Optimization of Carboxymethyl cellulase production by Bacillus sp. NAB37 and biocatalytic potential in saccharification of acid-alkali pretreated Parthenium hysterophorus

doi:10.21203/rs.3.rs-4242662/v1

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Research Article

Optimization of Carboxymethyl cellulase production by Bacillus sp. NAB37 and biocatalytic potential in saccharification of acid-alkali pretreated Parthenium hysterophorus

https://doi.org/10.21203/rs.3.rs-4242662/v1

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published 24 Sep, 2024

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Purpose

The primary objective of this study was to assess the feasibility of using unconventional Parthenium hysterophorus weed biomass for the production of carboxymethyl cellulase using Bacillus sp. NAB37.

Methods

Using P. hysterophorus as a substrate and submerged fermentation conditions in optimization studies helped strain NAB37 make more enzymes. The efficacy of different physiological factors was determined through a two-step approach: first, a one-factor-at-a-time (OFAT) investigation, and subsequently, employing the RSM-based CCD method in statistical design. Enzymatic saccharification of alkali-acid-pretreated P. hysterophorus was also used to determine the efficacy of crude cellulase.

Results

The isolate NAB37 was identified by molecular characterization of 16SrDNA. The maximum carboxymethyl cellulase production (5.38 U/ml) was obtained with a temperature of 40°C (A), a pH of 7.5 (B), a substrate concentration of 3.0% w/v (C), and a starch concentration of 1.0% w/v (D). The alkali-acid-pretreated P. hysterophorus biomass was hydrolyzed using the crude enzyme produced under optimal conditions. On utilizing the cellulase enzyme for biomass hydrolysis, a maximum 32.78% saccharification yield (of cellulose, 0.364 g/g) was achieved in 96 h when enzyme and substrate levels were 30 FPU/100 ml and 2% (w/v), respectively.

Conclusion

It is possible to hydrolyze P. hysterophorus biomass enzymatically, producing significant amounts of total reducing sugars. Thus, it can serve as a feedstock for the production of bioethanol.

Parthenium hysterophorus

Carboxymethyl cellulase

OFAT

RSM

Bacillus sp. NAB37

Saccharification

Relatively little research has been done on P. hysterophorus biomass as a substrate for the synthesis of carboxymethyl cellulase. We believe that the current study will provide useful information to overcome the challenges of bacterial cellulase production, as well as increase fermentable sugar hydrolysates for bioethanol production.

Parthenium hysterophorus is ranked as the eighth most destructive weed globally. This plant is a rapidly expanding annual herb that possesses a long taproot capable of reaching heights of up to 2 meters. Additionally, P. hysterophorus seed can be disseminated through several means, such as fooding, water currents, automobiles, cattle, feed, and wind [1]. The P. hysterophorus weed has been associated with allergic respiratory problems, contact dermatitis, and mutagenicity in humans and other animals. The agricultural productivity is significantly reduced due to the allelopathy. The rapid expansion of this plant also presents a danger to biodiversity. P. hysterophorus seeds have the ability to germinate throughout the year as long as there is enough moisture. They are capable of thriving in extremely severe settings. P. hysterophorus was renowned for its ability to persist over extended periods in diverse ecological conditions and settings [2]. Conventional physical, chemical, and biological treatments, as well as integrative strategies, have faced difficulties in effectively controlling this weed. Various microorganisms can efficiently exploit the lignocellulosic elements bound in their cell walls for cellulase synthesis. This will also present an effective technique for controlling this weed through its utilization. Recently, researchers have begun to take an innovative approach to weed control by utilizing weeds as a bioresource for multiple uses [3]. For the synthesis of endoglucanase [4], cellulase [5], xylanase [6], and laccase [7], P. hysterophorus has the potential to be a low-cost feedstock.

Cellulases are a class of enzymes found in fungi and bacteria that break down cellulose in the environment. According to Lingouangou et al. [8], bacteria produce cellulases, a class of enzymes that work together to hydrolyze cellulose into simple sugar. Cellulose is broken down by the enzyme cellulase, which uses it as a substrate. Three primary components make up cellulase: endoglucanases (EC3.2.1.4), β-glucosidases (EC 3.2.1.21), and exoglucanases (EC3.2.1.74) [9]. Bacteria are an efficient producer of novel cellulases with excellent process compatibility, and Bacillus subtilis is regarded as a powerful microorganism for the creation of novel enzymes because of its exceptional fermentation properties. A few of the principal bacterial species that produce cellulase are members of the Clostridium, Acidothermus, Rhodothermus, Bacillus, and Pseudomonas families. It has been observed that a diverse range of enzymes are produced by Bacillus members [10]. Cellulase enzymes have been produced in fermentation systems using a variety of methods. The two types of fermentation that are most frequently utilized are submerged and solid-state ones. These two types of fermentations vary from one another in terms of the ambient circumstances, especially the amount of free water in the medium. For the fermentation system to produce more enzymes, the process parameters must be optimized. Response surface methodology (RSM) and one factor at a time (OFAT) are the two methods utilized to optimize these parameters. The first method takes a lot of time and isn't thought to be very accurate, while the second method is popular because of its benefits [11].

