The Optimization of Culture Conditions for the Cellulase Production of a Thermostable Cellulose-Degrading Bacterial Strain and Its Application in Environmental Sewage Treatment

Abstract

A novel cellulose-degrading bacterial strain, D3-1, capable of degrading cellulose under medium- to high-temperature conditions, was isolated from soil samples and identified as Staphylococcus caprae through 16SrRNA gene sequencing. The strain’s cellulase production was optimized by controlling different factors, such as pH, temperature, incubation period, substrate concentration, nitrogen and carbon sources, and response surface methods. The results indicated that the optimal conditions for maximum cellulase activity were an incubation time of 91.7 h, a temperature of 41.8 °C, and a pH of 4.9, which resulted in a maximum cellulase activity of 16.67 U/mL, representing a 165% increase compared to pre-optimization levels. The above experiment showed that, when maize straw flour was utilized as a natural carbon source, strain D3-1 exhibited relatively high cellulase production. Furthermore, gas chromatography–mass spectrometry (GC-MS) analysis of products in the degradation liquid revealed the presence of primary sugars. The results indicated that, in the denitrification of simulated sewage, supplying maize straw flour degradation liquid (MSFDL) as the carbon source resulted in a carbon/nitrogen (C/N) ratio of 6:1 after a 24 h reaction with the denitrifying strain WH-01. The total nitrogen (TN) reduction was approximately 70 mg/L, which is equivalent to the removal efficiency observed in the glucose-fed denitrification process. Meanwhile, during a 4 h denitrification reaction in urban sewage without any denitrifying bacteria, but with MSFDL supplied as the carbon source, the TN removal efficiency reached 11 mg/L, which is approximately 70% of the efficiency of the glucose-fed denitrification process. Furthermore, experimental results revealed that strain D3-1 exhibits some capacity for nitrogen removal; when the cellulose-degrading strain D3-1 is combined with the denitrifying strain WH-01, the resulting TN removal rate surpasses that of a single denitrifying bacterium. In conclusion, as a carbon source in municipal sewage treatment, the degraded maize straw flour produced by strain D3-1 holds potential as a substitute for the glucose carbon source, and strain D3-1 has a synergistic effect with the denitrifying strain WH-01 on TN elimination. Thus, this research offers new insights and directions for advancement in environmental sewage treatment.

1. Introduction

As the most abundant resource on Earth, cellulose is a widely distributed complex polysaccharide, consisting of 8000 to 10,000 glucose residues linked by β-1,4 glycosidic bonds, but only 1% to 2% has been used [1,2,3]. Cellulose is mostly produced via photosynthesis, yielding about 180 billion tons of natural lignocellulose biomass annually [4,5]. Agricultural production produces significant amounts of crop residues and hulls every year; however, the main method of managing these agricultural waste products at present is to burn them in situ. Although this method saves time and effort, burning straw in situ not only fails to derive economic value from the straw for farmers but also seriously pollutes the environment and accelerates its deterioration [6]. The most efficient and environmentally friendly way to use natural cellulose is to degrade it into small molecules via cellulose enzyme, which can synergistically degrade cellulose into three components: endoglucanase, exoglucanase, and β-glucosidase [7].

Cellulase has a wide range of applications, as it can be used for fruit and vegetable processing, tea processing, oil crop processing, soy sauce brewing, beer production, etc. [8]. In the feed industry, adding enzymes can improve the growth production of broilers and laying hens [9,10]. Additionally, it can improve the feeling and appearance of fabrics in the textile industry [11,12]. In environmental engineering, it can be used to improve soil permeability by promoting plant cellulose decomposition, which can reduce heavy metal adsorption to the soil [13,14]. However, there is relatively little research into the application of cellulose as a carbon source after degradation; mainly, research has been focused on the fields of biosynthesis, bio-energy production, and edible fungus cultivation, with extensive applications [15].

