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Recent Experimental Studies in Soil

Stabilization with Bio-EnzymesA
Dr Mohd Raihan Taha
Professor and Head
Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia
Tanveer A. Khan
Ph.D. Student
Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia
Ibtehaj Taha Jawad
Ph.D. Student
Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia
Ali Akbar Firoozi
Ph.D. Student
Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia
Ali Asghar Firoozi
Ph.D. Student
Department of Civil & Structural Engineering, Universiti Kebangsaan Malaysia

Roads are vital for the economic growth of a country. Keeping balance between performance and cost
of the roads and at the same time fulfilling the environmental regulations is becoming a challenge for
public and private sector. Traditionally used soil stabilizers (lime, cement and bitumen) are becoming
costly. Gravel and sand, which are introduced to alter the soil properties are depleting and becoming
increasingly expansive. This scenario has led to an urgent need to identify and introduce new
materials to improve the roads performance and to keep the cost at an affordable level. In this respect
different types of stabilizers ranging from inorganic to organic have now been tried in laboratory and
in field to evaluate their suitability as soil stabilizer. Recently bio-enzymes have emerged as a new
chemical for soil stabilization. They are organic materials which are supplied as concentrated liquid.
It is claimed by bio-enzymes manufacturers that their products are effective, environmental friendly
(non-toxic), cost effective and convenient to use. They improve the compressive strength, reduce
compaction effort and increase the density thus reducing the permeability as well. But the studies so
far suggest that these claims must be verified through independent laboratory testing before applying
these bio-enzymes in the field.
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KEYWORDS: Enzyme, soil, stabilization, roads
Microbial geo-technology is an emerging branch of geotechnical engineering that deals with the
application of microbiological methods to improve the mechanical properties of soil to make it more
fitting or appropriate for construction and environmental purposes. In these regard two noteworthy
applications, bio-clogging and bio-cementation have been explored. Bio-clogging is the production
of pore-filling materials through microbial means so that the porosity and hydraulic conductivity of
soil can be reduced where as bio-cementation is the generation of particle binding materials through
microbial processes in situ so that the shear strength of soil can be increased [Ivanov & Chu 2008].
One promising application is the formation of the soil plugs by bacillus pasteurii in the medium
containing urea and calcium chloride. Bacteria produce enzyme urease that hydrolyzes urea by the
following reaction:
C0 +SB
0 2NB
+ BC0

Due to this enzymatic reaction, pH is increased and hydrocarbonate is produced. It is initiating
precipitation of calcium carbonate, which clogging the pores and binding soil particles [Kucharski et
al. 2005].
Enzymes are biological catalyst and are present in all living organisms. They are obtained from
plants and animals including microorganisms by extraction using suitable solvent (Prescott 2001).
Enzymes are large protein molecules which are more efficient than inorganic catalyst; the reaction
rate is often increased by a factor of 106 to 1012. They usually catalyze one particular reaction
therefore enzymes do not produce side reaction. They are temperature sensitive and work at mild
temperature (35
C), and loss their effectiveness at higher temperature. Also they are pH sensitive
too and work good around pH value 7 [Norris et al. 2011].
The specific substance (metabolite) that fits on the enzyme surface and is converted to
products(s) is called substrate. For example the enzyme urease catalyzes the reaction:
C0 + B
0 2NB
+ C0

