Isolation of alkali-thermotolerant cellulolytic bacteria from spent mushroom substrate

In this study, the isolation of new alkali-thermophilic bacteria with enhanced cellulase producing ability from the spent mushroom substrate was conducted. Repeated streaking was performed on the Microcrystalline-cellulose (MCC) to obtain a pure culture. Two thermophiles bacteria isolates were used for subsequent experiment. Morphological characteristic by gram staining and endospore staining also genetic identification of 16S rDNA was performed and the growth profile had been generated using Polymath Software. Filter paper assay (determination of enzyme activity), Bradford protein concentration were used in plotting the growth associated product curve. Based on the 16S rDNA sequencing results, the bacterium strains of Anoxybacillus geothermalis, incubated at 50˚C, pH 7 and Aeribacillus pallidus, incubated at 60˚C, pH 8 was successfully isolated. Both strains are gram-positive and endospore-forming bacteria. Based on the growth curve, the doubling time for Anoxybacillus geothermalis was calculated to be 49.5 minutes. Whereas, doubling time of Aeribacillus pallidus was 56.7 minutes. Both strains represent the growth assosiated product curve in which, when product formation is growth associated the specific rate of the product formation increases with specific growth rate and vice versa.


Introduction
Cellulosic or lignocellulosic compound is the organic matters that can be found abundantly in plants.
Microorganisms that producing cellulose enzyme are capable of hydrolyzing the cellulosic materials [1]. Cellulose is the components that makes of plants' cell wall. It is presumably the most plenteous organic compound on the earth since it is made by all plants [2]. Cellulose, (C6H10O5)n is a polysaccharide comprising a linear chain of several hundred to many thousands of β(1→4) linked Dglucose units [3]. It has numerous uses over being the primary building material for plants by its nature to make paper, explosives, film, and plastic depending on how it is treated [3].
Cellulose is a water-insoluble compound and does not deliquesce effectively in water. It is chiral in structure and is biodegradable. It can be separated chemically into glucose units by treating it with concentrated mineral acids at high temperature or can be hydrolyzed by the enzyme, cellulase [4]. Those microorganisms that have cellulase will degrade the cellulose into glucose. Cellulase is being generated by cellulolytic microorganisms which include yeast, bacteria, fungi, and actinomycetes. 1 To whom any correspondence should be addressed Primitively, fungi are the precedence once due to its capability to produce copious amounts of cellulases, which are less complex, can be easily extracted and purified than bacterial glycoside hydrolases. However, due to certain benefits such as higher affinity and feasibility towards genetic engineering, a higher growth rate of bacteria, and its synergy of their complex enzyme system, bacteria are preferred over fungi [5].
A spent mushroom substrate (SMS) is the co-product that was produced in almost unconfined amount after done harvesting the mushrooms, either in the production of white button mushroom (Agarius bisporus), shiitake (Lentinula edodes) or oyster mushrooms (Pleurotus spp.). The mushroom cultivating process had mass-produced the compost of organic medium, spent mushroom substrate. The term "spent mushroom substrate (SMS)" or in other words, it's also being called "spent mushroom compost (SMC)" portraying the agro-residues and fungal mycelium left after harvesting of mushrooms [6]. Spent mushroom substrate typically rich in cellulosic compound [7]. Thus, spent mushroom substrate is a good source for cellulose exploration.
The present study is focused on the isolation of alkali-thermotolerant cellulolytic bacteria from spent mushroom substrate. Alkaliphile means the ability of some groups of the bacteria that are capable of survival in alkaline environment, roughly at pH 8.5 to 11. These extraordinary adaptation mechanisms are exhibited by certain types of bacteria only. Thermotolerant is the ability of the microorganism to grow at the higher array of temperature. Thermophiles have cell membranes that are drenched with fatty acids. Thus it provides a hydrophobic atmosphere for the cell to maintain the cell rigidity at high temperatures [8]. From the biotechnological judgment, the dual properties of alkalophile and thermotolerant capability is attractive for applications such as household laundry detergents where enzymes and additives need to be active in callous washing conditions for improving fabrics softness and brightness [9].

Sample collection
Approximately 50 g of spent mushroom substrate was collected using a sterile microcentrifuge tube from Kaki Bukit, Perlis. The collected sample was sealed, labelled in right manner and stored at 4˚C for further use.