The goal of the current study is to increase the production of cellulase from Bacillus sp. NAB37 by using P. hysterophorus biomass as a substrate under submerged fermentation (SmF) conditions. The enzyme produced after optimization studies will be used to saccharify P. hysterophorus biomass. These factors were taken into consideration during the design of the study.

Microbial isolate

The bacterial isolate NAB 37 was isolated from the rock soil [12]. The culture was maintained at 30°C in nutrient agar medium (pH 7.0) containing (w/v) 0.5% peptone, 0.3% beef extract, 0.5% NaCl, and 2% Agar.

Molecular identification by 16S rDNA gene sequencing and phylogenetic analysis

The bacterial isolate NAB37 was characterized genetically using commercial service provided by Eurofins Scientific India Pvt Ltd, Bangaluru, Karnataka, India. The DNA was isolated from the culture using the standard protocol. From the isolated DNA, the fragment of 16SrDNA gene was amplified by polymerase chain reaction (PCR) using 8F and 1492R primers. The purified PCR amplicon free from contaminants was further subjected to DNA sequencing reaction using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. The 1545bp long 16S rDNA sequence obtained was compared against sequences of close relatives of the NAB37 isolate using EzTaxon e-server [13]. Multiple sequence alignments were performed using CLUSTALX version 2.1 [14], and alignment was edited manually using DAMBE (data analysis in molecular biology and evolution) in order to get unambiguous data [15]. The phylogenetic dendrogram was constructed by neighbor-joining end method using MEGA 7.0 (Molecular Evolutionary Genetics Analysis, version 7.0) software [16]. Bootstrap analysis was done with 1000 replicas. The sequence of the strain was submitted to the National Centre for Biotechnology Information (NCBI) with an accession no. MF663661 and was designated as Bacillus sp. NAB37.

Enzyme production and enzyme activity

For enzyme production, 1 ml of the activated culture was inoculated in 25ml of Mandel’s Medium (modified) (pH 7.0) containing 0.1% peptone, 0.14% (NH₄)2SO₄, 0.2% KH₂PO₄, 0.03% urea, 0.03% MgSO₄.7H₂O and 1ml of trace element solution containing 5mg/L FeSO₄.7H₂O; 1.6 mg/L MnSO₄,7H₂O; 1.4 mg/L ZnSO₄.7H₂O and 2mg/L CoCl₂. The incubation was done at 30°C at 180rpm for 4 days and the enzyme was extracted by centrifugation of the contents at 10,000rpm at 4°C for 15-20min followed with filtration through Whatman filter paper no.1. The filtrate obtained was used as the crude enzyme for further assays. Endoglucanase activity (or Carboxymethyl cellulase, CMCase) was determined according to the method by Ghose 1987 [17]. To determine endoglucanase activity, 0.5ml of appropriately diluted crude enzyme was mixed with 0.5 ml of 1% (w/v) substrate, i.e., carboxymethyl cellulose (prepared in 100 mM sodium acetate buffer, pH 5.0). The contents were incubated at 50°C for 10 min. The glucose released in the hydrolytic reaction was quantified using dinitrosalicylic acid (DNS) method [18]. One unit of the enzyme activity is defined as the amount of enzyme required for producing 1 µmol of glucose per ml per minute under the assay conditions. The enzyme activity was expressed as units per ml (U/ml).

Optimization of fermentation conditions

Under the shaking conditions, the effect of different variables was studied on the production of CMCase by the NAB37 isolate. The different parameters studied included the effect of biomass pretreatment (biomass pretreated in each method under their respective optimized conditions), substrate concentration (0.5–2.5% w/v), inoculum level (1–5% v/v), inoculum age (12-28h), pH (5.0–9.0), temperature (25-45^oC), incubation period (1-7days), carbon sources (glucose, fructose, galactose, cellobiose, lactose, sucrose, CMC, starch and mannitol), concentration of best carbon source (0.25-2.0% w/v), nitrogen source (urea, ammonium sulphate, ammonium nitrate, casein, peptone, beef extract, yeast extract and ammonium molybdenum), concentration of best nitrogen source (0.25–2.5% w/v) and shaking speed (100-180rpm).

