Microbial xylanase and pectinase are two extremely valuable enzymes, which have captivated much attention. This can be seen from the increased demand for these enzymes by many industrial sectors. This study investigates the isolation and screening of extracellular xylanopectinolytic enzymes-producing bacteria in a submerged fermentation (SmF). Samples are collected from the compost of empty fruit bunch (EFB) at Biocompost Pilot Plant, located at Biorefinery Plant, Universiti Putra Malaysia. From the experiment, out of 20 isolates, 11 isolates show xylanase or/and pectinase activity, and only one isolate (EFB-11) shows the concurrent activities of xylanase and pectinase. These activities are selected for enzyme production under submerged fermentation (quantitative screening). At the 72nd hour of incubation, xylanase and pectinase show the highest production, which ranges about 42.33 U/mL and 62.17 U/mL (with low amount of cellulase present), supplemented with 2% (w/v) of rice bran as carbon source at incubation temperature level, which is 30°C. Meanwhile, the pH of media is shifted to 8.42, which indicates that EFB-11 isolate is alkalotolerant bacteria and identified as
“Environment-friendly” technologies are demanded for industrial processes. The use of enzymes, especially, is gaining momentum in several frontier developments. Furthermore, xylanase and pectinase are the enzymes which are vital in the industrial sector, and they are widely used in the paper and pulp industry, animal feed, textile industry, oil extraction, tea and coffee fermentation, waste paper recycling, and the fruit juice industry [
One of the potential applications of xylanolytic and pectinolytic enzyme system is found in the pulp and paper industry, in which their enzymatic actions are involved in the removal of xylan and pectin from kraft pulp fiber. This consequently increases the brightness of pulp [
In order to degrade pectin on the pulp fibers, the treatment of pectinase is required, in order to weaken the chemical bond between cellulose and lignin before their further refinement during chemical bleaching. Pulp-treated pectinases introduce the separation of pulp microfibrils and pulp fibers, compared to the smooth surfaces of untreated pulp. Additionally, they also promote efficient loosening and swelling of pulp fibers, greater porosity, and loss in the compactness of the pulp fibers, when observed under scanning electron microscope (SEM). This indicates that only a low amount of chlorine compound is required, since enzymatic treatment renders the pulp fibers more accessible to chemical bleaching [
The replacement of conventional pulp bleaching could become a new fiber liberation technology in pulp and paper industry. This is because it uses high amount of chemical additives. Besides that, the additives involve the use of enzymatic bleaching, which is ecofriendly and cost-effective and has a product yield. Both xylanases and pectinases play an important role in the bleaching practice, which is the earliest step in the process of papermaking [
In general, bacterial-producing xylanopectinolytic enzymes can be found in environments rich in lignocellulosic and hemicellulosic material as their microbial sources. Examples of microbial sources are soil-enriched compost area, rotten and decayed wood, and agroresidual wastes [
Samples were collected from the compost of empty fruit bunch (EFB) at Biocompost Pilot Plant, which was located at Biorefinery Plant, Universiti Putra Malaysia, with surrounding temperature of 30°C.
A tenfold serial dilution (101 to 107 dilutions) was performed in 10 mL scale. 1 mL of each sample of the dilution was added into 9 mL of sterile 0.85% (w/v) NaCl solution, and it was serially transferred before getting thoroughly mixed. The amount of each serial dilution was 0.1 mL aliquots, and it was spread on a plate using hockey stick on sterile nutrient agar. Then, the dilution was incubated for 24 hours at a temperature of 30°C [
Primary screening was conducted on xylan agar medium and pectin agar medium. Basal agar medium was used for the screening of xylanopectinolytic enzymes-producing bacteria (g/L: peptone, 5; yeast extract, 5; MgSO4
Bacterial suspension (grown 24 hours in nutrient broth) samples (100
The plates were flooded with Gram’s iodine solution [
Following primary screening, the bacteria cultures were further narrowed down by secondary screening in submerged fermentation. Substrate (rice bran) was used as the main carbon source, while other components of media (basal medium) remained the same.