Isolaion of bacteria
Approximately 5 g of spent mushroom substrate was added in 150 mL of sterile deionized water. After that, 3 mL from the solution was transferred to 97 mL of MCC broth. The sample was incubated for 2 days at 2 different temperatures, 50˚C and 60˚C. Subsequently, 100 µL of the sample was pipetted and spread on MCC agar of different pH. One with original concentration and the other with 10x dilution. Then, both plates were incubated in the oven for 2 days at temperature 50˚C and 60˚C respectively. A single colony grown on spread plates was picked and streak repeatedly until obtaining the pure colonies. The plates of pure colonies were kept at 4˚C for preservation as well as further identification and characterization of cellulose-degrading bacteria. The isolates from the plate B2(2)-temperature

Morphological and genetic identification
There are three test were conducted under morphological which are colony appearance, gram staining, and endospore staining, then the isolates was sent for analysis of 16S rDNA identification [10]. Colony appearance was performed by observing the bacteria under microscope to study its elevation, opacity, edge, margin and color [11]. Gram staining was performed according to its distinctive principle of applying crystal violet, mordant, ethanol and lastly safranin [12]. While endospore staining also was performed accordingly by applying Malachite green then washed with water then safranin was applied [13].

Reagent preparation
Concentration of 0.05 M of citrate buffer was prepared by dissolving 5.25 g of citric acid monohydrate in 500 mL of distilled water. The pH was adjusted to pH 4.8. While, for preparation of DNS reagent, ten grams of sodium hydroxide (NaOH) was added to 1 L of volumetric flask. Subsequently, 10 g of dinitrosalicyclic acid (DNS), 2 g of phenol, 0.5 g of sodium sulphide and 500 mL of deionized water were added and stirred until all of the compounds dissolved. Finally, 200 g of potassium sodium tartrate was added and the deionized water was topped up to 1 L. This preparation was done under dark condition and stored in an amber bottle.

Growth profile construction
This being done by using two types of broth in which nutrient broth and CMC broth. Three percent of inoculums, was pipetted into each of 97 mL nutrient and CMC broth in a 250 mL flask. The flask then was incubated at 50°C for pH 7 and 60˚C for pH 8 at 150 rpm for 24 hr. Sampling was done for every 1 hour (for nutrient), 3 hours each (for CMC) and absorbance of 600 nm was determine using a UVvis spectrophotometer. The supernatant in CMC broth was indicated as crude enzymes. These crude enzymes were used in cellulase activity and protein content quantification experiments.

Quantification of cellulase activity
Cellulase activity was assayed by the determination of reducing sugar released from Filter Paper. The substrate was prepared by cutting the filter paper to 1.0 cm X 6.0 cm. Then, 1 mL of 0.05 M sodium citrate buffer with pH 4.8. was added to a test tube. Supernatant was collected after centrifugation of the culture from previous experiment at 10,000 rpm for 10 minutes. The supernatant indicated as the crude enzyme. An aliquot of 0.5 mL of crude enzyme then was transfered to different test tube. The substrate then was added into the test tube and incubated at 50˚C, 60 minutes. To stop the reaction, 3.0 mL of 3,5-dinitrosalicylic acid (DNS) was added. The test tube then was placed in a water bath at 95˚C for 5 minutes and was transferred into the cold water bath for 2 minutes. To measure the absorbance, 0.2 mL of the mixture was pipetted out to another new 14 mm of test tube and, 2.5 mL of distilled water was added. An aliquot of 1.0 mL of the diluted mixture was transferred to a cuvette and its absorbance was measured at 540 nm [14].

Quantification of protein content
Protein Bradford test used BSA (Bovine Serum Albumin) as standard and 30 µl of crude enzyme was transferred into test tube . Then,1.5 mL of Bradford Reagent was added into the solution. After 5 mins, 1 mL of each solution was pipetted into cuvette and the absorbance was read at 595nm.

Growth profile construction using POLYMATH software
The data from the nutrient broth from exponential phase region was used and was tabulated in the software for calculating of regression model used [15].

