




































 

 

 
11 

© 2021 Conscientia Beam. All Rights Reserved. 

DEVELOPMENT OF AN EFFECTIVE BIOCATALYZED ORGANIC FERTILIZER 
DERIVED FROM GLIRICIDIA SEPIUM STEM BIOCHAR   

 

 

 W. L. N.  Wasana1 

 R.T.K. 
Ariyawansha2 

 B.F.A. Basnayake3+ 

 

1Postgraduate Institute of Agriculture, University of Peradeniya, Peradeniya, 
Sri Lanka. 
2,3Solid Waste Management Research Unit, Department of Agricultural 
Engineering, Faculty of Agriculture, University of Peradeniya, Peradeniya, 
Sri Lanka. 

 
 

 
(+ Corresponding author) 

 ABSTRACT 
 
Article History 
Received: 12 January 2021 
Revised: 9 February 2021 
Accepted: 2 March 2021 
Published: 22 March 20201 
 

Keywords 
Biochar 
Biochar-biocatalyst 
Compost 
Gliricidia green leaves 
Market waste 
Michaelis-Menten kinetics 
Organic fertilizer. 

 

Biochar biocatalyst action could improve quality and reduce costs in producing 
biochar fertilizer. Objective of this study was to develop a high-quality organic 
fertilizer using biochar biocatalyst action. To activate Gliricidia stem biochar 
(produced at 400-500oC and 2.5 hours residence time), aqueous biocatalysts 
were prepared in two separate aerobic reactors containing 4L of water, 12.5 g of 
rock phosphate, and Gliricidia biocatalyst (GBC) with 1 kg of Gliricidia leaves 
and market waste biocatalyst (MWBC) with 1 kg of market wastes.  
Intermittently total 377.5 g and 525 g of biochar were added respectively to the 
reactors until they reach neutral pH. GBC showed higher total nitrogen (243 
mg/L) and available phosphorous (8,125 mg/L) contents. Four compost piles 
were prepared with fresh immature grass of 18 kg/pile and Gliricidia leaves of 2 
kg/pile. Produced biocatalysts were added at the beginning to three piles as 6% 
GBC, 3% GBC, and 3% MWBC and the control with 6% biochar on dw basis. 
N, P, K levels of all the compost piles after 8 weeks were within the 
recommended levels of compost. The highest total nitrogen (20.3 g/kg) and 
available potassium (83.71 g/kg) remained in 6% GBC and the highest available 
phosphorous (3.41 g/kg) measured in 3% MWBC. pH values of all piles ranged 
between 8.8-9.2. The made fertilizer is very suitable and cost-effective for acidic 
soils to improve soil nutrient status unlike the addition of lime. Michaelis-
Menten kinetics indicates that it is preferable to add market waste-like 
substances to GBC for optimizing the qualities.  
 

Contribution/Originality: This study documents a novel procedure of biochar activation using an aqueous 

mixture of Gliricidia sepium stem biochar, Gliricidia leaves and rock phosphate under aerobic conditions and also, 

production of high quality organic fertilizer with the addition of activated biochar during active phase of 

composting. 

 

1. INTRODUCTION 

Intensive farming practices lead to depleting soil carbon storage thus reduces its capacity to act as a carbon 

sink [1].  Conservation tillage, the addition of soil amendments with biosolids and organic wastes and improved 

crop-rotation have been identified as strategies to increase carbon sequestration in soils [2]. There is a potential to 

increase stored carbon in soils and to reduce greenhouse gas emissions by applying organic residues into 

Current Research in Agricultural Sciences 
2021 Vol. 8, No. 1, pp. 11-30. 
ISSN(e): 2312-6418 
ISSN(p): 2313-3716 
DOI: 10.18488/journal.68.2021.81.11.30 
© 2021 Conscientia Beam. All Rights Reserved. 

 
 
 

 
 
 

 

 
 
 
 

https://orcid.org/0000-0002-7663-2341
https://orcid.org/0000-0003-2207-8135
https://orcid.org/0000-0002-3384-2955
https://www.doi.org/10.18488/journal.68.2021.81.11.30


Current Research in Agricultural Sciences, 2021, 8(1): 11-30 

 

 
12 

© 2021 Conscientia Beam. All Rights Reserved. 

agricultural soils [3]. But the relatively fast rate of degradation which leads to the emission of carbon dioxide and 

becoming a carbon source to greenhouse gas emission rather than being a sink is a problem associated with the use 

of organic residues like green manures and compost [4]. Still, there is a possibility to convert organic residues into 

biochar which has a relatively slow rate of decomposition [5].  Biochar has been found to be more stable than 

composts and can be effectively used to improve carbon sequestering in soils [6-8]. By both physical and chemical 

fractionation studies, it has demonstrated that labile carbon fractions decompose faster than non-labile carbon 

fractions [9]. The physical and chemical structures including surface area, condensation grade, and particle size of 

biochar control their stability in soils [10-12]. 

Composting is one of very popular organic soil amendments which contributes to restoring soil quality and 

sequestering carbon in the soil upon use [13]. Compost is produced by aerobic microbial decomposition of 

biodegradable materials [14].  During the composting process, nutrient loss is an avoidable problem, specially the 

case for nitrogen [15]. Total nitrogen loss during the composting process ranges from 16% - 76%. This substantial 

loss of nitrogen results in the reduction of the nutrient value of the final compost products [16]. Therefore, 

reducing nutrient loss specially in the nitrogen loss during composting can produce compost with superior quality.  

As biochar and composts are organic soil amendments, there is a high potential to use both together and get 

improved results [17]. According to a study conducted by Hardy Schulz, it has shown that Avena sativa L. (Oat) 

plant growths have increased in sandy substrates than loamy substrates with the use of co-composted biochar. In 

addition, they have found that the application of co-composted biochar could only be a better way to enhance plant 

growth and soil nutrient levels if only applied at rates higher than 2.5 Mg/ha [18]. However, Blackwell and co-

workers have suggested that it has a potential to have better results by using loaded or activated biochar [19]. 

Biochar is proven to reduce nutrient losses from the soil by adsorbing nutrients. This phenomenon can also be 

applied in the composting process to minimize nutrient loss during composting, specially nitrogen loss. The 

addition of biochar during composting will reduce the loss of nitrogen and produce nutrient stabilized organic 

fertilizer [6].  

The addition of biochar can be further improved with biocatalyst. Biocatalyst is the entity which accelerates or 

catalyzes biochemical reactions in living cells [20]. Catalysis of biochar can be done either chemically or 

biologically [21]. But biologically activated biochar might be very suitable for agricultural purposes than 

chemically activated biochar.  Biocatalyzed biochar is the catalyzation of biochar with natural enzymes with the 

presence of microbial activities instead of using synthesized chemicals. Thus, a study was conducted to develop a 

procedure to produce a totally organic fertilizer with biochar biocatalyst, compost, and Eppawala rock phosphate 

(ERP) which will be rich in nutrients. 

 

2. MATERIALS AND METHODS 

Initially, two types of aqueous biochar biocatalysts using market waste and Gliricidia sepium (Gliricidia) green 

leaves were produced separately under aerobic conditions based on the study conducted by Gunasekara, et al. [22].  