In secondary screening, the isolates were carried out in 250 mL Erlenmeyer flasks containing 50 mL of basal medium. The basal medium was supplemented with 1% (w/v) of rice bran and incubated with 1% (v/v) of inoculums (18 h old, OD ~ 0.85), subjected to shaking condition (200 rpm) for a period of 144 hours at ambient temperature (30°C). Crude xylanopectinolytic enzymes were harvested every 25 hours, and they were used for the measurement of pH and the optical density of bacterial growth. Meanwhile, the cell supernatant (treated as crude enzyme) was used for xylanase, pectinase, and cellulose assay, protein estimation, and reducing sugar quantification. The secondary screening studies were tested in triplicate.
All the analytical methods were performed including the determination of enzymes assay, reducing sugar, and soluble protein as described by Bradford (1976).
The enzyme activities were measured according to the method described by Miller (1959). Substrates used in assaying activity of xylanase, pectinase, and cellulase were 1% (w/v) of beechwood xylan, 0.5% (w/v) of polygalacturonic acid, and 1% (w/v) of carboxymethyl cellulose, respectively. The reaction mixture which was 490
Enzyme activity is expressed in one unit and defined as the release of one
The selected isolate was grown in nutrient broth for 24 hours at 30°C, with shaking at 200 rpm. The extraction of DNA was done by using G-spin™ Genomic DNA Extraction Kit, provided by iNtRON Biotechnology, Korea. The extracted genomic DNA was purified using FavorPrep™ Genomic DNA Mini Kit (Favorgen Biotech Corp., Taiwan). A 16S rDNA fragment was amplified using i-Taq™ plus DNA Polymerase (iNtRON Biotechnology, Korea) with the universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′) [
The soil samples collected in this study consisted of soil from the compost (decayed organic materials) in Biocompost Pilot Plant, which was located at Biorefinery Plant, Universiti Putra Malaysia, Serdang, Malaysia.
In this study, the soil samples near compost sites were targeted because the xylanase and pectinase-producing microbes have been frequently reported to be isolated from the agricultural wastes and soils. These are where the hemicellulose materials were deposited. Besides that, the compost sites selection was also due to the nutrient rich environments [
The cultures were grown on nutrient medium and 20 isolates, with distinctive macroscopic morphology being selected and preceded with Gram’s stain. This was to verify the purity of a culture, instead of determining the Gram-negative or Gram-positive bacteria and their cellular morphologies. The cultural characteristics of 20 isolates are shown in Table
Morphological characteristics of isolates.
Isolate number | Cultural characteristics | Gram’s nature |
---|---|---|
EFB-1 | Flat, circular, entire | Gram-positive, |
EFB-2 | Convex, circular, entire | Gram-positive, |
EFB-3 | Flat, circular, entire | Gram-positive, |
EFB-4 | Convex, irregular, entire | Gram-positive, |
EFB-5 | Convex, circular, undulate | Gram-positive, |
EFB-6 | Flat, irregular, entire | Gram-negative, |
EFB-7 | Convex, irregular, entire | Gram-positive, |
EFB-8 | Convex, circular, entire | Gram-positive, |
EFB-9 | Convex, irregular, entire | Gram-negative, coccus |
EFB-10 | Convex, irregular, undulate | Gram-negative, coccus |
EFB-11 | Umbonate, circular, entire | Gram-positive, |
EFB-12 | Convex, circular, entire | Gram-negative, coccus |
EFB-13 | Convex, irregular, entire | Gram-positive, |
EFB-14 | Convex, circular, entire | Gram-positive, |
EFB-15 | Flat, irregular, entire | Gram-negative, |
EFB-16 | Umbonate, circular, entire | Gram-positive, |
EFB-17 | Convex, circular, entire | Gram-negative, coccus |
EFB-18 | Convex, circular, undulate | Gram-negative, coccus |
EFB-19 | Convex, circular, entire | Gram-positive, |
EFB-20 | Flat, irregular, undulate | Gram-positive, |
EFB-11 is a rod-shaped Gram-positive bacteria with a violet color of Gram’s stain. Based on the microscopic view of Gram’s stain slide, the constant identical features of bacteria’s shapes and colors demonstrated that the pure colony of EFB-11 was obtained from isolation techniques.