Isolation and screening of alkali-thermotolerant cellulolytic bacteria
From the isolation steps of spread and streak plate procedures, different colonies were identified and further streaked to obtain a single colony. After morphological identification, there were five plates of single colony obtained in which incubated at their respective condition of 50˚C, pH 7 (B2-2, A1), pH 7, 60˚C (A1-2, B) and 60˚C, pH 8 (A2). MCC agar plate was used from the beginning as a selective medium in which only cellulase producers can grow. The screening results based on filter paper assay revealed that sample 1 (B2-2) has liberated the high glucose content, (0.26 mg/0.5 mL at 12hrs sampling compared to other samples. Only one isolate has been obtained from conditions at 60˚C, pH 8. Based on the cellulase activity results, strain B2-B and A2 have been selected for the subsequent experiments.  Figure 1 and Figure 2 shows the dynamic of bacterial growth by plotting the graph of cell concentration versus time of incubation (h). Generally, bacterial growth curve is in sigmoid curve, and Anoxybacillus geothermalis (Figure 1) present the exact sigmoid curve. However, the graph observed for Aeribacillus pallidus (Figure 2) is in unsymmetrical sigmoid shape due to some constrain such as nutrient in the medium, temperature and pH of the nutrient broth itself [19]. This growth curve had been constructed successfully using alkalo-thermotolerant cellulolytic bacterium as it is incubating at 50˚C, pH 7 and 60˚C, pH 8. The data from exponential phase was further analyzed using Polymath software in order to measure its growth accurately.

Quantification of cellulase activity and protein content
The Figure 3 and Figure 4 shows the graph of growth assosiated product curve for all of the analysis performed. The result obtained indicate that when product formation is growth associated the specific rate of the product formation increases with specific growth rate and vice versa.   Figure 4. The graph of the cell concentration, cellulase activity of filter paper assay, protein concentration versus time, h for Aeribacillus pallidus incubate at 60˚C, pH 8.
According to the graph ( Figure 3) for Anoxybacillus geothermalis incubate at 50˚C, pH 7, and ( Figure 4) for Aeribacillus pallidus incubate at 60˚C, pH 8, increasing in the number of cell concentration resulting in increasing of protein concentration and cellulase activity over time. The highest value of the three test for both strains was in 18hr of incubation time, with 0.0766 mg/mL and 0.1348 U/mL of Anoxybacillus geothermalis and for Aeribacillus Pallidus, 0.0675 mg/mL and 0.0397 U/mL for its protein concentration and cellulase activity respectively. The graph verified that, product formation is growth associated the specific rate of the product formation increase with increasing of specific growth rate and vice versa. However, Anoxybacillus geothermalis was more stable compare to Aeribacillus pallidus as it cell concentration plotted, or bacterial growth curve was in symmetrically sigmoid curve. Radchenkova,N et al., (2013) had reported that the time for reaching maximum productions of exopolysaccharides (cellulase) for Anoxybacillus sp. is at 15hr and Aeribacillus sp. is at 20hr [16]. The less the time for maximum production, the more the product. The lag phase of the growth profile also shorter [20]. Thus, Aeribacillus pallidus isolated from spent mushroom substrate are more stable as it has shorter time to reach maximum activity. Meanwhile, Anoxybacillus geothermalis, its activity is a bit slower (less productive) and it may due to the different species of bacteria and also from previous study, it was isolated from hots spring in Bulgaria and the Anoxybacillus geothermalis was isolated from spent mushroom substrate at mild conditions.

Growth profile studies of growth using Polymath software
In this research, the growth of bacteria was studied using Polymath software. The absorbance reading from growth profile were used in this software to generate a non-linear graph based on selected equation. From the generated graph which shown in Figure 5 and Figure 6, the value inserted near linear line and this indicated the accuracy of the data based on equation provided (Eq.1). The rectangular pattern in the graph represent the inserted equation for the kinetic growth of non-linear pattern. On the other hand, round pattern represents the value from the absorbance which has been inserted into polymath software. For Anoxybacillus Geothermalis, the initial concentration was 0.0273 and its specific growth rate was determined to be 0.84 hr-1. A regression plot ( Figure 5), was established and its shows two lines. Xt calc represents the concentration of the biomass at the specific time calculated based on the equation of non-linear model (Equation 1). Meanwhile, the Xt exp indicates the experimental values obtained for the cell concentration. The plot was established by using the linear regression (R2) valued which was computed to be 0.9615 and its adjusted R2 valued was determined to be 0.9832 which is also very near to experimental R2 and the regression model is correct as the value close to 1. The value of m and variance were 0.8366 and 0.0050 respectively [15].