Reactor designing, fabrication, establishment, and evaluation were done at the Faculty of Agriculture, University of 

Peradeniya. Then compost was prepared by using produced biocatalysts with different treatments at the farm site of 

Meewatura, University of Peradeniya. Gliricidia sepium stem biochar production and collection of Gliricidia sepium 

leaves and garden waste were also done at the Meewatura farm. 

 

2.1. Biocatalysts Production and Evaluation 

2.1.1. Collection of Raw Materials And Preprocessing 

5 kg of fruits and vegetable waste from a marketplace, 5 kg of Gliricidia sepium stem biochar, 0.1 kg of 

Eppawala Rock Phosphate (ERP) were collected. The collected market waste was sorted into fruits and vegetables 

and then 2 kg of fruits and 2 kg of vegetables were chopped into small size particles using a grinder.  4 kg of green 



Current Research in Agricultural Sciences, 2021, 8(1): 11-30 

 

 
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© 2021 Conscientia Beam. All Rights Reserved. 

leaves was also chopped into small pieces using a chopping machine and then ground using a grinder separately to 

prepare a slurry solution. The collected raw materials were analyzed to determine the initial composition.  

Gliricidia sepium stem biochar was obtained from Meewathura farm which was produced in a batch pyrolytic 

reactor at a temperature range of 400 oC - 500 oC for 2.5 hours. Collected biochar was size reduced and sieved 

through a sieve (<4 mm) and particles less than 4 mm in size were used for the experiment. The preprocessed 

biochar was tested for moisture content (MC), total solids (TS), volatile solids (VS), and ash content by using 

APHA Method 2540-G, pH by using a pH meter (Thermo Scientific, model Orion 2 star), salinity, electrical 

conductivity (EC),  and total dissolved solids (TDS) by using a conductivity meter (Thermo Orient Model 145 A), 

particle size distribution by using the standard methods and instruments. Size reduced market waste and size 

reduced (<2 mm) Gliricidia sepium green leaves were analyzed separately to determine the MC, ash content, TS, and 

VS by using APHA Method 2540-G.  

 

2.1.2. Reactor Fabrication, Biocatalysts Preparation and Analysis 

Two aerobic reactors were fabricated using 15 L plastic containers and 4 mm diameter transparent flexible 

tubes.  Plastic containers were used without any modifications and the lids were kept open to facilitate gas 

exchanges and promote aeration (to insert aerator tube).  Aeration was done continuously using an aerator pump 

(SDA-2800). The mixture of shredded market waste was blended by adding a measured amount of water to obtain a 

solution.  The same chopping and blending procedure were used to preprocess Gliricidia sepium leaves separately to 

have another solution.  100 mL from each solution was kept separately for the characterization. The rest of the 

slurries were diluted up to 12 L by adding water to maintain organic matter: water ratio as 1: 4.  50 g of ERP was 

added to each solution while diluting with water.  The prepared slurries were put into the fabricated reactors. 

Aeration was also done continuously throughout the experimental period. Size reduced biochar (< 4 mm) was added 

intermittently to the reactors separately until the reactor pH reached a neutral pH value of 7. The added biochar 

was measured and weights were noted as given in Table 1. 

A representative sample from each reactor was taken and analyzed daily for 31 days for pH by using a pH 

meter (Thermo Scientific, model Orion 2 star), EC, salinity, and TDS concentration by using a conductivity meter 

(Thermo Orient Model 145 A), TS, and VS by using APHA Method 2540-G,   and dissolved oxygen (DO) by using 

a DO meter (Eutech DO 6+)  while available total nitrogen (N) content by using the Kjeldahl method, available 

potassium (K) (Exchangeable base method using a flame photometer), available phosphorous (P) by using the Olsen 

P method were measured, on 1st  day, 15th  day and on 31st  day. Scanned electron microscopic (SEM) (EVO LS15) 

view was taken at the end of the experiment (on 31st day).  

 
Table-1. Quantities of biochar added to the each biocatalytic reactor. 

Time (Days) Quantity of biochar added (g) 

Gliricidia biocatalyst Market waste biocatalyst 

1 50 50 

2 25 27 
4 32 35 

6 35 38 
8 50 62 

9 70 120 
10 77 100 

11 100 200 

12 120 300 
13 100 110 

15 500 600 
16 100 110 

17 100 120 
22 150 230 

Total 1,510 2,100 
 



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© 2021 Conscientia Beam. All Rights Reserved. 

2.1.3. Analysis of the Biocatalyst Production Process with the Application of Biochemical Transformation Kinetics 

Biochemical transformation kinetics [23] was used to evaluate the biocatalyst production process. Data were 

interpreted and analyzed by using an Excel sheet with the developed equations and extensions to Michaelis-Menten 

kinetics [23].  The concept of a unit production to describe the cyclic behavior of the reactions as stated in 

Equation 1 is the fundamental theory,  

 

S E + R
k1 k

1

k
r

E + S
k

1

k'
1

k
2

E + P

A B C

D

E

k
1

E
ex  

+ S
ex

 

Path X

Path Y

k
1

ES

k
2

                   (1) 

  where, Eex = Enzymes from external environment,  Sex = Substrates from external environment, S = substrate,  E 

= enzymes,  R = reactant, k1,
'

1
k , and k2 = rate constants, ES = enzyme-substrate complexes, P = products,  path X 

= substrates from internal body entity, path Y = substrates from food and nutrients supply.  

 

There are no alterations to the original Michaelis-Menten hyperbolic function, which is defined as given in 

Equation 2;  

 
 SK

Sv
v

m

m


       (2) 

where, v  = overall rate of reaction, mv  = maximum rate of reaction, mK  = Michaelis constant, and  S  = substrate 

concentration.   The substrate was defined as [product]3/4 [23] and cumulative VS concentration at time t was 

calculated for each catalyst production process and it was used as the product for this analysis. It has been 

confirmed that in all biochemical transformations, enzyme productions from body  'E can be defined as given in 

Equation 3 and 4;  

333.0' 2SE                                                        (3) 

and 
25.0' 2PE       (4) 

The classical Michaelis-Menten kinetics is applied to the considered cycle from C to D to E in Equation 1. By 

calling the initial concentration of enzyme Eo, 

     EESEo      (5) 

Furthermore, v can be expressed as given in Equation 6.  

 

 ESkv 2      (6) 

When v = vm, [ES] = Eo as given in Equation 7. 

 om Ekv 2      (7) 

And also, 

                                               

1

2
'
1

k

kk
Km


          (8)  



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15 

© 2021 Conscientia Beam. All Rights Reserved. 

Enzyme productions and formation of enzyme-substrate complexes derived from transformations given in 

Ariyawansha, et al. [23] were used to evaluate the biocatalyst production process (Supplementary material 1).   

 

2.2. Composting and Evaluation 

Next step was to produce an effective organic fertilizer by using produced biocatalysts. For that, collected and 

separated green garden waste (a mixture of Gliricidia sepium green leaves and buffalo grass (Bouteloua dactylodies) 

was chopped into small pieces with particle sizes less than 2 cm long using a household grinder.  Four windrow 

composting piles were established using preprocessed garden waste mixed with different ratios of biochar at the 

Meewathura farm Table 2.  The percentage of biochar incorporation was decided based on the results reported by 

Samudrika, et al. [17]. Amount of water that should be added to bring the MC of the pile to standard MC (40 - 60 

% (w/w)) of composting was calculated and added accordingly.  