Twenty bacteria isolates were successfully inoculated. The primary screening of xylanopectinolytic enzymes-producing bacteria was performed and resulted in the formation of clearance zone on xylan and pectin containing agar. The primary screening, which resulted in 11 isolates, displayed the zone of clearance on either xylan agar medium or pectin agar medium. However, only one isolate showed the concurrent reaction of zone clearance in xylan and pectin agar medium (Table
Primary screening of selected isolates on agar plate with clear zone diameter.
Isolate number | Clear zone size (mm) | |
---|---|---|
Xylan agar | Pectin agar | |
EFB-1 | nd | 14 |
EFB-6 | nd | 15 |
EFB-9 | nd | 14 |
EFB-12 | nd | 13 |
EFB-15 | nd | 14 |
EFB-3 | nd | 22 |
EFB-8 | 17 | nd |
EFB-9 | 13 | nd |
EFB-14 | 16 | nd |
EFB-19 | 16 | nd |
EFB-11 | 25 | 36 |
|
21 | 32 |
EFB-11 displayed higher clearance zone diameter for xylanase and pectinase (25 mm and 36 mm of size, resp.), as compared to the control-type strain
Even so, this result was not fully expressed during submerged fermentation (SmF) cultivation [
Culture broth’s pH was measured at every 24-hour interval, and the clear supernatant was used for the estimation of enzymes’ activity [
Growth profile and enzymes production by EFB-11 from initial screening.
Period of maximum enzyme production (hours) | Optical density | pH | Composition and enzymes activity of cell-free supernatant harvested at the hour of maximum enzyme production | ||||
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Soluble protein (mg/mL) | Reducing sugar (ug/mL) | Xylanase activity (U/mL) | Pectinase activity (U/mL) | Cellulase activity (U/mL) | |||
0 |
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24 |
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48 |
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72 |
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96 |
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120 |
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144 |
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Values are mean of triplicate ± standard deviation.
Catabolite repression on enzymes production apparently occurred at the hour of maximum enzyme production, which resulted in lowering of the reducing sugar levels. There are several reports regarding the catabolite repression of xylanase [
After 72 hours of growth, the amount of xylanase and pectinase activity began to slightly decline. It was possible that these enzymes were digested by protease released by the autolysis of cells [
During fermentation period, protein concentration did not fluctuate much in the culture medium. However, it slightly declined after 120 hours of fermentation period, while the pH of medium shifted towards alkaline values by 144 hours of growth. Compared to any other industrial applications, especially those related to pulp and paper industries, the usage of alkaline pH enzymes is preferable and very important properties compared to fungal enzymes, that is, slightly acidic, require extra processes in subsequent steps. This causes them to be less suitable for practical applications, as the requirement of its cost also increases. The presence of cellulase in preparation of crude enzyme could affect cellulose fibers. The negative effects on cellulase could destroy the cellulose structure, which results in serious viscosity drop of pulp due to cellulose hydrolysis. Consequently, paper quality will possibly be diminished [
Since EFB-11 was
Hence, the xylanase and pectinase production, which was displayed by isolate EFB-11, was comparable with the previous reports. Their production could be further increased by the optimization of fermentation conditions.
In NCBI database, BLAST showed significant alignments of top three highest similarities between
The construction of a phylogenetic tree (Figure
Phylogenetic relationship of (Δ)
Agricultural waste or compost (decayed organic materials) area has high populations of microorganisms, and it could serve as a source of microorganisms which produces xylanase and pectinase enzymes. These results indicated that xylanopectinolytic enzymes-producing microorganism can also be screened in different environments. The screening of the total of 20 isolates from the compost resulted in 11 isolates with xylanase or pectinase enzyme-producing bacteria. From the 11 isolates, only one isolate was found to be xylanase and pectinase enzymes producer. The mentioned isolate was identified as
Submerged fermentation
Solid state fermentation
Empty fruit bunch
The authors declare no conflicts of interest.
Muhammad Hariadi Nawawi and Wan Zuhainis Saad participated in manuscript preparation and data analysis and Rosfarizan Mohamad and Paridah Md. Tahir assisted in designing the research.
This research was financially supported by the ScienceFund grant from MOSTI, Malaysia.