http://dx.doi.org/10.11594/jtls.13.03.19
How to cite:
Ramadhani SI, Ardyati T, Sjofjan O (2023) Screening of Cellulolytic Bacteria from Sugarcane Waste (Bagasse) and Optimi- zation of Cellulase Activity as Animal Feed.Journal of Tropical Life Science 13 (3): 607 – 614. doi: 10.11594/jtls.13.03.19.
Research Article
Screening of Cellulolytic Bacteria from Sugarcane Waste (Bagasse) and Optimi- zation of Cellulase Activity as Animal Feed
Sulistya Ika Ramadhani 1*, Tri Ardyati 1, Osfar Sjofjan 3
1 Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Brawijaya, Malang, Indonesia
2 Department of Animal and Feed, Faculty of Animal Husbandry, Universitas Brawijaya, Malang, Indonesia
Article history:
Submission March 2023 Revised April 2023 Accepted May 2023
ABSTRACT
The Sugar Factory of Tjoekir Jombang is one of the sugar factories located in Jom- bang, East Java under PT Perkebunan Nusantara X. Sugarcane waste, also known as bagasse causes pollution of the environment. Some microorganisms are able to degrade cellulose-containing bagasse, because of cellulolytic enzymes produced by cellulolytic bacteria. This study aims to obtain cellulolytic bacteria isolates, screen the highest cellulolytic activity, identify the highest cellulolytic activity iso- lates, and optimize conditions (pH and temperature) for the highest cellulase activ- ity. Cellulolytic bacteria from bagasse were grown on a medium containing 1%
CMC. Several additional minerals were other than those in the CMC medium. The cellulase activity was assayed semi-quantitatively with the addition of 0.1% Congo red and quantitatively using the 3,5-Dinitro Salicylic Acid (DNS) method. Bacte- rial isolates with high cellulolytic activity were identified based on the 16S rDNA sequence. This research obtained 20 bacterial isolates, where isolate A1T4 had the highest cellulolytic index of 1.18 mm. Measurement of cellulase activity using the DNS method showed that isolate A2T2 had the highest cellulase activity of 2.19 U/mL. Hemolysis assay showed that from 12 isolates, only two isolates have γ- hemolysis activity (isolates A1T6 and A3T3). Those isolates were optimized in a CMC broth medium with temperatures of 30°C and 37°C and pH 5 and pH 6 to produce the highest cellulase activity. Isolate A1T6 and A3T3 were grown opti- mally at 30oC and pH 6. Isolate A1T6 was identified as Citrobacter amalonaticus with a similarity of 99.80%, and isolate A3T3 was identified as Pseudomonas men- docina with a similarity of 98.83%.
Keywords: Bagasse, Cellulolytic bacteria, Cellulase activity
*Corresponding author:
E-mail: [email protected]
Introduction
The rapid development of industry in Indone- sia cannot be separated from the problem of envi- ronmental pollution. Indonesia is the largest sugar- producing country in the world; therefore, the re- sulting sugarcane waste is quite large. Sugarcane production 2021 reached 2.36 million tons and in- creased annually by 2.58% from the previous year [1].
Bagasse is one of the wastes produced by the sugar industry and includes the residue of the sug- arcane juice extraction process. About 50% of ba- gasse produced in each sugar factory is used as
fuel [2]. Bagasse is biomass containing cellulose, the most abundant renewable resource [3]. Cellu- lose is also one of the most common organic pol- ymers, about 30% of the plant composition. It is insoluble in water and difficult to degrade [4], which is by handling waste so that its utilization is more optimal.
Cellulase is an enzyme produced by microor- ganisms and plays an important role in the biodeg- radation of cellulose into simpler compounds [5].
The cellulase group include endo-1,4-β-glucanase, exo-1,4-β-glucanase, and β-glucosidase, which
synergistically hydrolyze cellulose to glucose [6].
Cellulolytic bacteria can degrade cellulose by pro- ducing extracellular enzymes, namely cellulase.
Several genera of bacteria that are capable of pro- ducing cellulase are Pseudomonas, Enterobacter, Bacillus, Klebsiella, Paenibacillus, Rhodococcus, Cellulomonas, Streptomyces, Citrobacter, Nocar- dia, Kurthia, Vibrio, Flavobacterium, Neisseria, and Micrococcus [7] [8].