 

Table-2. Treatments of the composting experiment. 

Treatment Name 
Immature 
grass (kg) 

Gliricidia 
leaves (kg) 

% of raw biochar/ catalyst added from 
dry weight of the total stock material 

6% biochar 18 2 6% biochar 
6% Gliricidia catalyst 18 2 6% Gliricidia catalyst 
3% Gliricidia catalyst 18 2 3% Gliricidia catalyst 
3% market waste catalyst 18 2 3% market waste catalyst 

 

Temperature of the pile was measured daily throughout the experimental period using thermal sensors and did 

set the system to automatically record the data in a data logger. Representative samples from four piles were taken 

separately just after the preparation. And sampling from each composting pile was done daily for 53 days 

throughout the experimental period. For that, five sub-samples were obtained randomly from various places in each 

composting pile to obtain a representative sample. Sub-samples of each pile were placed into a container and mixed 

thoroughly to make a composite sample for analysis. The composite samples were analyzed daily for pH by using a 

pH meter (Thermo Scientific, model Orion 2 star), moisture content, total solids, volatile solids, and ash content by 

using APHA method 2540-G. Samples were analyzed for total N content by using the Kjeldahl method available P 

content by using the Olson method, and available K content by using the exchangeable base method using a flame 

photometer on weekly basis. 

 Michaelis-Menten kinetics and Michaelis-Menten equation were used to analyze the biochemical 

transformation kinetics of the composting process.  The substrate was defined as [product]3/4 [23] and cumulative 

VS% at time t was calculated for each composting treatment and it was used as the product for this analysis.  Then, 

v was calculated based on both rate and time perspectives as given in Equation 9.   

 

(9) 

Km and vm values for different composting treatments were calculated by drawing graphs using the Lineweaver-

Burk double reciprocal plot of 1/[S] vs 1/v [24].  Then using those Km and vm values and considering the preferred 

substrate concentration as the calculated [S] of 6% Gliricidia sepium catalyst added treatment, again v was 

calculated for each treatment and Lineweaver-Burk plots were drawn to identify inhibitive conditions.  

 

3. RESULTS AND DISCUSSION 

3.1. Characterization of Raw Materials 

Typically, biochar is alkaline and pH value is usually in the range of 7 - 10 [25]. Fast pyrolysis chars produced 

in the absence of steam tend to be very basic [26] but it varies mainly with the feedstock characteristics [27]. 

Gliricidia sepium stem biochar used for this experiment had a pH value of 8.02.  The moisture content (wb %) of 

 
dt

tdt

Sd
dvv .

. 



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© 2021 Conscientia Beam. All Rights Reserved. 

biochar was 6.59% and ash content was 4.66%. TDS concentration was 289.79 mg/L. EC was 590.35 µs/cm and 

salinity content was 0.318 PSU.  Biochar is considered as a soil nutrient regulator as it enriches soil similar to other 

fertilizers [28]. Total N content of used biochar was 8,184.0 mg/kg where available K concentration was 18,077.8 

mg/kg and available P concentration was 353.3 mg/kg.  Market waste which was used mainly comprised of 10.3% 

of pumpkin (Cucurbita maxima), 12.9% of brinjole (Solanum melongena), 9.6% of carrot (Daucus carota subsp. sativus), 

2.4% of long bean (Vigna unguiculata ssp. sesquipedalis), 7.2% of beans (Phaseolus vulgaris) and 7.5% of cabbage 

(Brassica oleracea var. capitata) as wasted vegetables  and it contained 17.1% of papaw (Carica papaya), 13.7% of 

banana (Musa spp.), 11% of water melon (Citrullus lanatus), 5.4% of pineapple (Ananas comosus), 2.88% of pomegranate 

(Punica granatum), and 1% of lime (Citrus aurantiifolia) as wasted fruits. Moisture content (wb %) of market waste 

mixture used was 90.5% while in Gliricidia sepium leaves it was 66.7%. Market waste had 9.5% of total solids, 65.0% 

of volatile solids and 35.0% ash content. Gliricidia sepium leaves had 33.3% of total solids, 55.0% of volatile solids, 

and 45.0% of ash content. Total solids content was much higher in Gliricidia sepium leaves when compared to that of 

market waste, which was comprised with more watery fruits and vegetables. But market waste had high volatile 

solids content, thus less total fixed solid contents (ash).  Therefore, the mineral content of Gliricidia sepium leaves 

could markedly influence the major differences in activation of biocatalysts.  TS content of Gliricidia slurry was 92.3 

g/L, VS content was 56.9 g/L, pH was 5.31, TDS concentration was 1.25g/L, EC content was 2.56 µs/cm, DO 

concentration was 2.31 mg/L, salinity was 1.39 PSU.  Whereas TS content of market waste slurry was 35.1 g/L, 

VS content was 33.3 g/L, pH was 4.11, TDS concentration was 1.09 g/L, EC was 2,225 µs/cm, DO concentration 

was 1.8 mg/L, salinity was 1.21 PSU. So, market waste slurry was more acidic than Gliricidia sepium slurry.  It can 

be due to lime, pineapple like acidic components of market waste slurry and also due to more acidic and readily 

degradable nature of fruits and vegetables compared to cellulose like compounds in Gliricidia sepium green leaves.  

The water content of the slurry in market wastes was much higher than Gliricidia sepium leaves.  

 

3.2. Evaluation of Biocatalyst Production Process  

As reported by Kastner, et al. [21] catalyzed or activated biochar has more advantages than raw biochar. Solid 

carbon catalysts are stable under both acidic and basic conditions, have very high surface areas (500-1,500 m2/g), 

can be used to generate active carbon, active material can be finely dispersed through the carbon structure and can 

be reused. In the first experiment, within a day there was a considerable decrease in pH value of Gliricidia catalyst 

from 5.31 to 4.76 and in market waste catalyst it reduced from 4.11 to 3.46 due to initial acidogenic reactions in the 

activation process.  But thereafter, pH values of both catalysts increased continuously at a slow rate, with the 

addition of biochar.  Biochar is characteristically alkaline in nature.  On the 24th day, Gliricidia catalyst reached near 

to a neutral pH value which was 7.07 while market waste catalyst was still acidic having a pH value of 5.54.  Total 

biochar addition rate was 377.5 g/kg Gliricidia leaves in Gliricidia catalyst, whereas it was 525 g/kg market waste 

in market waste catalyst.  In view of pH as a deciding parameter, Gliricidia catalyst is more suitable for agricultural 

applications and commercial purposes than market waste catalysts with very acidic nature.  Even though both of the 

catalysts were maintained up to 31 days, there was no remarkable changes in pH values after about 24th day Figure 

1(a). EC of Gliricidia catalyst was higher than market waste catalyst throughout the experimental period Figure 

1(b) except on 26th  day. It could be due to higher total fixed solid content of Gliricidia catalyst and readily 

degradable nature of market waste catalyst.  There was a fluctuating nature in EC of catalysts while gradual 

increasement was in market waste catalyst EC values. But there had been a distinguishable increasement in the final 

EC values of catalysts compared to initial values. Initial and final EC values of Gliricidia catalyst were 2.561 mS/cm 

and 7.007 mS/cm respectively while EC values of market waste catalyst changed from 2.225 mS/cm to 6.435 

mS/cm at the end of the experiment. It could be attributed to loss in TS content with time. Variations of TDS also 

showed the same pattern as EC, where Gliricidia catalyst had higher (1.225 g/L) and fluctuating values compared 

to low (1.091 g/L) and gradually increasing TDS of market waste catalyst Figure 2(a). There were continuous 



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© 2021 Conscientia Beam. All Rights Reserved. 

fluctuations in DO Figure 2(b) TS Figure 3(a) and VS Figure 3(b) contents of both catalysts throughout the time 

period.  It could be deduced that there was a decrease in biocatalysts properties after the 20th day and thereafter it 

was not at all useful to maintain catalysts up to 31st  day. 