Optimization of Endoglucanase Production by Response Surface Methodology (RSM)

RSM using the Central Composite Design (CCD) of experiments was used to develop a mathematical correlation between four variables on the production of CMCase by Bacillus sp. NAB37. The four independent variables chosen included temperature (^oC) (A), pH (B), concentration of substrate (%w/v) (C) and concentration of carbon source, starch (%w/v) (D). The production was carried out using steam pretreated biomass (found best in one variable at a time approach) under submerged fermentation (SmF) conditions. Each variable was set at two levels (-1 and + 1). The minimum and maximum ranges of variables have been listed in Table 1. A design of 30 experimental runs (Table 2) was developed using Design Expert, version 10.0.3.0. The mean endoglucanase production was taken as the responses (R1). A multiple regression analysis of the data was carried out for obtaining an empirical model that relates the response measured to the independent variables. A second order polynomial Eq. (1) for a four-factor system was obtained. The data was analyzed by ANOVA and the 3D response surface plots (showing interactions between two variables) were generated using the software.

Y = β₀ + β₁A₁ + β₂B₂ + β₃C₃ + β₄D₄ + β₁β₂AB + β₁β₃AC + β₁β₄AD + β₂β₃BC + β₂β₄BD + β₃β₄CD + β₁₁A² + β₂₂B² + β₃₃C² + β₄₄D² (1)

Table 1

Ranges of the four independent variables used in RSM for *Bacillus* sp. NAB37
Variable	Name	Coded Levels
Variable	Name	-1	0	+ 1
A	Temperature (^oC)	35	40	45
B	pH	6	7.5	9
C	Concentration of substrate (%w/v)	1.5	2	2.5
D	Concentration of starch (%w/v)	0.5	1.0	1.5

Table 2

Experimental plan for optimization of CMCase production using RSM, and the predicted and observed responses *Bacillus* sp. NAB37
Runs	Temperature (^oC)	pH	Substrate Concentration (%w/v)	Starch Concentration (%w/v)	CMCase activity (U/ml)
Runs	Temperature (^oC)	pH	Substrate Concentration (%w/v)	Starch Concentration (%w/v)	Observed	Predicted
1	40	7.5	2.0	2.0	1.67	1.60
2	45	9.0	2.5	0.5	2.008	2.10
3	35	9.0	2.5	0.5	2.56	2.58
4	40	7.5	2.0	1.0	3.39	3.49
5	45	6.0	1.5	0.5	1.25	1.28
6	40	7.5	3.0	1.0	5.38	5.16
7	40	7.5	2.0	0.0	1.29	1.20
8	40	7.5	2.0	1.0	3.45	3.49
9	40	7.5	2.0	1.0	3.51	3.49
10	45	6.0	1.5	1.5	1.5	1.52
11	35	6.0	2.5	0.5	2.81	2.89
12	45	9.0	1.5	1.5	1.44	1.46
13	45	6.0	2.5	1.5	2.61	2.75
14	35	9.0	2.5	1.5	2.67	2.79
15	35	6.0	1.5	1.5	1.34	1.35
16	40	7.5	2.0	1.0	3.62	3.49
17	40	7.5	2.0	1.0	3.42	3.49
18	30	7.5	2.0	1.0	1.29	1.22
19	50	7.5	2.0	1.0	1.00	0.906
20	35	6.0	2.5	1.5	3.07	3.15
21	35	6.0	1.5	0.5	1.16	1.19
22	40	10.5	2.0	1.0	1.35	1.36
23	45	9.0	2.5	1.5	2.34	2.35
24	45	6.0	2.5	0.5	2.28	2.37
25	40	4.5	2.0	1.0	1.88	1.71
26	40	7.5	2.0	1.0	3.55	3.49
27	35	9.0	1.5	1.5	1.33	1.27
28	45	9.0	1.5	0.5	1.37	1.32
29	40	7.5	1.0	1.0	2.52	2.58
30	35	9.0	1.5	0.5	1.19	1.198

Biomass Saccharification

The P. hysterophorus biomass pretreated by alkali-acid pretreatment, having 56% (w/w) cellulose content compared to 36.11% (w/w) in the native biomass [19], was used for saccharification. The reaction employed crude enzyme produced from Bacillus sp. NAB37 under optimized conditions. For the hydrolysis reaction, the biomass was mixed with 100 mM citrate buffer (pH 4.8) and crude enzyme. The mixture was also added with 0.03% sodium azide to avoid microbial contamination. The incubation was done at 50°C at 120 rpm. The effect of substrate concentration was studied by varying the amount of pretreated biomass from 1.0–7.0% (w/v). To study the effect of enzyme loadings, the amount of the enzyme was varied in reaction volume (100 ml) in FPU and total volume was made with citrate buffer. The progress of hydrolysis was monitored for 4 days (96 h) at an interval of 24 h. After completion of the reaction, the hydrolysate was subjected to centrifugation at 10,000 rpm for 15–20 min and the supernatant was assayed for total reducing sugars (TRS) released in hydrolysis process [18]. The saccharification yield was calculated using the formula:

where 0.9 is the conversion coefficient from glucan to reducing sugars.