Urea is the substrate in this reaction as it fits onto the surface of enzyme. Water reacts with the
urea only when urea is bond to the enzyme surface. The catalyst activity of enzyme is measured in
enzyme activity which is the number of moles of substrate converted to product per minute. The
complicated folding of the polypeptide chain of enzyme gives pocket on the surface of the enzyme
known as active site. The substrate binds itself on the active site where catalysis takes place. The
active site has specific amino acid side-chains which form weak bonds with substrate. The substrate
in this manner is kept in place so that other molecules can react with it in the right orientation.
[Norris et al. 2011].
Most clay has a molecular structure with a net negative charge. To maintain the electrical
neutrality, cations (positively charged) are attracted to and held on the edges and surfaces of clay
particles. These cations are called exchangeable cations because in most cases cations of one type
may be exchange with cations of another type. When the cation charge in the clay structure is weak,
the remaining negative charge attracts polarized water molecules, filling the spaces of the clays
structure with ionized water. As a consequence a movement of moisture from areas of low cation
concentration to areas of high cation concentration is produced to achieve the equilibrium of the
cation concentration. Cations are unable to disperse freely in the soil structure because of the
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attractions of the negatively charged surface of the clay particles. This creates an osmotic pressure
gradient, which tries to equalize the cation concentration. As a consequence a movement of moisture
from areas of low cation concentration to areas of high cation concentration is produced to achieve
the equilibrium of the cation concentration [Scholen 1992].
The flow of cations through the clay deposits gives the shrinking and swelling properties of the
soils; when a stabilizer solution is added in to the soil, the magnitude of the effect depends on the
characteristics of the particular cation. In general there are two main characteristics, the valence of
the cation or number of positive charges and the size of the cation [Rauch et al. 2002].
The size determines the mobility of the cation; smaller ones will travel a greater distance
throughout the soil structure (the hydrogen ion is the smallest one). With respect to the valence, the
hydrogen ion is doubly effective affecting the clay structure because even though it has only a single
charge, the hydrogen ion produces an effect of valence of two due to its high ionization energy.
These hydrogen cations exert a stronger pull on the clay layers pulling the structure of the soil
together and removing the trapped moisture permitted by the single sodium and potassium cations.
The loss of moisture results in a strengthening of the molecular structure of the clay [Scholen 1992].
Enzymes speed up a chemical reaction, that otherwise would happen at a slower rate, without
becoming a part of the end product. The enzyme combines with the large organic molecules to form
a reactant intermediary, which exchange ions with the clay structure, breaking down the lattice and
causing the cover-up effect, which prevents further absorption of water and the loss of density. The
enzyme is regenerated by the reaction and goes to react again. The enzymes are absorbed by the clay
lattice, and then released upon exchange with metals cations. They have an important effect on the
clay lattice, initially causing them to expand and then to tighten. The enzymes can be absorbed also
by colloids enabling them to be transported through the soil electrolyte media. The enzymes also
help the soil bacteria to release hydrogen ions, resulting in pH gradients at the surfaces of the clay
particles, which assist in breaking up the structure of the clay [Scholen 1992].
The idea of using enzyme stabilization for roads was developed from enzyme products used for
treatment of soil to improve horticultural applications. A modification to the process produced a
material, which was suitable for stabilization of poor ground for road traffic. When is added to a soil,
the enzymes increase the wetting and bonding capacity of the soil particles. The enzyme allows soil
materials to become more easily wet and more densely compacted. Also, it improves the chemical
bonding that helps to fuse the soil particles together, creating a more permanent structure that is
more resistant to weathering, wear and water penetration (Marasteanu et al. 2005).
The basic effect of the action of the enzyme into the structure of the soil can be summarized as,
initially, the film of absorbed water is greatly reduced and in fact entirely broken, as shown
schematically in Figures 1 and 2.The film of water enveloping the particles, which ultimately
governs the expansion and shrinkage of colloidal soil constituents, cannot be completely eliminated
by purely mechanical methods. However, by means of temperature effects, addition or removal of
water with mechanical pressure, it is possible to vary the amount of water held in this manner. Such
variations are attended by swelling or shrinkage. This provides an ideal point of operation for the
enzyme (The Carbon Group).
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Figure 1: Absorbed water in the structure of soil (The Carbon Group )