 

Figure-1. Variations of electrical conductivity and pH of catalysts with time (a.) Electrical conductivity, (b.) pH. 

 

Figure-2. Variations of TDS and dissolved oxygen levels of catalysts (first experiment) with time (a) TDS, (b) dissolved oxygen. 
 

 
Figure-3. Variation of TS and VS concentration of the catalysts (first experiment) (a) TS concentration; (b) VS concentration. 

 

mK was lower than the value of the substrate  S  during the first 18 days in the Gliricidia biocatalyst (Figure 

4(a)). In the market waste biocatalyst mK was lower than the value of the substrate  S  within the first 5 days then 

suddenly increased Figure 5(a).   It indicates that there were adequate substrate and microbial activity during this 

period.  Kinetics of biochemical transformation is built based on the formation of enzyme-substrate (ES) complexes. 

[ES] complexes Figure 4(c) and differentials of d[ES]/dt Figure 4(d) drastically increased on the 8th  day in 

Gliricidia catalyst and on the 5th  day in the market waste biocatalyst Figure 5 (c) and Figure 5(d) which manifests 

higher catalytic activity potential. Such an increase accompanies higher production of enzymes.  It indicates 

exceedingly high productions as shown in the differentials from -ve liquid phase to +ve d[ES]/dt solid phase of all 

the treatments, where dS/dt approaches zero. Thus, there is a potential for producing biocatalyst within a shorter 



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© 2021 Conscientia Beam. All Rights Reserved. 

period of time. Quantity of biochar required to augment the pH can be added to the reactor within the first three 

days.  

 

 
Figure-4. Analysis of Gliricidia biocatalyst production process with the applications of biochemical transformation kinetics (a.) Substrate (S) 
concentration and Km variations with time (b.) d[S]/dt variations with time (c.) Substrate concentration, [Eo], [ES], and [E] variations with 

time (d.) d[ESʹ]/dt, d[ESʹʹ]/dt, d[ESʹʹʹ]/dt, and d[ES]/dt variations with time (e.) Lineweaver-Burk plot for different composting treatment for 
the preferred substrate concentration of 6% Gliricidia catalyst treatment. 

 

 
Figure-5. Analysis of market waste biocatalyst production process with the applications of biochemical transformation kinetics (a) Substrate 

concentration and Km variations with time (b) Substrate concentration, [Eo], [ES], and [E] variations with time (c) d[ESʹ]/dt, d[ESʹʹ]/dt, 

d[ESʹʹʹ]/dt, and d[ES]/dt variations with time.  

 



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The nutrient contents of the two catalysts were dependent on the degree of aeration throughout experimental 

period.  When comparing initial nutrient contents of both catalysts, total N content of Gliricidia catalyst (50.96 

mg/kg) was almost twice that of the market waste catalyst (21.28 mg/kg) and as expected, Gliricidia has very high 

levels of nitrogen compared to most plant materials as given in Table 3. Gliricidia was chosen especially due to its 

richness of nitrogen and abundance when compared to most of the other plants and wood biochar which can be 

produced or purchased at low cost. On the other hand, market waste catalyst had initially high levels of available P 

(57.06 mg/kg) and available K (1,200 mg/kg) compared to initial available P (38.43 mg/kg) and available K (1,000 

mg/kg) of Gliricidia catalyst. In Gliricidia catalyst, available total N content was 243.04 mg/kg and available K 

concentration was 8,125 mg/kg on the 15th day which were the maximum reported values and thereafter decreased 

towards the 31st day.  It can be due to high microbial population and activities towards the middle of the experiment 

and then decreasing microbial activities with gradual death of microbes due to lack of substrate. As mentioned 

before, the pH values of the catalysts reached to its maximum pH value on the 10th  day and thereby it can be 

deduced that 10-15 days would be enough to activate Gliricidia catalyst with maximum total N and available P. 

Market waste catalyst had its maximum total N level of 21.28 mg/kg on the 1st  day and thereafter it decreased to 

4.48 mg/kg towards the 15th day and then again increased to 5.60 mg/kg on the 31st day.  The decrease in total N 

level on the 15th day could be attributed to increase in microbial utilization of it during that period and releasing 

some of it towards the 31st day, which could be due to higher levels of microbial respiration, thus addition of some of 

the microbial nitrogen to the catalyst. Variations of available P levels of both catalysts showed same pattern. 

Available P concentration of Gliricidia catalyst was 38.43 mg/kg on the 1st day and on the 15th day it was 16.15 

mg/kg, on the 31st  day it was 75.62 mg/kg.  On 1st day P concentration of market waste catalyst was 57.06 mg/kg, 

on the 15th day it was 53.4 mg/kg and on the final day it was 110.4 mg/kg. Not like N, P uptake was more because 

of microbial growth and their activities and utilization of more nutrients in the earlier period and then activities 

decreased towards the end, increasing P. Solubilization of ERP happens very slowly but microbial utilization 

happens at high rates during initial period and it could be the possible reason for the decrease of available P on 15th 

day.  But thereafter, due to dying of microbes and slow rate of P utilization compared to ERP solubilization 

available P has increased on the 31st  day in both catalysts.   

 
Table-3. Nutrient contents variation of catalysts. 

Parameter 
Gliricidia catalyst Market waste catalyst 

Initial 
(1st day) 

Middle 
(15th  day) 

Final 
(31st  day) 

Initial 
(1st day) 

Middle 
(15th  day) 

Final 
(31st  day) 

Total N 50.96 243.04 109.76 21.28 4.48 5.6 
Available P 38.43 16.15 75.62 57.06 53.4 110.4 
Available K 1000 8125 1140 1200 1625 1320 

 

 

There are distinct differences between the SEM view of raw biochar and bio-catalyzed biochar Figure 6. 

Biochar from both catalysts have greater smoother surfaces than raw biochar, implying occupation of active sites.  

 

3.3. Performances of Composting Piles 

3.3.1. Variations of Temperature, ph, Electrical Conductivity, and Volatile Solid Content with Time 

According to the temperature observations of composting piles, they have not passed thermophilic stage. 