Molecular identification of NAB37 isolate

A 1545bp long sequence of 16S rDNA from isolate NAB37 was obtained. On the basis of nucleotide homology and phylogenetic analysis, the bacterial culture NAB37 was found to be closely related to the members of the genus Bacillus. The strain NAB37 showed the highest similarity (100%) with Bacillus tequilensis KCTC 13622(T) and more than 99% similarity with other Bacillus strains. Figure 1 illustrates the phylogenetic tree that shows the evolutionary relationship between Bacillus sp. NAB37 and the closely related strains (type).

Optimization of process parameters for carboxymethyl cellulase production

Effect of pretreatment

Bacillus sp. NAB37, under shaking conditions (Fig. 2.1), produced the maximum CMCase enzyme (0.628 ± 0.016 U/ml) when steam-pretreated biomass was used. The alkali-pretreated biomass produced an amount of enzyme (0.445 ± 0.019 U/ml) comparable to that synthesized in the presence of untreated biomass (0.459 ± 0.015 U/ml). The enzyme production, however, decreased in the medium incorporated with acid- and alkali-acid-pretreated biomass. Generally, the biomass subjected to a pretreatment has a larger surface area. The researchers have reported increase in the enzyme adsorption sites with the increase in the surface area, regardless of the biomass [20]. As a consequence, the titers of the free cellulases are low due to adsorption of the produced cellulases over the biomass particles. Oke et al. [21] have seen maximum endoglucanases synthesis by Bacillus aerius S5.2 using untreated lignocellulose.

Effect of substrate concentration

The substrate concentration is one of the important parameters playing an effective role in the induction of microbial cellulases. In the present work, the study on the effect of P. hysterophorus biomass (steam-pretreated) concentration on cellulase production by Bacillus sp. NAB37 showed a 2% (w/v) concentration optimum for the highest CMCase activity under shaking conditions (0.934 ± 0.011 U/ml) (Fig. 2.2). The enzyme synthesis increased steadily with the increase in the amount of substrate in the fermentation medium until it reached optimum concentration, followed by a decrease with a further increase in the biomass levels. The low activity at lower substrate levels may be accounted for by the dilution of the substrate required for the induction of cellulases encoding genes. Goyal et al. [22] reported that the presence of pretreated rice straw at 0.75% and 1% concentrations under shaking and stationary conditions, respectively, stimulated cellulase production from Bacillus sp. 313SI.

Effect of Inoculum level

The inoculum size was optimized for maximizing endoglucanase production by Bacillus sp. NAB37 because it is one of the important factors governing the synthesis of cellulases. An increment in the CMCase synthesis, from 0.929 ± 0.014 U/ml to 1.216 ± 0.011 U/ml, was achieved by raising the inoculum size from 1–2% under shaking conditions (Fig. 2.3). The reduction in enzyme synthesis with the rise in inoculum level beyond optimum value is correlated with decreased microbial growth as a result of competition for space and nutrients [23].

Effect of Inoculum Age

The appropriate age of the inoculum is also an essential requirement for the synthesis of enzymes at optimal concentrations. Therefore, the effect of inoculum age was studied on CMCase production by Bacillus sp. NAB37 by varying age from 12h to 18h. It was found that Bacillus sp. NAB37 produced the highest level of endoglucanase (1.551 ± 0.015 U/ml) using 16-h old culture under shaking conditions (Fig. 2.4). Gautam and Sharma [24] found the 24-hour age of the inoculum suitable for the highest cellulase production by Bacillus subtilis under SmF conditions.

Effect of pH

The effect of pH was investigated on cellulase production by Bacillus sp. NAB37 by varying the initial pH of the fermentation medium over a range of 5.0 to 9.0. Maximum CMCase activity was seen at pH 7.5 under shaking (1.917 ± 0.016 U/ml) conditions (Fig. 2.5). The activity was relatively low at pH levels lower or higher than the optimum. This indicates that the isolate needed a neutral-alkaline environment for enhanced cellulase synthesis. Faiz Rasul et al. [25] have achieved the highest cellulase production by Bacillus sp. at pH 7.0.

Effect of Temperature

In the isolate Bacillus sp. NAB37, maximum endoglucanase activity was achieved when the temperature was maintained at 40°C under shaking conditions. Under shaking conditions, the endoglucanase production increased sharply when the temperature was raised from 25°C (CMCase activity 0.781 ± 0.009 U/ml) to 30°C (CMCase activity 1.866 ± 0.008 U/ml) and reached a maximum of 2.113 ± 0.023 U/ml at 40°C (Fig. 2.6). However, a decline in cellulase synthesis was detected beyond 40°C, which could result from the denaturation of the enzyme at a higher temperature [26].