Figure 2: Elimination of absorbed water in the soil (The Carbon Group )
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Lowering the dipole moment of the water molecule by the enzyme results in dissociation in a
hydroxyl (-) and a hydrogen (+) ion. The hydroxyl ion in turn dissociates into oxygen and hydrogen,
while the hydrogen atom of the hydroxyl is transformed into a hydronium ion. The latter can accept
or reject positive or negative charges, according to circumstances. Normally the finest colloidal
particles of soil are negatively charged. The enveloping film of absorbed water contains a sufficient
number of positive charged metal ions - such as sodium, potassium, aluminum and magnesium,
which ensure charge equalization with respect to the electrically negative soil ion. The positive
charges of the hydronium ion or of the negatively charged hydroxyl ion will normally combine with
the positively charged metal ions in the water adhering to the surface of the particles. Because of the
effect of the enzyme formulation in reducing the electric charge of the water molecule, there is
sufficient negative charge to exert adequate pressure on the positively charged metal ions in the
absorbed water film. As a result of this, the existing electrostatic potential barrier is broken. When
this reaction occurs, the metal ions migrate into the free water, which can be washed out or removed
by evaporation. Thus the film of absorbed water enveloping the particles is reduced. The particles
thereby lose their swelling capacity and the soil as a whole acquires a friable structure. The hydrogen
ions, which are liberated in the dissociation of the water molecules, can once again react with free
hydroxyl ions and form water along the gaseous hydrogen. It is important to note that the moisture
content of the soil affects the surface tension and is thus a factor affecting compaction. The enzyme
reduces surface tension making the soil compaction easier to perform. After the absorbed water is
reduced the soil particles tend to agglomerate and as a result of the relative movement between
particles, the surface area is reduced and less absorbed water can be held, which in turn reduces the
swelling capacity ((The Carbon Group).
Unlike the standard stabilizers such as Portland cement, lime and bitumen, non-standard
stabilizers have no laboratory tests that can be used to predict their field performance. Because of the
lack of communication between the manufacturers (unfamiliar with the road design process) and the
engineers the considerable benefits of the non-standard stabilizers remain undiscovered or not clear
[Scholen 1992].
Some of the recent experimental studies of soil stabilization with bio-enzymes are presented
Lacuoture & Gonzalez (1995) studied the effect of the TerraZyme (TerraZymesoil stabilizer is
an enzyme produced in the United States by Nature Plus, Inc. and is available worldwide through
local distributors or directly from NPI) soil stabilizer product on sub-base and sub-grade soils.
Variation in properties was observed and no significant improvement was reported during the early
days but progressive improvement was observed.
Hitam et al. (1999) of Palm Oil Research Institute of Malaysia conducted field studies on
improvement on plantation roads. Road section of 27.2 km was treated with TerraZyme and it was
observed that road remained good after two monsoon seasons, which had serious problems due to
monsoon before.
Bergmann (2000) concluded through studies that Bio-Enzymes need some clay content to
strengthen the soils. It was observed that at least 2% clay is needed for successful stabilization
whereas 10 to 15% clay gave very good results.
Shukla et al. (2003) used Bio-Enzymes to stabilize five different types of soil ranging from low