Average temperature of the 6% biochar added pile was 25.5± 1.7 °C and it was 25.4± 2.2 °C in the 3% Gliricidia 

catalyst   mixed pile.  And the average temperature of 6% Gliricidia catalyst added pile was 26± 2.2 °C and it was 

25.2± 1.9 °C in the 3% Gliricidia catalyst mixed pile during the experimental period. 34 °C was the highest 

temperature recorded in the 3% Gliricidia catalyst mixed pile from among four treatment piles. The temperature 

values were not very much higher than the environmental temperatures, since the average temperature of the 

environment was 25.6±1.4 °C during the experimental period. 



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20 

© 2021 Conscientia Beam. All Rights Reserved. 

 
Figure-6. Scanned electro microscopic view of raw biochar and each biocatalyzed biochar (a) Raw biochar; (b) Gliricidia 
catalyst; (c) Market waste catalyst. 

 

Even though the optimum pH for compost is 7, pH of 6.5 - 8 are the most common range of values of 

composting [29].  In the initial stages, hydrolysis and acidogenic reactions occur lowering the pH. Thus at the 

beginning, all four piles had slightly acidic pH values for first few days as shown in Figure 7(a).  Then, it suddenly 

increased to values around pH 9.  It must have been the result of mixing biochar biocatalysts with Gliricidia leaves 

and immature grass.  It would have produced ammonia because of high nitrogen content in the starting materials.  

Thereafter pH values fluctuated around pH 8-9 throughout the experimental period. High pH fertilizers have the 

advantage of neutralizing more frequently encountered acidic soils caused by applying inorganic fertilizers. 

Continuous use of inorganic fertilizers in modern agriculture has detrimental effects of groundwater contamination, 

surface water pollution and eutrophication of surface water bodies, development of soil acidity, and human health 

problems [30]. It also results in deficiencies in micronutrients, imbalance of soil physiochemical properties and 

unsustainable crop production [31].  In fact, liming is a common practice in commercial agriculture to minimize the 

soil acidity. But this increases the atmospheric C by releasing carbon dioxide from calcium carbide in the process of 

breaking down of acids in soil [32]. Since the manufactured compost was moderately alkaline and will be a 

promising solution to reduce soil acidity while increasing soil nutrient status. During the initial period, EC values 

fluctuated in all four treatments Figure 7(b).   It could be due to increases and decreases in decomposition reactions.  

But EC values became high, more stable, and less fluctuating in latter period of experimentation as most of the 

decomposition had taken place and more labile fractions were available. At early stages, volatile solids contents 

were very high Figure 7(c) and it could be due the presence of more microbial population but it has decreased 

towards the end as microbes utilize more nutrients with the time and converting it into fixed solids.  Total solids 

are the summation of volatile solids and fixed solids, thus when there is an increase in the fixed solids, volatile solids 

tend to decrease. 

 



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0

500

1000

1500

2000

2500

0 5 10 15 20 25 30 35 40 45 50 55

E
C

 (
µ

s
/c

m
)

Time (days)

6% Biochar 3% Gliricidia

6% Gliricidia 3% Market waste

b

5

6

7

8

9

10

0 5 10 15 20 25 30 35 40 45 50 55

p
H

Time (days)

6% Biochar 3% Gliricidia

6% Gliricidia 3% Market waste

a

0

20

40

60

80

100

0 5 10 15 20 25 30 35 40 45 50 55

V
S

 (
%

) 

Time (days)

6% Biochar 3% Gliricidia

6% Gliricidia 3% Market waste

c

 
Figure-7. Variations of pH, electrical conductivity, and volatile solids content with time of the 
composting piles (a.) pH, (b.) Electrical conductivity, (c.) Volatile solids content. 

 

3.3.2. Nutrient Contents of Composting Piles  

Initially, 6% Gliricidia biocatalyst added pile had the highest total nitrogen content of 25.56 g/kg Figure 8(a) 

and it can be due to the high amount of nitrogen it gained from immature grass, Gliricidia leaves, and from the 

Gliricidia catalyst as well.  3% market waste biocatalyst added pile had the lowest total nitrogen content of 15.10 

g/kg and it may possibly be due to low nitrogen content it received from the market waste catalyst. There were 

peaks and steep gradients in total nitrogen content throughout the research period without any distinguishable 

pattern.  Peaks might have occurred because of decomposition and releasing of nitrogen in raw materials and 

microbes.  Steeps could be due to microbial utilization and volatilization of nitrogen in raw materials.  At the end, 

6% Gliricidia catalyst added pile had the highest total nitrogen content of 20.9 g/kg of dry compost while 3% 

market waste catalyst added pile had the lowest value of 12.6 g/kg of dry compost.  Initially, 6% biochar added 

compost pile had the highest amount of total available phosphorous of 1.22 g/kg of dry compost while 3% market 

waste catalyst added pile had the lowest amount of 0.53 g/kg of dry compost.  But at the end, 3% market waste 

catalyst added pile had the highest available phosphorous; amounting to 0.75 g/kg of dry compost and 6% Gliricidia 

catalyst added pile had the lowest value of 0.67 g/kg of dry compost Figure 8(b).  Initially, all four treatments had 

the same available potassium levels approximately 50 g/kg of dry compost.  But at the end 6% Gliricidia catalyst 

added pile had the highest total potassium level of 83.7 g/kg of dry compost Figure 8(c).  

Replacement with high nutrient content is essential because agriculture is accounted as the major means of 

nitrogen (N) and other nutrients loss to the environment [33]. Doubling the world food production in 1965, led to 

increase N fertilizer usage by 6.9-fold [33] so that the other fertilizers phosphorous (P) and Potassium (K) were 

increased. As emphasized before, synthetic fertilizer usage is crucial to control groundwater contamination by 

leaching of excessive nitrogen [34] surface water eutrophication by runoff of excessive P and N.  According to 

[34] improvements in soil N were observed in compost treatments compared to raw manure and synthetic fertilizer 

treatments. Also, compost treatments have showed high yields and improvements in soil C and N while synthetic 

fertilizer added treatments showed only high yields [34]. 

 

 

 



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Figure-8. Variation of nutrient contents of composting piles with time (a) Total Nitrogen; (b) Available phosphorous; (c) Available potassium 
level. 

 

3.3.3. Application of Biochemical Transformation Kinetics 

According to the obtained Km and vm values for different compost treatments, the highest Km value indicated in 

6% biochar treatment while the lowest Km value indicted in 3% market waste catalyst treatment as given in Table 4.  

The highest vm value indicated in 6% biochar treatment and the lowest vm value was in 3% Gliricidia biocatalyst 

added treatment.  According to the Lineweaver-Burk plot, Km is the substrate concentration when v is equal to half 

of the vm.  Therefore, in order to have a highest v value at a low substrate level vm should be high and Km should be 

low for a given treatment.  Therefore, better results could be obtained by mixing market waste and Gliricidia leaves 

together to make the catalyst. 

 
Table-4. Km and vm values for each compost treatment obtained from Lineweaver-Burk plot. 

Treatment Km vm 

6% Biochar 47.38 76.92 

6% Gliricidia catalyst 40.43 71.43 

3% Gliricidia catalyst 36.76 58.82 

3% Market waste catalyst 31.75 62.5 
 

 

Even though 6% Gliricidia catalyst treatment was best according to the nutrient contents, 6% biochar 

treatment was also very closer to its performance.  It is evident that in-competitive inhibition manifested according 

to Figure 9. 