Cellulolytic bacteria isolated from bagasse and their cellulolytic activity involved in cellulose degradation of bagasse, have not been studied yet.
Therefore, this research aims to obtain cellulolytic bacteria isolates, screen the highest cellulolytic ac- tivity, identify the highest cellulolytic activity iso- lates, and optimize conditions (pH and tempera- ture) for the highest cellulase activity.
Material and Methods
Sampling and physicochemical parameters measurement of sugarcane waste (bagasse)
Bagasse was taken from the sugar factory in- dustry located in Cukir, Jombang Regency, East Java. Samples were taken about 2500 g at the bot- tom of the bagasse heap with three replications each. Physicochemical parameters measurements included temperature, moisture content, pH, cellu- lose, organic matter, organic C, N total, and C/N ratio.
Isolation of cellulolytic bacteria
A 25 g bagasse sample was taken and sus- pended in 225 mL of 0,85% NaCl, and serial dilu- tion was carried out from 10-1 to 10-6 then, 0.1 mL of suspension was poured plated with 1% CMC agar medium. Samples were incubated at 30oC for 72 hours. The CMC agar medium (g/L) consisted of 10 CMC and 0.2 MgSO4. 7H2O; 4 KH2PO4; 0.004 FeSO4. 7H2O; 0.001 CaCl2. 2H2O; 4 Na2HPO4; 4 yeast extract, and 15 agar with a pH of 6 [9].
Screening of cellulolytic bacteria semi-quantita- tively assay of cellulolytic bacteria
The purified bacterial isolates were inoculated into 20 mL of 1% CMC broth with optical density (OD600 = 0.6). A 100 µL of bacterial culture was added to a blank disk (0.5 mm). After that blank disk put on to 1% solidified 1% CMC agar me- dium and then incubated at 30oC for 72 hours.
After incubation was completed, the agar medium was flooded with 2 mL of 0.1% Congo red reagent for 15 minutes, then rinsed with 1 M NaCl. The clear zone around the colony was measured using a caliper [10]. Calculation of the cellulolytic index was determined by:
Cellulolytic Index = clear zona diemeter−colony diameter Colony diameter
Cellulase activity assay
The purified bacterial isolates capable of pro- ducing clear zones in CMC agar medium were prepared for cellulase activity assay. Each bacte- rial isolate (OD600 = 0.6) was grown on 20 mL 1%
CMC broth medium and incubated at rotary shaker 30°C, 120 rpm for 72 hours. The bacterial culture was then centrifuged at 4°C at 10,000 rpm for 10 minutes. 200 µL of crude Enzyme was in- cubated in 1800 µL 50 mM phosphate buffer (pH 7) at 50°C for 30 minutes. The reaction was stopped by adding 2 mL of DNS solution, then boiling at 100°C for 5 minutes [11]. The sample was cooled at room temperature, and the absorb- ance value was measured at a wavelength of 540 nm [12]. Cellulase activity that has been converted to the glucose standard curve is determined by:
AE = 𝐶
𝐵𝑀 𝐺𝑙𝑢𝑘𝑜𝑠𝑎 × H note: 𝐸
AE = Enzyme activity (U/mL) C = Glucose concentration BM Glucose = 180 g/mol
H = Total volume of substrate (mL) E = Enzyme volume (mL)
Hemolysis assay
One ose of bacterial isolates was streaked on a blood agar medium and then incubated at 30°C for 24 hours. If a clear zone around the colony was observed, the bacteria could lyse the blood cells in the medium [13]. This hemolysis assay aims to de- termine the pathogen in bacteria.
Optimization of different culture parameters measurements for cellulase activity
The bacterial isolates that were unable to lyse red blood cells will then be cultured at 30oC and 37oC, pH 5 and 6. Cellulase activity was measured at a wavelength of 540 nm.
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Identification of bacteria based on 16S Rdna Bacterial chromosomal DNA was extracted using the Quick-DNATM Kit (ZYMO RE- SEARCH, USA). The 16S rDNA sequence was amplified by Polymerase Chain Reaction (PCR) using the universal primers 27f (5'-AGAGTTT- GATCCTGGTCCAG-3') and 1492r (5'- GGTTACCTTGTTACGACTT-3') in the PCR program: pre-denaturation at 94oC for 5 minutes, followed by denaturation at 94oC for 30 seconds, annealing at 55oC for 30 seconds, and extension at 72oC for 30 seconds with 35 cycles and final elon- gation at 72oC for 5 minutes. Amplicons of 16S rDNA were confirmed by 1.5% agarose gel elec- trophoresis and visualized using a UV transillumi- nator. A 16S rDNA amplicon was purified and se- quenced at First BASE, Malaysia. Bacterial 16S rDNA sequences were aligned to reference strains from the GenBank database using the MEGA 11 program with the Neighbor-Joining algorithm and the Tamura-Nei model with bootstrap 1000 [14].