clay content to very high clay content, engineering properties and strength characteristics were
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determined and it was found that there is little to high improvement in physical properties. Little
improvement could be due to soil constituent, which has low reactivity with Bio-Enzymes. There
was improvement in CBR and unconfined compression strength of soils like silty soil to sandy soil.
Milburn & Parsons (2004) conducted different tests (freeze-thaw, wet-dry, leach testing,
Atterberg limits and strength tests) on soils (classified as CH, CL, ML, SM, and SP) stabilized with
lime, cement, Class C fly ash, and Permazyme 11-X. Compaction, Unconfined compression,
stiffness, freeze-thaw, wet-dry and leaching tests were conducted on two silty soils (ML and SM)
treated with Permazyme 11-X at a dosage recommended by the supplier. ML and SM soils had fines
88 and 30%, LL 30 and 20% and PI 7 and 3% respectively. Compaction test for treated soils was
carried out at moisture content 1% less than the optimum. But only 4% and 1% increase in dry
density was found for ML and SM soils respectively. The soil samples for two soils after 28 days of
curing were tested for stiffness and no improvement was recorded. Similarly for freeze-thaw very
modest improvement and for wet-dry and leaching tests no improvement was observed.
Marasteanu et al. (2005) conducted resilient modulus and tri-axial tests on two soils which were
stabilized with two different enzymes. Soil-I has 96% of fines (75% of clay) a SPG of 2.73 and
plasticity index of 52%. Soil-II has 60% of fines (14.5% of clay) and plasticity index of 9.4%.
Chemical analysis of only one enzyme (A) was conducted, as the supplier of the other enzyme (B)
did not agree for this. The chemical analysis for the enzyme included pH, metals concentrations
(e.g., Ca, Fe, and Al), total organic carbon concentration, and inorganic anion concentrations (e.g.,
Cl-, NO
and SO
). The pH of product A was 4.77 and had very high concentration of potassium
(K), and moderate to high concentrations of calcium (Ca), magnesium (Mg), and sodium (Na). The
metal concentration and inorganic anion concentration are given in Tables 1 and 2. The tests were
conducted on a base (Base-1) as well to compare the results.
The protein concentration in the undiluted product A was 9230 mg/L. The presence of protein
alone does not indicate that the solution will exhibit enzymatic activity therefore enzyme activity
tests were conducted and it was found that the product A exhibited no detectable enzymatic activity
for the used substrates. This indicates two possibilities:
Product A is a highly purified enzyme solution that contains only a single enzyme or group of
enzymes that catalyze reactions not tested for in our experiments or
Product A may not stabilize soil via enzymatic activity but rather via some other mechanism,
possibly due to their surfactant-like characteristics.
Therefore surface tension test was carried out and it was found that enzyme A was more
effective in reducing the surface tension of water than a common surfactant, sodium dodecyl sulfate
(SDD). Comparison among the two enzymes and SDD is presented in Figure 3. The results from
quantitative surface tension testing and qualitative observations suggest that product A behaves like
a surfactant, which may play a role in its soil stabilization performance. Base-1, on the other hand,
contains high concentrations of sodium and silicon, which suggests that it acts like cement by
forming hydrated calcium silicate when added to soil.
Resilient modulus tests were run with different confining (2, 4, 6 and 8 psi) and deviator stress
(4, 7, 10 and 14 psi). It is difficult to discuss all the results but brief comparison is presented here:

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Table 1: Metal concentration in product-A and Base-1
Concentration, mg/L
A Base-1
Al 2.74 60.4
Ca 719 420
Fe 24.1 3.19
K 7800 1.55
Mg 337 2.13
Mn 2.11 <1
Na 169 31000
P <1 2.94
Rb 11 <1
Si 318 63000
Zn 3.05 <1

Table 2: Inorganic anions in product-A and Base-1
Concentration, mg/L
A Base-1
Cl- 1150 14.5
Not detected Not detected
664 27.8

Figure 3: Surface tension test results of products A, B and SDD
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For example with 7 psi deviator stress the increase in resilient modulus in soil-I was 3 % to 10 %
for enzyme-A and 55 % to 85 % for enzyme-B. Similarly for the same deviator stress the increase
was 51 % to 61 % with enzyme-I and 57 % to 137 % with enzyme-B for soil-II. It shows that
enzyme-A was not very effective in highly expansive soil (PI of soil-I =52%) whereas enzyme-B
produced significant improvement in both soils.
Tri-axial shear tests were carried out on two confining pressures (4psi and 8psi). On average
Enzyme-A increased the shear strength of soil-I by 9%, and by 23% the shear strength of soil II. On
the other hand enzyme B increased the strength by 31% for soil I and 39% for soil II. It was also
concluded that resilient modulus for all combinations of soil (I and II) and enzyme type (A, B)
increases with curing time. They recommended that more mixtures of soils and enzymes be tested
and laboratory data should be compared with field data. They also recommended 4 months curing
time to achieve improvement in shear strength.
Shankar et al. (2009) studied the effect of different dosages of Bio-Enzymes on Lateritic soil of
Dakshina Kannada (district of India), having liquid limit and Plasticity Index more than 25% and 6%
respectively. Tests were conducted on lateritic soil by adding different percentages of sand as well.
They concluded that there is medium improvement in physical properties of lateritic soil. Therefore
it was suggested that effect of Bio-Enzyme on soil should be examined in laboratory before actual
field application. Higher dosage (200ml/2m
of soil) produced 300% increase in CBR, 450% in
unconfined compressive strength and permeability was reduced by 42% after four weeks of curing.
It was also observed that enzyme is not effective for cohesion less soil.
Venkatasubramanian & Dhinakaran (2011) conducted tests on three soils with varied properties
and different dosages of Bio-Enzyme. Three soils had liquid limits of 28, 30 and 46% and plasticity
index of 6, 5 and 6%. Increase in unconfined compressive strength and CBR after 4 weeks of curing
was reported as 152 to 200% and 157 to 673% respectively.
Rauch et al. (2003), measured effects of three liquid stabilizers (Ionic Stabilizer, Enzyme
Stabilizer, Polymer Stabilizer) on five different soils. Two soils were natural clays of high plasticity
and three were composed of predominately one clay mineral: kaolinite, illite, and sodium
montmorillonite. The enzyme was proprietary, concentrated, biodegradable, nonbacterial, multi-
enzymatic formulation claimed to increase soil density, reduce compaction effort, improve bearing
capacity, and lower soil permeability. The analytical tests to characterize the enzymes included pH,
potentiometric titration, total organic carbon (TOC), Fourier transform infrared spectroscopy (FTIR),
nuclear magnetic resonance (NMR), high-performance liquid chromatography-mass spectroscopy
(HPLC/MS), fast atom bombardment (FAB), and UV/Vis spectroscopy.
The pH and conductivity for the enzyme stabilizer diluted 1:10,000 were 3.26 and 0.791 mS/m,
respectively. This value of conductivity is in the range of high purity water. It was found through
potentiometric titrations that the stabilizer was a weak acid with an acidity constant greater than Ka
=10-5. Sample consisted of a 0.26N acid. The TOC of the concentrated enzyme stabilizer was
370,000 mg/L. This result suggests a very large organic carbon fraction in the enzyme stabilizer.
FTIR was conducted to identify organic carbon functional groups present in the enzyme stabilizer
and results shown in Table 3 are based on the J ournal of Optical Society (1988), which was further
verified by NMR. HPLC/MS, FAB, UV/Vis spectroscopy suggested the presence of polyethylene
glycol (C2H4O).
The treated and untreated clay materials were characterized using BET surface area analysis,
cation exchange capacity (CEC), environmental scanning electron microscopy (ESEM) and X-ray
diffraction (XRD).
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BET surface area technique quantifies external surface area and pore size. BET nitrogen
adsorption results for treated and untreated samples showed a significant reduction in surface area
for all of the clay minerals and soils tested. These results suggest that the enzyme stabilizer caused a
substantial amount of agglomeration of the soil particles regardless of the nature of the soil material,
which again is consistent with the proposed mechanism. ESEM images for the treated and untreated
samples also confirmed that the treated samples appear more aggregated than the corresponding
untreated sample, and the clay features are less visible.
Ionized water can then form linkages between packed particles to provide a cementation effect.
In the presence of the enzyme stabilizer the d-spacing of the montmorillonite sample is similar to the
d-spacing obtained for an untreated glycolated sample. This result suggests that the enzyme has
penetrated the inner layer of the montmorillonite and forced it into an expanded state in accordance
with the proposed mechanism. Whereas there was no change in the d-spacing for kaolinite and illite
which means enzyme was not able to penetrate into the inner layer. This result suggests that upon
application of the enzyme to expansive clay such as sodium montmorillonite, the clay layers fully

Table 3: Interpretation of peaks in FTIR spectra for the enzyme stabilizer
(J ournal of Optical Society, 1988)
Wavelength (cm
) Functional Group
1,120 C-O
1,220 tertiary butyls
1,350 C-OH
1,300 CH2=CH, CH-OH
1,460 C-H bend