Effect of Incubation Period

The effect of time course on endoglucanase production was studied by incubating the Bacillus sp. NAB37 isolate for various time intervals under shaking conditions. In shaking conditions, the isolate produced maximum endoglucanases (CMCase activity 2.334 ± 0.013 U/ml) on 2 days of incubation (Fig. 2.7). The decrease in enzyme activity after the optimum incubation period, however, might result from changes in pH, the accumulation of inhibitory metabolites, and the depletion of nutrients in the fermentation medium [27].

Effect of Carbon Source and its Concentration

The inductive effect of various carbon sources was tested individually by incorporating each carbon source separately into the production medium. The results obtained showed that Bacillus sp. NAB37 could utilize a range of carbon sources under shaking conditions (Fig. 2.8). Among different carbon sources, starch induced the highest levels of enzyme synthesis by the bacterium. In the presence of starch, Bacillus sp. NAB37 produced 2.624 ± 0.014 U/ml CMCase under shaking conditions. In addition, galactose, lactose, CMC, and mannitol also enhanced endoglucanase production in the bacterium under shaking conditions. On studying the effect of concentration of carbon source, it was found that the presence of 1% starch stimulated maximum CMCase production by Bacillus sp. NAB37 under shaking (2.631 ± 0.014 U/ml) conditions (Fig. 2.9). A drop in the activity was seen on further increase in the concentration. Irfan et al. [28] have also found starch as one of the carbon sources showing an enhanced effect on cellulase production by Cellulomonas sp. ASN2.

Effect of Nitrogen Source and its Concentration

While studying the effect of different nitrogen sources, it was observed that Bacillus sp. NAB37 produced the highest amounts of CMCase when peptone was used as the only nitrogen source in the medium under shaking (2.942 ± 0.011 U/ml CMCase activity) conditions (Fig. 2.10). Only peptone showed a positive effect under shaking conditions. Several other studies have also reported that organic nitrogen sources are more favorable for cellulase production compared to inorganic sources [29].

In the shaking conditions, a 1.75% (w/v) concentration of peptone increased the CMCase activity to 3.272 ± 0.051 U/ml compared to 2.596 ± 0.007 U/ml activity in the control without any nitrogenous additive in the fermentation medium (Fig. 2.11). Several other workers have also recorded the inductive effect of peptone on cellulase production by various microorganisms, including bacteria. Paudel and Qin [30] have also noticed maximum cellulase production by Bacillus sp. K1 at a 1% peptone concentration.

Effect of shaking speed

The agitation speed has a significant impact on the growth as well as the enzyme production by various microorganisms. To study the effect of shaking speed, the rpm was varied from 100 to 180 rpm. The isolate Bacillus sp. NAB37 produced a similar amount of CMCase at 120 (3.421 ± 0.028 U/ml) and 150 rpm (3.424 ± 0.019 U/ml) (Fig. 2.12). The activity was, however, significantly low at 100 and 180 rpm. In a study by Deka et al. [31], maximum enzyme production was achieved at 120 rpm. An increase in agitation speed further reduced the synthesis of enzymes.

Statistical study for enhanced production of CMCase by RSM

The statistical response surface methodology (RSM) is a useful model for studying the effect of several factors influencing the process of enzyme production. Therefore, the RSM study was conducted using the Central Composite Design (CCD) of experiments for bacterial strain of Bacillus sp. NAB37. The four independent variables chosen were temperature (^oC) (A), pH (B), concentration of substrate (pretreated biomass) (%w/v) (C) and concentration of starch (%w/v) (D). Each variable was set at two levels (-1 and + 1). The minimum and maximum ranges of variables have been listed in Table 1. A set of 30 experiments was carried out (Table 2). The analysis of variance (ANOVA) yielded the following regression equation in terms of the CMCase enzyme production (Y₁) as a function of temperature (A), pH (B), substrate concentration (C) and concentration of starch (D):

Y₁ = 3.45 − 0.083 × A – 0.087 × B + 0.65 × C + 0.10 × D + 4.250E-003 × AB − 0.16 × AC + -0.013 × AD − 0.074 × BC − 0.018 × BD + 0.030 × CD − 0.60 × A² − 0.49 × B² + 0.097 × C² − 0.52 × D² (2)