During the sewage treatment process, denitrification is an important process, and the hydraulic retention time (HRT) usually starts at 4 h, depending on a variety of factors, including oxygen concentration, carbon source adequacy, temperature, pH, and nitrate concentration. Low C/N is an important limiting factor, and the addition of a carbon source is the most mature and stable method used for nitrogen removal at present. As heterotrophic organisms, denitrifying bacteria need a sufficient carbon source to grow, so current sewage treatment plants often use additional carbon sources, such as acetic acid, sodium acetate, or glucose, materials that are expensive compared with the sugars in the liquid products degraded by cellulase, which therefore have important application value in the denitrification of sewage. These products produce soluble sugars through cellulose degradation, which provides the necessary carbon source for the denitrification process, thereby improving denitrification efficiency. In addition, studies showed that some cellulose-degrading bacteria, such as Bacillus and Paracoccus, also have denitrifying properties, and their synergistic effects with denitrifying bacteria showed more efficient denitrification capacity, significantly reducing the nitrate content of sewage [16]. In general, it is a win–win situation in that the external carbon source for the denitrification of sewage can be produced from agricultural waste straw, as this not only improves the sewage treatment efficiency but also realizes the recycling of agricultural waste resources, thereby providing a new approach to sustainable sewage treatment.

In this experiment, we isolated a heat-resistant cellulolytic bacterium, D3-1, which was identified as S. caprae, from soil. Reports on the cellulose-degrading properties of this type of strain, which can be used in environmental ecology, are rare [17]. In order to better acclimatize the strain to environmental treatment, its culture conditions were optimized to achieve maximum cellulase production. We also determined its degradation products and found that they are applicable as a carbon source in the denitrification of sewage. Ultimately, we proved that the strain’s degradation products have the potential to replace the glucose carbon source in the sewage treatment process. It is of great significance that agricultural waste can be efficiently used as a carbon source for cellulose-degrading bacteria.

2. Materials and Methods

2.1. Sample Sources and Culture Media

The samples were derived from humus soil along the Xunsi River in Wuhan, Hubei Province, China. The environmental sewage for the application experiments was collected from the Huangjiahu Municipal Sewage Treatment Plant in Wuhan, Hubei Province, China. The relevant indicators (nitrate nitrogen—NO₃-N, TN, and chemical oxygen demand—COD are shown in

Table 1. Related water quality index of municipal sewage.

Water Quality Index	NO3-N (mg/L)	TN (mg/L)	COD (mg/L)
Municipal Sewage	31	43	80–100

.

According to the related literature [18], we prepared different culture media for bacterial growth and cellulose production, (The reagents used in this experiment were all provided by Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), including (A) Reasoner’s 2A (R2A) agar, (B) Luria–Bertani (LB) broth, (C) carboxymethyl cellulose (CMC) agar, (D) cellulose production broth, (E) basal culture medium, and (F) denitrification medium. Their compositions were as follows:

(A)

R2A agar (g L⁻¹) contained 0.5 g yeast extract, 0.5 g peptone, 0.5 g starch, 0.5 g MnSO₄, 0.5 g casein hydrolysate, 0.5 g glucose, 0.3 g K₂HPO₄, 0.3 g sodium pyruvate, 15 g agar, and distilled water to 1 L total volume.

(B)

LB broth (g L⁻¹) contained 10 g peptone, 5 g yeast extract, 5 g NaCl, and distilled water to 1 L total volume.

(C)

CMC agar (g L⁻¹) contained 5 g CMC, 1 g NaNO₃, 1 g K₂HPO₄, 1 g NaCl, 0.5 g MgSO₄, 0.5 g yeast extract, 15 g agar, and distilled water to 1 L total volume.

(D)

Cellulase production broth (g L⁻¹) contained 10 g CMC, 10 g peptone, 7.5 mg FeSO₄·7H₂O, 2.5 mg MnSO₄·H₂O, 2.0 mg ZnSO₄, and distilled water to 1 L total volume.

(E)

Basal culture medium (g L⁻¹) [19] contained 0.5 g CaCl₂·2H₂O, 0.4 g FeSO4·7H₂O, 1 g K₂HPO₄, 0.05 g MgSO₄·7H₂O, 2 g NaCl, 1.5 g NaHCO₃, 1 mL trace element solution, and distilled water to 1 L total volume.