ESEM images for the treated and untreated samples of the clay minerals showed that all of the
clay and soils analyzed except possibly the kaolinite, the treated samples appear more aggregated
than the corresponding untreated sample, and the clay features were less visible.
Cation exchange capacity (CEC) was measured for untreated and treated samples of
montmorillonite at three application mass ratios: 1:4, 1:1,000 and 1:6,000. There was no impact of
the enzyme stabilizer on the CEC of the montmorillonite sample at any of the application mass ratios
examined indicating that the expanded inner layer is still accessible for cation exchange.
Overall (in terms of Atterberg limits, density, shear strength and swell potential) no marked
improvement was observed, yet there were improvements in individual cases. It was suggested that a
higher dosage than recommended by the supplier might produce significant benefits.
Brandon et al. 2010_ENREF_2_ENREF_2 conducted Atterberg limits, density, strength and
R-value test on a commercially available enzyme called Permazyme on six single source and three
blended soils. It was suggested by the enzyme company that the optimum temperature is above 10
for a curing time of 72 hrs and that the temperature above 120
F reduces the enzyme activity. Also
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the presence of strong bases and acids reduce the enzymes effectiveness. The gradation curves for
the six single source soils are shown in Figure 4.
The summary of Atterberg limits, increase in dry density, friction angle, cohesion and R-value
are given in the Table 4.
The liquid limit and plastic limit tests for treated and untreated soils were carried out only on
three soils (soil-1, soil-2, and soil-6). There was decrease in plasticity index for soils-1 and soil-2.
Whereas an increase of 44% in plasticity index of soil-6 was observed. By looking at the Table 2 it
can be easily said that there is no definite trend in the treated and untreated soil properties except for
cohesion. The increase in cohesion was observed between 6 to 64%. This increase in cohesion
advocates the claim of the enzymes supplier that these products agglomerate the soil particles.

Table 4: Summary of laboratory test results
L.L P.L P.I.
Angle of
1* 2* 3* 1 2 3 3 3 3 3 3
1 A-2-4/ GC 26.2 24.3 -7.3 17.6 19 8 -38.4 5.2 -3.7 6 27.3
2 A-2-4/ GP 24.2 -- -- 17.4 -- -- -- -7.7 12 -5 -43.5
3 A-2-4/ SW-SC 22.1 -- -- 16.9 -- -- -- 0.5 -6.8 14.7 -23.3
4 A-2-4/ GW 22.5 -- -- 16.2 -- -- -- -0.1 -0.2 36.2 1.4
5 A-2-4/ SP 29.3 28.7 -2 19.3 20 3.6 -13 -4 6.7 20.5 --
6 A-2-4/ GW-GC 26.9 26.7 -0.7 21.5 18.9 -12.9 44.4 1.6 -10.5 64.2 7.7
50 % Soil 5 +50%
class-II AB
-- -- -- -- -- -- -- 1.4 -3.7 20.1 14.7
75 % Soil 6 +25%
class-II AB
-- -- -- -- -- -- -- 4.4 -4 38.9 -13.2
50 % Soil 1 +50%
class-II AB
-- -- -- -- -- -- -- 0.6 -10.7 59.5 -10
1* untreated, 2* treated and 3* % increase

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Figure 4: Gradation summary
Peng et al. (2011) conducted unconfined compression tests on three soils; fine-grained, silty
loam and coarse grained textures named as Soil I, Soil II and Soil III respectively. Three soils were
stabilized with quicklime and an enzyme (Perma-zyme). The samples were cured up-to 60 days in
two different conditions; air-dry and in sealed container. In air-dry curing the samples were allowed
to dry at room temperature where as in sealed container the moisture was preserved in the samples
during the curing time. Figures 5 and 6 summarize the results for two stabilizers for air-dry and
sealed curing. The enzyme was found more effective in air-dry curing for Soil I and Soil II than
quicklime where as it was not effective for Soil III in air-dry curing and for three soils in sealed
curing too. In sealed containers, the quicklime was found more effective than the enzyme as the
water in the specimens was not allowed to evaporate which promoted the further hydration of

Vol. 18 [2013], Bund. R 3892

Figure 5: Relation of unconfined compressive strength and curing time under air-dry

Figure 6: Relation of unconfined compressive strength and curing time in sealed glass

The above studies show varying results from very modest improvement to significant
improvement. Therefore the published claims or results by the manufacturer of these enzymes
cannot be trusted without independent laboratory testing. But at the same time the laboratory tests
are criticized for not replicating the field conditions. But if the product does not show significant
results in the controlled laboratory conditions then it is more difficult to attain desired results in
lesser favorable conditions in the field. Its only when laboratory testing shows significant results
then the next question would be how much improvement is required to validate its use in the field.

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2013, EJ GE