The mean predicted response based on above polynomial equation and the observed responses have been shown in Table 2. As shown in Table 3, ANOVA of regression model indicated the value of determination coefficient (R²) to be 0.9938, which implies that 99.38% of the experimental data of the CMCase production was compatible with the data predicted by the model. The R² value is always between 0 and 1.0. An R² value closer to 1.0 shows that the model is stronger. The value of the adjusted determination coefficient (adjusted R²) was 0.9881, which indicates greater significance of the model. A low value of coefficient of variation (C.V., 5.60%) demonstrated better precision and reliability of the executed experiments. The adequate precision value measures signal to noise ratio, and a ratio > 4.0 is desirable. In the present model, higher ratio of 52.229 indicated an adequate signal, and also proved that model could be used to navigate the design space. The F-value of 172.41 denotes that the model was significant. There is only 0.01% chance that a “model F- value” so large could occur due to noise. ANOVA analysis also indicated that the model terms linear temperature (P < 0.0500), pH (P < 0.0500), substrate concentration (P < 0.0500), starch concentration (P < 0.0500), quadratic temperature A² (P < 0. 0500), pH B² (P < 0.0500), substrate concentration C² (P < 0. 0500), starch concentration D² (P < 0. 0500) and two interaction terms (AC, BC) were significant. Values of "Prob > F" less than 0.0500 indicate that the model terms are significant, while values greater than 0.1000 indicate that the model terms are not significant.

Table 3

ANOVA for response surface quadratic model *Bacillus* sp. NAB37
Source	Sum of Squares	df	Mean Square	F Value	p-value Prob > F
Model	32.04	14	2.29	172.41	< 0.0001
A-Temp	0.15	1	0.15	11.48	0.0041
B-pH	0.20	1	0.20	14.81	0.0016
C-Substrate Conc	9.99	1	9.99	752.98	< 0.0001
D-Starch Conc	0.25	1	0.25	18.57	0.0006
AB	1.369E-003	1	1.369E-003	0.10	0.7525
AC	0.36	1	0.36	27.39	0.0001
AD	5.329E-003	1	5.329E-003	0.40	0.5359
BC	0.10	1	0.10	7.62	0.0146
BD	8.464E-003	1	8.464E-003	0.64	0.4370
CD	9.604E-003	1	9.604E-003	0.72	0.4084
A²	10.05	1	10.05	757.17	< 0.0001
B²	6.53	1	6.53	491.75	< 0.0001
C²	0.25	1	0.25	19.01	0.0006
D²	7.46	1	7.46	562.15	< 0.0001
Residual	0.20	15	0.013
Lack of Fit	0.16	10	0.016	2.16	0.2043
Pure Error	0.037	5	7.480E-003
Cor Total	32.24	29
Std. Dev.	0.12	R-Squared		0.9938
Mean	2.27	Adj R-Squared		0.9881
C.V.%	5.06	Pred R-Squared		0.9694
Press	0.99	Adeq Precision		52.229

The 3D response surface plots were used to understand the effects of variables on CMCase production (Fig. 3). In each set, two variables varied within their experimental range, whereas the other two variables remained constant at zero level. The model predicted maximum CMCase production (5.16 U/ml) using 3% (w/v) substrate (pretreated Parthenium biomass), 1% (w/v) concentration of starch, at temperature 40^oC and a of Ph 7.5. Figure 3(a) depicts the effect of interaction between pH and temperature on CMCase production by Bacillus sp. NAB37, when substrate and starch concentration were kept constant as 2% and 1% (w/v) respectively. The enzyme production increased with the increase in the initial pH upto the value of 7.5 at all temperatures, after which further rise in the pH resulted in a decline in the CMCase production. The highest enzyme synthesis was observed at temperature 40^oC and pH 7.5. Figure 3(b) shows the interaction between temperature and the substrate concentration. When the pH value of 7.5 and starch levels of 1% were kept constant, the CMCase synthesis by the bacterial strain increased with the increase in the substrate concentration over entire temperature (35 to 45^oC) range observed during the experiment. The activity, however, was maximum at temperature 40^oC and substrate concentration of 3% (w/v). At 3% substrate levels, rise in the temperature beyond 40^oC showed a gradual reduction in the production of the enzyme. Figure 3(c) represented the relationship between the starch concentration and temperature. The highest enzyme synthesis was observed at 40^oC temperature and the starch concentration of 1% (w/v). The interaction between the substrate concentration and initial pH has been shown in Fig. 3(d). The surface plot revealed that higher substrate levels of 3% (w/v) were favorable for achieving high CMCase production at all pH levels, being maximum at pH 7.5. Figure 3(e) shows the effect of interaction between starch levels and pH, while keeping temperature as 40^oC and substrate concentration 2%. The CMCase synthesis was maximum at pH 7.5 and starch concentration of 1%. Figure 3(f) indicated that high substrate levels induced high CMCase production. The maximum enzyme activity was recorded when substrate and starch concentration in the fermentation medium were 3% and 1% (w/v) respectively. The model was validated experimentally by carrying out CMCase production by Bacillus sp. NAB37 under conditions optimized by RSM. The experiments were performed in triplicates and the average CMCase production was seen. The maximum CMCase activity, 5.38U/ml was in good agreement with the value (5.16U/ml) predicted by the regression model. These results indicated that the developed model was accurate and reliable for predicting CMCase production by the Bacillus sp. NAB37.