(F)

The denitrification medium (simulated sewage) contained 0.324 g L⁻¹ KNO₃, added to the basal medium as the only nitrogen source; the carbon source was added separately as an experimental variable.

2.2. Screening, Isolation, and Identification

We diluted a 2.5 g soil sample with 50 mL of sterile water, incubated at 30 °C with shaking for 60 min; the suspension was then spread on R2A agar and incubated at 30 °C for 48 h. Cellulase-producing strains were screened using the Gram iodine solution method [20]. Target strains were selected based on colony morphology, size, and color, then picked out and cultured in 5 mL of LB broth (30 °C, 150 rpm) for 24 h, after which 1 mL of each culture was retrieved and centrifuged (8000 x g, 10 min); the pellets were resuspended in sterile water, inoculated into 100 mL of enzyme production medium (0.1 M citrate buffer, pH5), and fermented (30 °C, 150 rpm, 4 days). The selected single colonies were purified 3 times for further analysis.

For bacterial identification, genomic DNA was amplified via PCR using universal primers 27F (5’-AGAGTTTGATCATGGCTCAG-3’) and 1492R (5’-TACCTTGTTACGACTT-3’). The 25 µL reaction mixture contained 0.5 µL DNA, 2.5 µL 10×Buffer (Mg²⁺), 1 µL dNTPs, 0.2 µL DNA polymerase, 0.5 µL of each primer (10 µmol/L), and ddH₂O. PCR conditions included initial denaturation (94 °C, 4 min), 34 cycles of denaturation (94 °C, 30 s), annealing (54 °C, 30 s), extension (72 °C, 1.5 min), and final extension (72 °C, 10 min). The products were sequenced by Sangon Biotech (Shanghai, China), and sequences were aligned using NCBI. A phylogenetic tree was constructed in MEGA7.0.26 via the neighbor-joining method.

2.3. Quantification of Cellulase Activity

The determination scheme for cellulase activity was developed according to the reference method [21]. A 1 cm × 6 cm strip of filter paper, 1 mL of moderately diluted crude enzyme solution, and 1 mL of 0.1 M citric acid buffer pH4.8 were added to a test tube. After reaction at 50 °C for 30 min, 1.5 mL of DNS reagent was added [22] to terminate the reaction, and then the mixture was placed in a boiling water bath for 5 min. The mixture was then cooled by adding 25 mL of distilled water, and then a colorimetric analysis was conducted at a 540 nm wavelength to determine the cellulase activity, defining 1 µg of reducing sugar generated by 1 mL of enzyme solution per minute as 1 enzyme activity unit (U/mL).

2.4. Single-Factor Optimization of Cellulase Production Conditions

Based on the enzyme-producing medium, we systematically investigated the optimal single-factor culture conditions for cellulase production by this strain through sequential variations in incubation time (24 h, 48 h, 72 h, 96 h, 120 h, 144 h), temperature (20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C), pH (3.0, 5.0, 7.0, 9.0, 11.0), carbon source (rice husk, corn flour, CMC-Na, wheat straw, rice straw, maize straw, peanut shell), nitrogen source (tryptone, soy peptone, urea, yeast extract, ammonium chloride, ammonium sulfate), and inoculation amount (1%, 2%, 3%, 4%, 5%). We inoculated 1.5 ml of the bacteria into 100 mL of the culture medium and changed only one condition each time. For each culture condition, three replicate experiments were conducted, and the cellulase activity in the culture medium was measured to quantitatively assess the enzyme-producing capability of the cellulose-degrading bacteria.

2.5. Optimization of Cellulase Production Through Response Surface Methodology

The optimal ranges of parameters for cellulase production were initially determined through a series of preliminary single-factor experiments; subsequently, the production conditions for D3-1 were statistically optimized using a three-level, three-variable Box–Behnken Design (BBD). The variables selected for optimization were culture time (A), temperature (B), and pH value (C), with cellulase activity serving as the response variable. The experimental design is detailed in

Table 2. Box–Behnken design factor levels.

