for the best yield of oligosaccharides.
xi) The optimization of the substrate was conducted by treating 45 units and 60 units of
enzyme cellulase with cellulose. The CBR values gradually increased with variation of
substrate for both 45 units and 60 units. The highest CBR (63%) was obtained at 0.3 g of
substrate corresponding to 45 units of enzyme. Therefore the maximum production of
84
oligosaccharides for maximum increase in soil strength is prepared by treating 45 units of
enzyme cellulase with 0.3g of cellulose for 1 hour. The CBR curve of 60 units was slightly
lower than that of 45 units with the maximum CBR (61%) at 0.35g of cellulose.
xii) The best supernatant of oligosaccharides was supplemented with mucopolysacharides in
different quantities in reference to theory 3 of soil modification by termites. 10ml of the
resultant mixture was dissolved in 1000ml of water and treated with in-situ soil samples to
determine dry CBR and plasticity readings at 4 days curing. The mucopolysacharides led to
an increase in soil CBR treated with oligosaccharides from 63% to 75% with the addition of
100mg of mucopolysacharides. The mucopolysacharides reduce plasticity of the soil treated
with oligosaccharides. In conclusion, 100mg of mucopolysacharides were chosen to be the
ideal quantity for the best effect of strength improvement.
xiii) The constitution of model compound was accomplished by addition of 100mg of
mucopolysacharides to oligosaccharides produced from 45 units of enzyme cellulase treated
with 0.3g of cellulose for 1 hour from and then treating the solution with water in a ratio of
1:100. Two compounds were produced. Compound one was produced by mere addition of
mucopolysacharides to the solution of oligosaccharides and the mixture was left to stand
overnight before treatment with the soils. Compound two was produced and immediately a
stabilizer was added to inhibit any possible reaction between the oligosaccharides and
mucopolysacharides. The two samples were used to treat in-situ soils from Lubowa road
and both plasticity and strength values were recorded. Lower CBR values (50%) were
exhibited by un-stabilized compound compared to the stabilized compounds (50%) possibly
because of the formation of complexes between oligosaccharides and mucopolysacharides.
xiv) The TERMABOND road construction system is a cheaper option compared to the single,
double and asphalt concrete surfacing systems, with figures standing at 356,114,500 for a
TERMABOND system, UGX 420,000,000/=, 580,000,000/= and 1,690,000,000/= for single
surface dressing, double surface dressing and asphalt concrete surfacing options. It is more
expensive than the gravel system however the life cycle cost analysis shall render the latter
option more expensive. At a large scale, a total saving of UGX 64,000,000/= is made on a
TERMABOND system compared to the single surface dressing. Interventions to waive taxes
and reduction in the importation and shipping costs shall further lower the construction costs
of the TERMABOND road system.
85
CHAPTER 6: RECOMMENDATIONS
From the above study, the following recommendations can be drawn:
i) It is possible to achieve higher strength values using this compound should the
concentration of oligosaccharides be increased. The stabilization to some degree dilutes
the concentration of the oligosaccharides and affects the complex formation mechanism. A
higher concentration of oligosaccharides would therefore check the above.
ii) The model compound has been tested on only one type of in-situ soil. It is therefore
important to look at the performance of soils from other regions. This should involves
determining the optimum / best stabilizer to give best performance, from which a detailed
cost analysis can be made. There is also need from the bigger study to determine the best
type of mucopolysacharides that can result into improved performance. The type of
mucopolysacharides used in this study only aided to prove the concept of gluing
mechanism of most of the mucopolysacharides.
iii) It is major recommendation from this study that the results from this study be verified by
field applications by putting up test sections in some of the areas with problematic soils
around Uganda. Comparisons from the above studies can be used to correlate the above
findings. In addition, a firm base should be protected from the adverse effects of weather
by installation of a sealant to stop ingression of water into the deeper layers that would
reduce its structural integrity.
iv) It is suggested further to this study, that the above test sections should be constructed
using the TERMABOND road system and results compared with the conventional /
traditional road construction systems. The road sections should be setup in the same areas
where the different roads should experience same traffic and environmental conditions.
Thereafter, monitoring of the above sections should commence with and last for at least 1
year. The shelf life of the stabilizer, TERMABOND also needs to thoroughly be investigated
to determine the time this should be expire and strategies to preserve it longer
improvised.
86
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