Data analysis
Semi-quantitatively data of cellulolytic bacte- ria and cellulase enzyme activity test were ana- lyzed based on One-Way ANOVA. The results of the optimization of cellulase activity were ana- lyzed based on Two-Way ANOVA. Followed by the Tukey test using the SPSS 20 program.
Results and Discussion
Physicochemical parameters measurements of bagasse and isolation of cellulolytic bacteria
Research on cellulolytic bacteria from bagasse obtained 20 isolates with a bacterial cell density of 2.7 × 105 CFU/g. Several physicochemical param- eters influence the abundance of these bacteria (Table 1). Temperature can affect the speed of
growth of microorganisms. The increased produc- tion of organic acids causes the pH value. Mois- ture content is important in increasing the rate of respiration in the form of CO2 by microorganisms.
Organic matter plays an important role in the pH and abundance of microorganisms. Organic C and N total levels include macromolecules that have a structural and functional role in components of bacterial cells as well as a food source for micro- organisms. Cellulose is the largest component in plants, especially bagasse, and the amount is very abundant.
Screening cellulolytic bacteria semi-quantita- tively assay of cellulolytic bacteria
The cellulolytic index showed that 12 bacterial isolates were able to produce clear zones. There are three potential isolates consisting of A1T4, A1T7, and A1T6 with cellulolytic indices of 1.18;
0.95; and 0.83 mm (Figure 1). Other studies re- lated to cellulolytic bacteria from termite intesti- nes produced clear zones with a range of 1.07-1.32 mm [15]. Cellulose contained in the medium will Table 1. Bagasse physicochemical parameters and
bacterial abundance
Parameters Value
Temperature (°C) 30 ± 0
pH 6.18 ± 0.15
Water content (%) 8.95 ± 0.52 Organic matter (%) 71.95 ± 0.62
C/N Ratio 72.42 ± 2.75
C-organic matter (%) 41.75 ± 0.36
N total (%) 0.57 ± 0.01
Cellulose (%) 42.70 ± 0.30
Density of cellulolytic bacteria
(CFU/g) 2.7 × 105
Figure 1. Cellulolytic index. Different notations show significant differences between bacterial isolates and cellulolytic index (p <0.05)
abcd abcde f
def ef
bcde bcde abc
a
cdef bcde
ab 0.0
0.2 0.4 0.6 0.8 1.0 1.2 1.4
Cellulolytic index
Isolates
be hydrolyzed due to cellulase enzymes produced by bacteria. The hydrolysis process has a clear zone in the medium due to the reaction between Congo red and the 1,4-glycosidic contained in the cellulose polymer [16]. The principle of this stain- ing is that the dye will diffuse into the agar me- dium, where the larger the clear zone produced, the higher the solubility level of an enzyme [17].
The difference in the value of the cellulolytic in- dex is due to the ability of each isolate to hydro- lyze the cellulose contained in the medium due to the secretion of the enzyme endo-β-1,4-glucanase (CMC-ase) produced by cellulolytic bacteria by breaking the β-1,4 glycoside bond in the medium CMC [18].
Cellulase activity assay
The results of cellulase enzyme activity showed three potential bacterial isolates consist- ing of A2T2, A1T4, and A1T6, respectively 2.19;
1.60; and 1.57 U/mL (Figure 2). Other research
related to cellulase activity from sugar bioethanol fermentation resulted in the highest endoglucanase activity of 1.56 U/mL in isolate S-6 [19]. Extracel- lular cellulase enzymes can break down β-glyco- sidic bonds from cellulose to glucose, which these bacteria use as a carbon source for growth and de- velopment [20] [21]. The cellulase activity was tested using the Dinitro Salicylic Acid (DNS) method [22].