		Level
Factors	Symbols	−1	0	1
Time/h	A	48	96	144
Temperature/°C	B	35	40	45
pH	C	3	5	7

, where A, B, and C denote time, temperature, and pH, respectively, while −1, 0, and +1 represent the lower, intermediate, and upper levels of each factor. To minimize the impacts of potential variability in the response, the experiments were conducted in a randomized order.

The production conditions of cellulase were fitted through multiple regression using Design Expert 13 software under aerobic conditions. The model’s accuracy was verified by repeating each experiment three times.

2.6. Product Analysis of Maize Straw Flour Degradation by Strain D3-1

In order to preliminarily investigate the practical application and enzyme-producing characteristics of the selected cellulose-degrading bacterial strain D3-1, we analyzed this strain’s production using GC-MS [23]. Strain D3-1 was fermented in liquid enzyme culture medium, with maize straw as a carbon source, under optimal conditions (30 °C, 150 rpm, pH 5), and fermentation broth samples were taken at 0 h and 96 h.

2.7. Denitrification Application of Degradation by Strain D3-1 with Carbon Source

2.7.1. The Denitrification Performance of Different Bacterial Agents in Simulated Sewage Under Different Carbon Sources

Cellulase-producing strain D3-1 was fermented under optimal conditions (40 °C, 150 rpm, pH5). The fermentation production after degradation was used as the initial carbon source for MSFDL. For each denitrification process, 40 mL of simulated sewage was placed into a 50 mL conical flask, after which 1.44 mL of glucose solution (50 g L⁻¹), 180 mg of maize straw flour, and 0.83 mL of MSFDL were added separately as carbon sources to make the initial C/N ratio in the simulated sewage 6:1. The denitrification experiment was divided into 9 groups, 3 for each carbon source.

Bacterial agent WH-01, bacterial agent D3-1, and their combination were used at proportions of 5% to conduct three groups of parallel experiments for each variable. The culture was carried out in a closed manner under conditions of 35 °C and 140 r/min for 24 h. Samples were taken to detect nitrate nitrogen (NO₃-N), TN, and dissolved total nitrogen (DTN). The reductions from the initial values were calculated to determine the denitrification performance of the different agents in different carbon source media. The experiments were conducted in three parallel ways; the arrangement is shown in

Table 3. Experimental design for simulating sewage denitrification process.

Group	Carbon Source	Nitrogen Source	C/N	Bacterial Strain
1	maize straw	The nitrogen source used for all experimental groups was nitrate nitrogen.	6/1	D3-1
2	maize straw			WH-01
3	maize straw			D3-1 + WH-01
4	MSFDL			D3-1
5	MSFDL			WH-01
6	MSFDL			D3-1 + WH-01
7	glucose			D3-1
8	glucose			WH-01
9	glucose			D3-1 + WH-01

Note: WH-01 is the denitrifying bacterial strain that was previously selected in our laboratory.

. The general flow chart of the experiment is shown in Figure 1.

2.7.2. The Design of Denitrification Performance of MSFDL as a Carbon Source in Municipal Sewage

As above, 40 mL of municipal sewage with activated sludge was placed into a 50 mL conical flask for each denitrification process; 94.8 mg of maize straw flour, 0.44 mL of MSFDL, or 0.7 mL of glucose solution (50 g L⁻¹) was added to make the initial C/N ratio 6:1. The sewage solutions were cultivated in a closed manner under the conditions of 35 °C and 140 r/min, without any other bacterial agent. Since the actual denitrification time of most sewage treatment plants is about 2 to 6 h, a 4 h reaction liquid was taken from the actual municipal sewage plant to measure and detect NO₃-N, TN, and COD in order to determine MSFDL’s nitrogen reduction capacity. In addition, in order to detect the persistent stability of the carbon source in MSFDL, samples were taken and tested for NO₃-N, TN, and COD after 4 h, 12 h, 24 h, 48 h, and 72 h, to determine its nitrogen reduction capacity; the experiment was conducted in three parallel replicates.