This produced organic fertilizer is a good and promising solution to fertilizer related problems in commercial 

agriculture.  It is indeed an enormous step in agriculture towards organic farming by introducing this nutrient-rich 

organic fertilizer mainly to uplift rural farmers in replacing inorganic fertilizers. And, farmers can produce this 

nutrient-rich organic fertilizer by their own.  At present, soil acidity has become a major issue in the world, 

particularly so in the tropics because of intensive agricultural practices.  Instead of using lime, peat-like alkaline soil 

amendments, applying this alkaline biocatalyst compost may minimize soil acidity while improving soil nutrient 

status too. The made compost is very suitable and perhaps cost-effective for acidic soils to improve soil nutrient 

status unlike the addition of lime.   



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0.01

0.015

0.02

0.025

0.03

0.035

0.04

0.045

0 0.005 0.01 0.015 0.02 0.025 0.03 0.035

1
/v

1/S

6% Gliricidia 3% Gliricidia

3% Market waste 6% Biochar

 
Figure-9. Lineweaver-Burk plot for different composting treatment for the preferred substrate 
concentration of 6% Gliricidia catalyst treatment. 

 

4. CONCLUSIONS  

The prepared Gliricidia and market waste slurries had acidic pH values initially while biochar had a very 

alkaline pH value.  Market waste slurry had a very low pH value compared to the Gliricidia slurry.  But gradually 

both slurries reached neutral pH levels by adding biochar to the catalysts intermittently.  All pH, salinity, EC, and 

TDS values of both catalysts increased with time due to the addition of biochar and microbial degradation and 

mineralization of biomass. Even though aeration was done using an aerator, DO levels of both catalysts reduced 

with time due to increase of slurry density by addition of biochar, creating preferential flow without dissolving in 

slurry and due to microbial utilization. SEM views also show the depth of biochar activation and it has occurred in a 

satisfactory way in both catalysts.  In contrast, biochar activation and filling of pores on possible activated sites by 

microbes is very clear in short period biocatalyst than the long duration one. All four compost treatments were 

within the range of compost standards while 6% Gliricidia catalyst added treatment had the highest total nitrogen 

and total phosphorous levels.  Therefore, all the treatments can be considered acceptable to produce biocatalyzed 

organic fertilizer. The attempt to make an effective biocatalyst organic fertilizer derived from biochar was very 

successful but mathematical methods must be developed to explain precisely the reasons for the variations of total 

N, P, and K levels of catalyst and composting piles. Such changes, particularly of P and K were not very clear 

because we cannot account for losses and gains of those nutrients.  Further studies should also be undertaken using 

both market waste and Gliricidia leaves together to optimize the final product quality.  It is necessary to design and 

develop an effective industrial scale reactor to produce the catalysts and select the best type of mixing device, like a 

turner to incorporate the biocatalyst into compost piles during the active phase of composting. 

 

Funding: This investigation received financial support from the Faculty of Agriculture, 
University of Peradeniya. 
Competing Interests: The authors declare that they have no competing interests.  
Acknowledgement: The authors are very much grateful to D. D. K. Galahitiyawa, D. M. A. S. 
Wasantha, Department of Agricultural Engineering, G. Herath, Department of Crop Science, 
Faculty of Agriculture, University of Peradeniya for their support throughout the study 
period. 

 

REFERENCES 

[1] R. Lal, "Soil carbon sequestration impacts on global climate change and food security," Science, vol. 304, pp. 1623-1627, 

2004. Available at: https://doi.org/10.1126/science.1097396. 

[2] A. Sissoko and A. K. Kpomblekou, "Carbon decomposition in broiler litter-amended soils," Soil Biology and Biochemistry, 

vol. 42, pp. 543-550, 2010. Available at: https://doi.org/10.1016/j.soilbio.2009.10.007. 



Current Research in Agricultural Sciences, 2021, 8(1): 11-30 

 

 
24 

© 2021 Conscientia Beam. All Rights Reserved. 

[3] M. Bustamante, D. Said-Pullicino, C. Paredes, J. Cecilia, and R. Moral, "Influences of winery–distillery waste compost 

stability and soil type on soil carbon dynamics in amended soils," Waste Management, vol. 30, pp. 1966-1975, 2010. 

Available at: https://doi.org/10.1016/j.wasman.2010.03.012. 

[4] S. Godbout, M. Verma, J. Larouche, L. Potvin, A. Chapman, S. Lemay, F. Pelletier, and S. Brar, "Methane production 

potential (B0) of swine and cattle manures–a Canadian perspective," Environmental technology, vol. 31, pp. 1371-1379, 

2010. Available at: https://doi.org/10.1080/09593331003743096. 

[5] R. Kookana, A. Sarmah, L. Van Zwieten, E. Krull, and B. Singh, "Biochar application to soil: Agronomic and 

environmental benefits and unintended consequences," Advances in Agronomy, vol. 112, pp. 103-143, 2011. Available at: 

https://doi.org/10.1016/B978-0-12-385538-1.00003-2. 

[6] N. S. Bolan, A. Kunhikrishnan, G. Choppala, R. Thangarajan, and J. Chung, "Stabilization of carbon in composts and 

biochars in relation to carbon sequestration and soil fertility," Science of the Total Environment, vol. 424, pp. 264-270, 

2012. Available at: https://doi.org/10.1016/j.scitotenv.2012.02.061. 

[7] K. N. Palansooriya, Y. S. Ok, Y. M. Awad, S. S. Lee, J.-K. Sung, A. Koutsospyros, and D. H. Moon, "Impacts of biochar 

application on upland agriculture: A review," Journal of Environmental Management, vol. 234, pp. 52-64, 2019. Available 

at: https://doi.org/10.1016/j.jenvman.2018.12.085. 

[8] A. Karthik, S. A. H. Hussainy, and M. Rajasekar, "Comprehensive study on biochar and its effect on Soil properties: A 

review," International Journal of Current Microbiology and Applied Science, vol. 9, pp. 459-477, 2020. Available at: 

https://doi.org/10.20546/ijcmas.2020.905.052. 

[9] M. P. Bernai, C. Paredes, M. Sanchez-Monedero, and J. Cegarra, "Maturity and stability parameters of composts 

prepared with a wide range of organic wastes," Bioresource Technology, vol. 63, pp. 91-99, 1998. Available at: 

https://doi.org/10.1016/S0960-8524(97)00084-9. 

[10] D. A. Wardle, M.-C. Nilsson, and O. Zackrisson, "Fire-derived charcoal causes loss of forest humus," Science, vol. 320, 

pp. 629-629, 2008. Available at: https://doi.org/10.1126/science.1154960. 

[11] W. Kwapinski, C. M. Byrne, E. Kryachko, P. Wolfram, C. Adley, J. J. Leahy, E. H. Novotny, and M. H. Hayes, "Biochar 

from biomass and waste," Waste and Biomass Valorization, vol. 1, pp. 177-189, 2010. Available at: 

https://doi.org/10.1007/s12649-010-9024-8  

[12] J. Titova and E. Baltrėnaitė, "Physical and chemical properties of biochar produced from sewage sludge compost and 

plants biomass, fertilized with that compost, important for soil improvement," Waste Biomass Valor, 2020. 