The RSM technique has been used by various researchers to optimize cellulase production by different microorganisms and has also led to successful enhancement in the enzyme synthesis by knowing the interactions between influencing factors. The RSM study by Singh et al. [32] for the optimization of cellulase production from marine Bacillus VITRKHB has revealed maximum enzyme synthesis under optimized conditions of xylose 5.0%, beef extract 6.9%, NaCl 1.17%, pH 7.83 and temperature 25.84^oC, after 24h of incubation. The statistical optimization of medium components for cellulase production by Enhydrobacter sp. ACCA2 using RSM method increased the CMCase synthesis up to 2.39-fold compared to that under unoptimized conditions when the CMC, peptone, (NH₄)₂SO₄ and K₂HPO₄ were used at 15.00 g/l, 14.45 g/l, 0.45 g/l and 1.50 g/l concentrations [33].

Saccharification

In the case of the saccharification of pretreated P. hysterophorus biomass using bacterial (Bacillus sp. NAB37) cellulase, the highest hydrolysis yield was reached in 96h (Table 4, Fig. 4). Furthermore, the increase in the substrate concentration from 0.5–1% (w/v) resulted in an increase in the TRS from 0.167 ± 0.009 g/g biomass (≈ 26.83% saccharification yield) to 0.206 ± 0.009 g/g (≈ 33.11% saccharification yield). The amount of TRS obtained was 0.204 ± 0.012 g/g of the biomass when 2% substrate concentration as used (≈ 32.78% saccharification yield) (Table 4, Fig. 4). This value was insignificantly different from the value obtained when the substrate level was 1%. The saccharification process is considered good when it can hydrolyze higher loads of the biomass [34]. Therefore, the subsequent experiments were carried out using 2% (w/v) concentration of the substrate as the optimized level. The increase in the substrate levels from 2–7% resulted in a gradual decrease in the yield of TRS. Other studies have also documented that the increase in biomass concentration increases the viscosity of the system which interferes in the mass transfer of the enzymes and also the increased concentration of sugars tend to inhibit the enzyme [34, 35].

Table 4

Effect of substrate concentration on the TRS yield from hydrolysis of pretreated *P. hysterophorus* biomass using cellulase enzyme from *Bacillus* sp. NAB 37
Time (h)	Total reducing sugars (g/g biomass) at different substrate concentrations (%w/v)
Time (h)	0.5	1.0	2.0	3.0	5.0	7.0
12	0.067 ± 0.011	0.078 ± 0.015	0.076 ± 0.014	0.069 ± 0.015	0.065 ± 0.013	0.062 ± 0.011
24	0.099 ± 0.009	0.111 ± 0.018	0.112 ± 0.009	0.097 ± 0.011	0.094 ± 0.018	0.089 ± 0.013
48	0.121 ± 0.014	0.145 ± 0.009	0.143 ± 0.016	0.131 ± 0.011	0.126.008	0.121 ± 0.016
72	0.144 ± 0.015	0.173 ± 0.010	0.170 ± 0.017	0.158 ± 0.012	0.153 ± 0.013	0.148 ± 0.007
96	0.167 ± 0.011	0.206 ± 0.009	0.204 ± 0.012	0.178 ± 0.012	0.171 ± 0.016	0.167 ± 0.013
● Hydrolysis conditions: 50°C temperature, pH 4.8, 120 rpm, enzyme loading: 30FPU per 100ml (or 0.3 FPU/ml)

On studying the effect of bacterial cellulase enzyme loadings on the saccharification of the biomass, it was recorded that the amount of TRS released in the process increased significantly when the enzyme loading was increased from 5FPU to 30FPU in the reaction volume (100ml) and the hydrolysis time was 96h (Table 5). Maximum 0.204 ± 0.012g TRS per gram of the biomass was released (≈ 32.78% saccharification yield) in 96h when the enzyme was used at a concentration of 30FPU. Further rise in the enzyme loading to 40FPU resulted in an insignificant increase in the TRS, i.e., 0.208 ± 0.014g per gram of the biomass (Table 5, Fig. 5). Because a saccharification process involving use of the minimum amount of enzyme is most economical, therefore, 30FPU concentration of the enzyme was the optimized level in the current study. Furthermore, on studying the effect of hydrolysis time, it was found that the rate of hydrolysis increased almost linearly with the increase in the incubation time up to 96h (Fig. 5). The saccharification yield increased from 22.98% at 48h to 32.78% achieved in 96h. The stability of the enzyme, end product inhibition, and concentration of enzyme and substrate are among several factors influencing the rate of the saccharification process [36]. One of the probable reasons for the trend of hydrolysis rate in the present experiment could be the overall lower yield of sugars in the process.