Hemolysis assay
This assay is important to determine the cellu- lolytic bacteria obtained are pathogenic. Hemoly- sis testing of 12 potential bacterial isolates was performed using a blood agar medium to obtain two bacterial isolates with the codes A1T6 and A3T3 (Table 2). The results of this reaction show γ-hemolysis, which does not lyse red blood cells by indicating that there is no clear zone around the colony, so it is possible that the two isolates do not have pathogenic properties against living things [23].
Optimization of different culture parameters for cellulase activity
The isolates were selected based on the results of the hemolysis assay, where isolates A1T6 and A3T3 were unable to lyse red blood cells (γ-he- molysis). Cellulase activity test on bacterial isolate A1T6 at 30°C pH 5 was 1.33 U/mL and 30°C pH 6 was 1.50 U/mL. At a temperature of 37°C pH 5 is 1.32 U/mL, and a temperature of 37°C pH 6 is 1.42 U/mL The optimum bacterial isolate A3T3 produced cellulase activity at a temperature of 30°C pH 5 of 1.54 U/mL and a temperature of 30oC pH 6 of 1.70 U/mL. At 37°C pH 5 was 1.55 Table 2. Hemolysis Assay of Cellulolytic Bacteria
Isolates Hemolysis Assay
A1T1 β-hemolysis
A1T2 β-hemolysis
A1T4 β-hemolysis
A1T6 γ-hemolysis
A1T7 β-hemolysis
A2T1 β-hemolysis
A2T2 β-hemolysis
A2T3 β-hemolysis
A2T4 β-hemolysis
A2T6 β-hemolysis
A2T7 β-hemolysis
A3T3 γ-hemolysis
Figure 2. Cellulase activity of cellulolytic bacteria. Different notations show significant differences between bacterial isolates and cellulase activity (p<0.05)
a a
ab ab
bc ab
c
a a a ab
a
0.00 0.50 1.00 1.50 2.00 2.50
Cellulase activity (U/mL)
Isolates
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U/mL, and at 37°C pH 6 was 1.60 U/mL (Figure 3). Temperature and pH influence enzymatic reac- tions. Cellulase enzymes are able to work opti- mally at a temperature range of 20–50oC and in- crease the speed of the chemical reaction of the en- zyme, but a high temperature can cause denatura- tion of the enzyme [24]. Neutral pH (pH 5-7) changes in pH affect enzyme activity through in structure of amino acids that function in binding to substrates [25].
Species of cellulolytic bacteria
Cellulolytic bacterial isolates that did not lyse red blood cells from bagasse were identified based on 16S rDNA sequences. The code bacteria A1T6 was identified as Citrobacter amalonaticus with a similarity value of 99.80% (Figure 4). Citrobacter amalonaticus is a bacterial that has rarely been re- ported as a human pathogen and samples from hospitalized patients [26].
Figure 3. Optimization of temperature and pH of cellulase activity. Different notations show the interaction of temperature and pH on cellulase activity (p<0.05)
Figure 4. Phylogenic tree of isolate A1T6 based on similarity of 16S rDNA sequences according to the Neigh- bor Joining method with the Tamura-Nei algorithm and 1000 times boostrap
a
b
a b a
b a
a
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
5 6 5 6 5 6 5 6
30°C 37°C 30°C 37°C
A1T6 A3T3
Cellulase activity (CFU/mL)
pH Temperature
Isolates
The A3T3 code identified Pseudomonas mendocina with a similarity value of 98.83% (Fig- ure 5). Pseudomonas mendocina is a cellulolytic bacteria isolated from termite intestines [27] and isolated from rotting straw samples in China [28].
Pseudomonas mendocina exhibited biodegradative capabilities of 95% in filter paper degrading bacteria from the guts of Coptotermes formosanus [29].
Conclusion
Cellulolytic bacteria from bagasse have the potential to degrade cellulose. There are 20 iso- lates and code A1T4 has the highest cellulolytic index of 1.19 mm. However, the highest cellulase activity was 2.20 U/mL in A2T2 bacterial isolates.
Bacterial isolates were unable to lyse red blood cells in A1T6 and A3T3. The optimum condition isolates A1T6 at 1.50 U/mL and A3T3 at 1.70 U/mL at 30°C and pH 6. The A1T6 code identified Citrobacter amalonaticus with a similarity 99.80%
and bacterial isolate A3T3 as Pseudomonas men- docina with a similarity of 98.83% and it has po- tential to fermentate bagasse and rice bran to ani- mal feed.
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