[13] S. De Neve, S. Sleutel, and G. Hofman, "Carbon mineralization from composts and food industry wastes added to soil," 

Nutrient Cycling in Agroecosystems, vol. 67, pp. 13-20, 2003. Available at: https://doi.org/10.1023/A:1025113425069. 

[14] L. Cooperband, The art and science of composting: A resource for farmers and compost producers,” Centre for Integrated 

Agricultural Systems. University of Wisconsin-Madison, 2002. 

[15] H. T. Soudejani, H. Kazemian, V. J. Inglezakis, and A. A. Zorpas, "Application of zeolites in organic waste composting: 

A review," Biocatalysis and Agricultural Biotechnology, vol. 22, p. 101396, 2019. Available at: 

https://doi.org/10.1016/j.bcab.2019.101396. 

[16] S. Barrington, D. Choinière, M. Trigui, and W. Knight, "Effect of carbon source on compost nitrogen and carbon 

losses," Bioresource Technology, vol. 83, pp. 189-194, 2002. Available at: https://doi.org/10.1016/S0960-8524(01)00229-

2. 

[17] K. Samudrika, R. Ariyawansha, B. Basnayake, and A. Siriwardana, "Optimization of biochar additions for enriching 

nitrogen in active phase low-temperature composting," Organic Agriculture, vol. 10, pp. 449-463, 2020. Available at: 

https://doi.org/10.1007/s13165-020-00287-2. 

[18] H. Schulz, G. Dunst, and B. Glaser, "No effect level of co-composted biochar on plant growth and soil properties in a 

greenhouse experiment," Agronomy, vol. 4, pp. 34-51, 2014. Available at: https://doi.org/10.3390/agronomy4010034. 



Current Research in Agricultural Sciences, 2021, 8(1): 11-30 

 

 
25 

© 2021 Conscientia Beam. All Rights Reserved. 

[19] P. Blackwell, S. Shea, P. Storer, Z. Solaiman, M. Kerkmans, and I. Stanley, "Improving wheat production with deep 

banded oil mallee charcoal in Western Australia," in In Proceedings of Agchar Initiative Conference, Terrigal, Australia, 

2007. 

[20] W. Hartmeier, Immobilization biocatalysts: An introduction. Switzerland: Springer Nature, 2012. 

[21] J. Kastner, J. Miller, R. Ormsby, J. Locklin, and K. Fries, "Down to earth energy”,  In: Monroe, G.A. (Eds.) Catalytic 

properties of biochar for chemical synthesis," presented at the IBE.  Biofuels/Biomaterials Conference, Cambridge, 

MA, 2010. 

[22] N. S. Gunasekara, B. F. A. Basnayake, and R. T. K. Ariyawansha, "Development of an effective organic fertilizer 

through a novel approach of biocatalyst derived from biochar," in In: Proceedings of Faculty of Agriculture Undergraduate 

Research Symposium, University of Peradeniya, Sri Lanka, 2014, p. 173. 

[23] R. Ariyawansha, B. Basnayake, A. Karunarathna, and M. Mowjood, "Extensions to Michaelis-Menten kinetics for 

single parameters," Scientific reports, vol. 8, pp. 1-11, 2018. Available at: https://doi.org/10.1038/s41598-018-34675-2. 

[24] K. A. Johnson, "A century of enzyme kinetic analysis, 1913 to 2013," FEBS Letters, vol. 587, pp. 2753-2766, 2013. 

Available at: https://doi.org/10.1016/j.febslet.2013.07.012. 

[25] J. Lehmann, M. C. Rillig, J. Thies, C. A. Masiello, W. C. Hockaday, and D. Crowley, "Biochar effects on soil biota - a 

review," Soil Biology and Biochemistry, vol. 43, pp. 1812-1836, 2011. Available at: 

https://doi.org/10.1016/j.soilbio.2011.04.022. 

[26] J. E. Amonette and S. Joseph., Physical properties of biochar,” In: Lehmann, J., Joseph, S. (Eds.) Biochar for environmental 

management. Science and Technology: Earthscan, London, 2009. 

[27] F. Verheijen, S. Jeffery, A. C. Bastos, M. Van Der Velde, and I. Diafas, Biochar application to soils: A critical scientific 

review of effects on soil properties, processes and functions,” EUR 24099 EN. Luxembourg: Office for the Official 

Publications of the European Communities. 

[28] R. T. K. Ariyawansha, B. F. A. Basnayake, D. M. G. P. Vijenayaka, N. S. Waduge, and D. A. S. Gamage, "Biochar 

applications for enhancing nutrient availability in tropical soils," in In: Proceedings of the International Conference on Solid 

Waste Innovation in Technology and Management, Hong Kong SAR, P.R. China, 2013, pp. 676–679. 

[29] C. Sundberg, S. Smårs, and H. Jönsson, "Low pH as an inhibiting factor in the transition from mesophilic to 

thermophilic phase in composting," Bioresource Technology, vol. 95, pp. 145-150, 2004. Available at: 

https://doi.org/10.1016/j.biortech.2004.01.016. 

[30] C. Dissanayake and R. Chandrajith, "Phosphate mineral fertilizers, trace metals and human health," Journal of the 

National Science Foundation of Sri Lanka, vol. 37, pp. 153-165, 2009. Available at: 

http://doi.org/10.4038/jnsfsr.v37i3.1219. 

[31] P. Jayathilake, I. Reddy, D. Srihari, and K. Reddy, "Productivity and soil fertility status as influenced by integrated use 

of n-fixing biofertilizers, organic manures and inorganic fertilizers in onion," The Journal of Agricultural Sciences, vol. 2, 

pp. 46-59, 2006. 

[32] B. Liang, J. Lehmann, D. Solomon, J. Kinyangi, J. Grossman, B. O'neill, J. O. Skjemstad, J. Thies, F. J. Luizão, and J. 

Petersen, "Black carbon increases cation exchange capacity in soils," Soil Science Society of America Journal, vol. 70, pp. 

1719-1730, 2006. Available at: https://doi.org/10.2136/sssaj2005.0383. 

[33] R. H. Socolow, "Nitrogen management and the future of food: lessons from the management of energy and carbon," 

Proceedings of the National Academy of Sciences, vol. 96, pp. 6001-6008, 1999. 

[34] P. Hepperly, D. Lotter, C. Z. Ulsh, R. Seidel, and C. Reider, "Compost, manure and synthetic fertilizer influences crop 

yields, soil properties, nitrate leaching and crop nutrient content," Compost Science & Utilization, vol. 17, pp. 117-126, 

2009. Available at: https://doi.org/10.1080/1065657X.2009.10702410. 

 

 

 

http://doi.org/10.4038/jnsfsr.v37i3.1219


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 APPENDIX  

Supplementary Materials 1  

Analysis of the biocatalyst production process with the application of biochemical transformation kinetics  

A formulism based on the unit productions of enzymes and reactants in cyclic events to comply with mass 

action law to form enzyme-substrate complexes was developed by Ariyawansha, et al. [23].  This formulism 

supports the successful application of Michaelis–Menten kinetics in all biochemical transformations of all single 

parameters.  So, we analyzed the initial stage of different environments using this model. 