3. Results

3.1. Screening and Identification

Thirteen bacterial strains were isolated from collected soil samples and cultured on CMC agar. Following incubation, the plates were stained with Gram’s iodine solution, and five strains exhibiting distinct clear zones were selected (Figure 2). All five selected strains demonstrated cellulose degradation capabilities.

Supplementary screening tests were carried out on five strains of cellulose-degrading bacteria obtained from preliminary screening. After incubation at room temperature, a crude cellulase solution was collected from the cellulase production broth, and each strain’s cellulase activity was measured (

Table 4. Cellulase activity of cellulolytic bacterial strains in supplementary screening tests.

Strain	Cellulase Activity (U/mL)	p-Value
N4-1	3.88 ± 0.06	p = 0.18
N1-1	4.37 ± 0.09	p = 0.07
N1-3	4.54 ± 0.13	p = 0.06
A1-1	4.57 ± 0.05	p = 0.30
D3-1	4.73 ± 0.08	p = 0.11

Note: “p-Value” was used to determine whether the observed differences in enzyme activity were statistically significant.

). At room temperature, the cellulase activity of strain D3-1 was the highest, 4.83 ± 0.06 U/mL; thus, the conditions of cellulase production by strain D3-1 were optimized.

3.2. Molecular Identification of the Cellulase-Producing Strain

As shown in Figure 3, phylogenetic analysis revealed that strain D3-1 is most closely related to Staphylococcus species. A sequence homology comparison, conducted on NCBI, demonstrated that the sequence similarity exceeded 99%. Consequently, strain D3-1 was identified as S. caprae.

3.3. Preliminary Single-Factor Experiments

Strain D3-1’s cellulase production at various incubation times is illustrated in Figure 4a. Throughout the fermentation process, cellulase activity progressively increased [24], reaching a peak of 6.28 ± 0.17 U/mL; after 96 h, activity it declined sharply, suggesting that cellulase production ceased or was markedly reduced. Consequently, an optimal cultivation duration of 96 h was recommended.

Temperature is a critical parameter influencing microbial enzyme production. Strain D3-1 cellulase production exhibited an initial increase, followed by a decrease as the temperature rose. The peak enzyme activity, of (9.31 ± 0.02) U/mL, was observed at a cultivation temperature of 40 °C (Figure 4b); these findings suggest that strain D3-1 may possess potential thermotolerance.

The effects of different pH values on strain D3-1 cellulase production are shown in Figure 4c. When the pH was 5.0, after 96 h of culture, the cellulase yield of strain D3-1 was the highest, at 11.06 ± 0.73 U/mL. The strain is more inclined to secrete cellulase in a weakly acidic environment, and, when the pH increases to 11.0, the strain still demonstrates a good cellulase production capacity.

After culturing with CMC agar as the carbon source for 96 h, the cellulase yield of strain D3-1 was the highest (9.14 ± 0.14 U/mL). The enzyme production of strain D3-1 with maize straw as the natural carbon source was better, 7.7 ± 0.90 U/mL at 96 h. The weight loss rate of the natural carbon source was between approximately 20% and 30%; the weight loss rate of maize straw was 26.76% (Figure 4d).

When yeast extract was used as the nitrogen source, after 96 h of culture, the cellulase yield of strain D3-1 was the highest (13.66 ± 0.75 U/mL), and the substrate weight loss rate of yeast extract as the nitrogen source (11.70%) was second only to that of urea (12.07%) (Figure 4e).

As shown in Figure 4f, strain D3-1 showed better cellulase generation at inoculation amounts of 1% and 4%, with cellulase activities of 9.44 ± 0.29 U/mL and 10.41 ± 0.28 U/mL, respectively.