Table 5

Effect of enzyme concentration on the TRS yield from hydrolysis of pretreated *P. hysterophorus* biomass using cellulase enzyme from *Bacillus* sp. NAB 37
Time (h)	Total reducing sugars (g/g biomass) at different enzyme loadings (FPU)
Time (h)	5.0	10.0	20.0	30.0	40.0	50.0
12	0.019 ± 0.015	0.048 ± 0.013	0.066 ± 0.011	0.076 ± 0.014	0.079 ± 0.014	0.077 ± 0.014
24	0.029 ± 0.010	0.063 ± 0.011	0.086 ± 0.014	0.112 ± 0.009	0.111 ± 0.015	0.110 ± 0.015
48	0.041 ± 0.011	0.097 ± 0.012	0.121 ± 0.016	0.143 ± 0.016	0.146 ± 0.010	0.144 ± 0.010
72	0.055 ± 0.012	0.119 ± 0.011	0.157 ± 0.013	0.170 ± 0.017	0.176 ± 0.009	0.174 ± 0.009
96	0.073 ± 0.016	0.134 ± 0.011	0.175 ± 0.013	0.204 ± 0.012	0.208 ± 0.014	0.207 ± 0.012
● Hydrolysis conditions: 50°C temperature, pH 4.8, 120 rpm, 2% substrate (pretreated) concentration
● The amount of the enzyme (in FPU per 100ml) was adjusted by adding corresponding volume of the enzyme in the reaction mixture

Premalatha et al. [33] enhanced the cellulase production (8.86U/mL) from Enhydrobacter sp. ACCA2 by optimization of various parameters. When the same bacterial isolate was used for the saccharification of various biomass, it showed saccharification yield of > 40% in the sorghum leaves, sorghum stem and cumbu stem, > 50% in the cumbu leaves and the highest yield, i.e., > 60% in the bamboo. Alrumman [36] also optimized the cellulase production by Geobacillus stearothermophilus using alkali pretreated date palm waste and on using the same for optimization of the saccharification of alkali pretreated biomass observed pH 5.0, 50°C temperature, 4% biomass loading, 30FPU/g enzyme loading and hydrolysis time of 24h optimum for achieving saccharification yield of 71.03%. The yield, however, increased to 94.88% by the removal of the hydrolysate after 24h by using a two-step hydrolysis. Nargotra et al. [37] used the cellulase obtained from Bacillus subtilis SV1 (produced under optimized conditions) for the hydrolysis of ionic liquid pretreated pine needle biomass in a one pot consolidated bioprocess and was successful in obtaining the saccharification efficiency of 65.9%.

By employing the RSM approach to optimize the cultural conditions, the production of carboxymethyl cellulase from Bacillus sp. NAB37 was increased by 1.5-fold. Additionally, because RSM assumes random errors and reduces the number of experiments required, it is a superior experimental technique to OFAT. Conversely, OFAT is thought to be a challenging and time-consuming method. The lignocellulosic biomass was able to be saccharified by the crude enzyme derived from Bacillus sp. NAB37. This study highlights the potential of our crude enzyme for lignocellulosic biomass conversion systems.

Ethics approval and consent to participate: Not Applicable.

Consent for publication: Not Applicable.

Availability of data and materials: All obtained data during this work are included in this manuscript.

Competing interests: The authors declare no competing interests.

Funding: Not Applicable.

Authors' contributions: Anita Saini: writing original draft, editing writing, review and editing, formal analysis, resources Naveen Kumar: editing writing, review and editing, formal analysis, resources; Neeraj K. Aggarwal: writing review and editing, visualization, supervision.

Acknowledgements: The authors would like to thank the Department of Microbiology, Kurukshetra University, Kurukshetra, Haryana, India, for providing the lab facility for performing the experimentation.

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Optimization of Carboxymethyl cellulase production by Bacillus sp. NAB37 and biocatalytic potential in saccharification of acid-alkali pretreated Parthenium hysterophorus

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Abstract

Figures

Statement of Novelty

Introduction

Materials and methods

Microbial isolate

Molecular identification by 16S rDNA gene sequencing and phylogenetic analysis

Enzyme production and enzyme activity

Optimization of fermentation conditions

Optimization of Endoglucanase Production by Response Surface Methodology (RSM)

Biomass Saccharification

Results and Discussion

Molecular identification of NAB37 isolate

Optimization of process parameters for carboxymethyl cellulase production

Effect of pretreatment

Effect of substrate concentration

Effect of Inoculum level

Effect of Inoculum Age

Effect of pH

Effect of Temperature

Effect of Incubation Period

Effect of Carbon Source and its Concentration

Effect of Nitrogen Source and its Concentration

Effect of shaking speed

Statistical study for enhanced production of CMCase by RSM

Saccharification

Conclusions

Declarations

References

Supplementary Files

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