Michaelis-Menten Equation 

Michaelis-Menten equation is given in Equation 1   

                                                    (1)                                      

                 (1) 

 

Where, v = overall rate of reaction, S = substrate, Km = Michaelis-Menten constant, vm = maximum rate of 

reaction.  

vm and Km was calculated for the experimental data of each environment by using Lineweaver-Burk reciprocal 

plot and Eadie-Hostess plot as given below.  

The Lineweaver-Burk double reciprocal plot is given in Equation 2. 

                      

mm

m

vSv

K

v

11
                               (2) 

VS concentration  of each catalytic reactor  at time t throughout the 1st experimental period were used for this 

kinetic analysis.  Cumulative VS concentration at time t was calculated and it was considered as P for the study. 

Then the substrate concentration was defined as: 

4
3

PS                                          (3) 

Then, overall rate of reaction was calculated using this equation. 

                             

                                                                              (4) 

Derivation of Enzyme Generations 

The cyclic nature of enzyme-substrate complexes and products can be used as the basis for developing the 

proposed scheme. The concept of a unit production to describe the cyclic behaviour of the reactions as shown 

Equation 5.  Equation 5  was used to calculate the generation and utilization of enzymes and enzyme-substrate 

complexes through analysis of path X and path Y as reported by Ariyawansha, et al. [23].  

 

S E + R

k1 k
1

k
r

E + S
k

1

k'
1

k
2

E + P

A B C

D

E

k
1

E
ex  

+ S
ex

 

Path X

Path Y

k
1

ES

k
2

  (5) 

 
t

t

dS
dvv

0

 
 SK

Sv
v

m

m






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27 

© 2021 Conscientia Beam. All Rights Reserved. 

where, R = reactant, k1,
'

1
k , and k2 = rate constants, path X = substrates from internal body entity, path Y = 

substrates from food and nutrients supply. 

Analysis of Path X - Internal Body Entity 

We do not frequently encounter the differential of v with respect to  S  because v   is defined as the 

overall rate constant, and there was no necessity to dwell with rates of changes of  v  and  S .  But it is the 

foundation of this analysis as reported by Ariyawansha, et al. [23] , since 

 Sfv 
                                                  (6) 

Substrate S can be simulated by using Equation 7 

  mmm KtKvS 
                                 (7) 

'
mv  and '

mk  at time t  was calculated by using Equation 8 and Equation 9.  

   S
t

dt

Sd
Km 










5.0

'
   (8) 

 
'

2

'

.
5.0

m

m

K
dtSd

t
v









    (9) 

Calculated '
mv  and '

mk  at time t  values were inserted  into Equation 10 to estimate overall rate of reaction of path 

X, v’.   

 
 SK

Sv
v

m

m




'

'
'

      (10) 

Then, the amount of enzymes and enzyme-substrate complexes generations from the path X was derived by 

using Equation 11, 12, and 13. 

 
2

'
'
0

k

v
E m      (11) 

 
2

'
'

k

v
ES       (12) 

     ''
0

' ESEE       (13) 

Path Y - External Food and Nutrients Supply 

By rearranging Michaelis-Menten Equation 1,  

 vfS 
     (14),  

 

 



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28 

© 2021 Conscientia Beam. All Rights Reserved. 

Equation 15 was obtained.  

 
 vv

vK
S

m

m


      (15)  

We can deduce the differential of  S  with respect to v, 
 
dv

Sd
 as given in Equation 16, which is equal to t 

according to Equation 4, thus we can obtain specific ''
mv  and ''

mK  values from;  

 
 

t
vv

Kv

dv

Sd

m

mm 



2''

''''

                               (16) 

Then, overall rate of reaction can be simulated by using following equation. 

t

Kv
vv mm

m

''''
'' 

    (17) 

The differential of Equation 17 is given in Equation 18;  

5.1

''''
5.0

t

Kv

dt

dv mm
      (18)   

Both ''
mv

 
and 

''
mK

 
at time t  can be  calculated from the following equations which were derived by rearranging 

Equation 18 as given in Equation 19 and Equation 20.  

 

v
dt

dvt
vm 








 .

5.0

''
     (19) 

 
''

2''
''

m

m
m

v

vvt
k


      (20) 

Calculated 
''

mv
 
and 

''
mK   at time t  values were inserted  into Equation 21 to estimate overall rate of reaction of path 

Y, v’’.   

 
 SK

Sv
v

m

m




''

''
''

     (21) 

Then, the amount of enzymes and enzyme-substrate complexes generations from the path Y can be derived by 

using Equation 22, 23, 24. 

 
2

''
''

0
k

v
E m      (22) 

 
2

''
''

k

v
ES       (23) 



Current Research in Agricultural Sciences, 2021, 8(1): 11-30 

 

 
29 

© 2021 Conscientia Beam. All Rights Reserved. 

     ''''
0

'' ESEE      (24) 

Total Generation of Enzymes 

The total generated enzymes from two pathways were calculated using Equation 25, 26, 27 

 

                                                        (1) 

 

                                                  (26)  

  

                                                        (27) 

 

 

Derivation of Combined Function  

As reported by Ariyawansha, et al. [23] that the two quadratic functions (Equation 7, 17) are responsible for 

the generations of enzymes, whereas, the hyperbolic function (Equation 1) expresses utilization of the enzymes.  In 

other words, the total of enzymes (      '''''' EEE  ) and enzyme complexes (      '''''' ESESES  ), thus  

     ''''''
ooo EEE   are the total generations giving another set of '''

mv  
and '''

mK ,  since 
'''''' vvv  .  As reported 

by Ariyawansha, et al. [23], their investigations in all cases, vv '''
 at all times, thus manifest inhibitions of the 

generated enzymes.  The inhibited values of rate of the reaction v comprise of both these avenues supplying 

enzymes and enzyme complexes that were formed.  Therefore, we can presume the existence of proportionate 

values of v from 
'v and 

''v  persisting in the production of enzymes  E
 
and  ES  complexes.  Such that; 

vvv        (28)  

Where,  

)( '''

'

vv

v


      (29) 

)( '''

''

vv

v


      (30)  

Where  and  values are proportionate values.  Therefore, proportionate values of ES , E , and oE  and 

ES , E , and 
oE can be found using Equation 28, 29, 30. 

Calculation of proportionate values of enzymes and enzyme-substrate concentrations  

In applying Equation 28, the proportionate ES complex can be found, where; 

 
2k

v
ES


       (31) and  

     ''''''
ooo EEE 

     '''''' ESESES 

     '''''' EEE 



Current Research in Agricultural Sciences, 2021, 8(1): 11-30 

 

 
30 

© 2021 Conscientia Beam. All Rights Reserved. 

     
 '

'

ES

ES
EE 

      (32)  

Thus giving  

      EESEo      (33).  

Similarly  

 
2k

v
ES


 

     

(34), 

     
 ''

''

ES

ES
EE 

 
    

(35) and  

      EESEo      (36). 

      ESESES      (37)   

      EEE      (38)  

      ooo EEE 
    

(39)   

 

Then, the variations of kinetic parameters, Eo, ES, E generations and utilization with time and environmental 

parameters of each pile were analyzed, interpreted to evaluate the performances of each catalytic production 

process.  

 

 

 

 

 

 

 

 

 

 

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