3.4. The Cellulase Production Conditions of D3-1 Were Optimized Using Response Surface Methodology (RSM)

Based on the parameter ranges that were initially determined through single-factor experiments for cellulase production, CMC-Na served as the carbon source, and yeast extract as the nitrogen source. Incubation time (A), temperature (B), and pH value (C) were selected as the key variables, with cellulase activity serving as the optimization objective. The cellulase production conditions were analyzed through multiple regression modeling using Design Expert 13 software. According to the Box–Behnken design (BBD), 17 experimental runs were conducted in total, and the corresponding responses are presented in

Table 5. Treatment combination and response values of each factor level.

Run	(A) Time/h	(B) Temperature/°C	(C) pH	Cellulase Activity/(U/mL)
1	48	35	5	8.35
2	144	35	5	6.68
3	48	45	5	12.67
4	144	45	5	10.04
5	48	40	3	9.21
6	144	40	3	9.05
7	48	40	7	9.75
8	144	40	7	7.04
9	96	35	3	9.4
10	96	45	3	12.30
11	96	35	7	8.84
12	96	45	7	12.34
13	96	40	5	16.30
14	96	40	5	16.30
15	96	40	5	16.30
16	96	40	5	16.30
17	96	40	5	16.30

.

3.4.1. Analysis of Variance (ANOVA)

The response values obtained through the Box–Behnken experimental design are presented in

Table 6. Analysis of variance in response surface mode.

Source	Sum of Squares	df	Mean Square	F Value	p-Value
Model	198.21	9	22.02	269.12	<0.0001
A-Time	6.40	1	6.40	78.16	<0.0001
B-Temperature	24.79	1	24.79	302.87	<0.0001
C-pH	0.4932	1	0.4932	6.03	0.0438
AB	0.2066	1	0.2266	2.77	0.1401
AC	1.64	1	1.64	20.00	0.0029
BC	0.0889	1	0.0889	1.09	0.3320
A2	82.01	1	82.01	1002.10	<0.0001
B2	25.36	1	25.36	309.92	<0.0001
C2	41.14	1	41.14	502.73	<0.0001
Residual	0.5729	7	0.0818
Lack of Fit	0.5729	3	0.1910
Pure Error	0.0000	4	0.0000
Cor Total	198.79	16
R-Squared	0.9971
Adj. R-Squared	0.9934
Pred. R-Squared	0.9539
Adeq. Precision	42.4843

. The p-value of the model was less than 0.0001, indicating that the model is highly significant and statistically robust. The p-value of the misfitting term was 0.5729, suggesting that it is not significant. The misfitting term reflects the degree of fit between the model and the experimental data, and its insignificance indicates a good fit between the model and the experiment, with high reliability. The “Adeq. Precision” signal-to-noise ratio of the model was 42.4843, which is considered an ideal value for this model.

The determination coefficient (R²) of the model equation was 0.9971, the adjusted determination coefficient was 0.9934, and the predicted determination coefficient was 0.9539. The differences among these coefficients are all less than 0.2, suggesting that the polynomial model demonstrates sufficient accuracy and broad applicability. The coefficient of variation (CV%) was extremely low, at 2.47, indicating high precision and reliability of the experimental values. In summary, the use of this model’s equation to predict optimal cellulase production conditions is sufficiently reliable.

In this study, A and B are significant model terms; judging from the results, the constructed response surface model is reasonable for the cellulase production optimization.

The final regression model equation, based on the encoding factor, is as follows:

Cellulase activity (U/mL) = 16.3 − 0.8942 × A + 1.76 × B − 0.2483 × C − 0.2380 × AB − 0.6397 × AC + 0.1491 × BC − 4.41 × A² − 2.45 × B² − 3.13 × C²

The final regression model equation, considering practical factors, is as follows:

Cellulase activity (U/mL) = −193.6595 + 0.4212 × A + 8.2264 × B + 7.7339 × C − 0.0009 × AB − 0.0066 × AC + 0.0149 × BC − 0.0019 × A² − 0.0981 × B² − 0.7814 × C²

3.4.2. Optimization of Cellulase Production

In this study, we used Design Expert 13 to plot the response surface in order to evaluate the influences of different parameters and their interactions on cellulase production.

Figure 5 shows three three-dimensional response surface diagrams and their respective contour maps.

By solving the regression equation and analyzing the contour map of the response surface, the optimal cellulase production conditions (A = 90.7 h, B = 41.8 °C, C = 4.9) were estimated. Under the optimized operating conditions, the cellulase activity was predicted to be 16.67 U/mL based on the fitting equation; subsequently, three additional experiments were conducted to confirm the prediction. The average cellulase activity obtained from the experiments in the independent laboratory was 16.04 ± 0.41 U/mL, which was in good agreement with the predicted response value.

3.5. Product Analysis of Maize Straw Degradation by Strain D3-1

The products of maize straw flour degradation by strain D3-1 were removed at time 0 h and the optimal enzyme production at time (96 h) and then detected using GC-MS. The results are shown in

Table 7. Analysis of product sample content.

Detected Substance	0 h (ug/mL)	96 h (ug/mL)	Fold Change
Sucrose	0.00225	0.00018	0.072
D-Sorbitol	0.00106	0.00042	0.396
D-Arabinose	0.00168	0.00052	0.309
D-Fructose	0.00395	0.00012	0.030
D-Galactose	0.00060	0.00039	0.65
D-Galacturonic acid	0.00041	0.00017	0.414
Glucose	0.00969	0.00015	0.015
D-Glucuronic acid	0.00031	0.00042	1.355
Inositol	0.01044	0.00043	0.041
Levoglucosan	0.00028	0.00037	1.321
L-Rhamnose	0.00028	0.00044	1.571
D-Ribono-1,4-Lactone	0.00003	0.00020	6.667
D-Ribose	0.00010	0.00049	4.9
Raffinose	N/A	0.00013	>1
Cellobiose	0.00085	0.00033	0.388
Phenylglucoside	0.00014	0.00023	1.643
Maltose	0.02074	0.00016	0.008
Trehalose	0.00283	0.00032	0.113
Xylitol	0.00042	0.00049	1.167
D-Arabinitol	0.00287	0.00032	0.111
D-Xylose	0.00027	0.00036	1.333
1,5-Anhydroglucitol	0.00030	N/A	<1
L-Fucose	0.00016	N/A	<1
D-Mannose	0.00086	N/A	<1
2-Acetamido-2-deoxy-D-glucopyranose	0.00051	N/A	<1

Note: The symbols are defined as follows: not applicable (no yield) (N/A); increase in substance concentration (>1); decrease in substance concentration (<1).

, which shows that the main products were D-arabinose, xylitol, ribose, L-rhamnose, and D-sorbitol, all of which are monosaccharides.

3.6. Denitrification Application Results

3.6.1. The Denitrification Performance of Different Bacterial Agents in Simulated Sewage with Different Carbon Sources

In Figure 6a, only the denitrifying bacterial agent WH-01 was used in the three experiments; the processing object was simulated sewage. When glucose was used as the carbon source, WH-01 had the most significant removal effect on NO₃-N, with removal reaching 93 mg/L; when MSFDL was used as the carbon source, the removal rate was 81 mg/L; when maize straw was used as the carbon source, the removal rate was only 23 mg/L (Figure 6a). In terms of TN removal, the conditions of glucose and MSFDL remained at around 70 mg/L, while removal under the maize straw carbon source was only 15 mg/L. The removal trend of DTN was consistent with the above indicators.

In Figure 6b, only bacterial agent D3-1 was used in the three experiments; the processing object was simulated sewage. When maize straw was used as the carbon source, the TN reduction ability of strain D3-1 was relatively weak; when glucose was used as the carbon source, strain D3-1 TN reduction was approximately 50 mg/L; when MSFDL was used as the carbon source, TN reduction was approximately 45 mg/L. Under the same conditions, D3-1 is obviously weaker than WH-01; it is speculated that strain D3-1 has weak denitrification ability, but for NO₃-N reduction, when MSFDL was used as the carbon source, the NO₃-N reduction rate of strain D3-1 was far greater than those with the other carbon sources. MSFDL may be more conducive to the growth and metabolism of strain D3-1, thereby increasing the NO₃-N reduction.

In Figure 6c, the denitrifying bacterial agents WH-01 and D3-1 were both used in the three experiments; the processing object was simulated sewage. Figure 6c shows the effects of the dual-bacterial combination on nitrogen removal under different carbon source conditions, with the results showing that all three carbon sources promoted the removal of NO₃-N, TN, and DTN by the two bacteria. Among them, the experimental group with glucose as the carbon source showed the best nitrogen removal effects, and the reduction rates of NO₃-N, TN, and DTN reached 118 mg/L, 125 mg/L, and 128 mg/L, respectively. MSFDL as a carbon source produced the second best result, with the reduction rates of NO₃-N, TN, and DTN reaching 104%, 88%, and 89% of those of glucose, respectively, while the removal efficiency of maize straw as a carbon source was the lowest. Furthermore, the dual-bacteria combination exhibited a synergistic effect in the glucose group. The reductions in NO₃-N, TN, and DTN were 21%, 43%, and 32% higher, respectively, compared to those achieved by glucose combined with the single bacterial strain WH-01, thereby further enhancing nitrogen removal efficiency.

3.6.2. The Denitrification Performance of MSFDL as a Carbon Source in Municipal Sewage

(1)

The nitrogen removal effects in municipal sewage for 4 h under different carbon sources are as follows:

The results are presented in Figure 7. As the processing object was municipal sewage, there was no denitrifying bacterial agent used in the experiments. When maize straw was used as a carbon source, the reduction in nitrogen did not exceed 10 mg/L, indicating limited effectiveness. Glucose demonstrated the best performance as a carbon source, with reductions in NO₃-N and TN both exceeding 15 mg/L, and its treatment efficiency was set as the reference (100%). When MSFDL was used as a carbon source, the reductions in NO₃-N and TN were approximately 70% of those achieved with glucose; these findings suggest that, as an external carbon source, MSFDL can be effectively utilized in the denitrification of municipal sewage, but it is slightly inferior to glucose.

(2)

The nitrogen removal effects in municipal sewage for 72 h under different carbon sources are as follows:

Continuing with the above experiment, in order to detect the persistent stability of MSFDL as a carbon source throughout the denitrification process, samples were taken and tested for NO₃-N, TN, and COD after 4 h, 12 h, 24 h, 48 h, and 72 h to determine its nitrogen reduction capacity. As shown in Figure 8a, the NO₃-N removal rate in the glucose group was the fastest. The NO₃-N concentration decreased from 31 mg/L to 5 mg/L within 24 h and was nearly completely removed after 72 h. The MSFDL group was the second most successful, with the reduction in NO₃-N at 12 h close to 80% of that of glucose, and nitrate was nearly completely removed after 72 h. Maize straw worked at a slower rate compared to the other two groups, but all three were almost completely effective after 72 h.

As shown in Figure 8b, the TN removal effects of the glucose group were the best; the TN concentration dropped to 9 mg/L after 24 h and was lower than 5 mg/L after 48 h. Although MSFDL’s nitrogen reduction ability was not as good as that of glucose within 4 h, it was almost identical after 24 h. While the effects of maize straw were not ideal, TN was reduced to 10 mg/L within 48 h; however, it rose after a further 48 h.

The decrease in COD indicates the consumption of carbon sources. As shown in Figure 8c, the COD with glucose as the carbon source decreased rapidly within 12 h, indicating that glucose was utilized rapidly. MSFDL gradually approached the level of the glucose group at 24 h and thereafter, approaching 100%, indicating that cellulose gradually releases soluble sugars through microbial degradation, providing a continuous carbon source for denitrification and demonstrating the potential to replace glucose as a carbon source. After 48 h, the COD under all three conditions was reduced to below 50 mg/L.

4. Conclusions and Discussion

In the field of environmental engineering, identifying a bacterial strain that can degrade cellulose enzymes is beneficial for resource recycling. A strain designated D3-1, with relatively high cellulase production activity, was screened; this strain, identified as S. caprae, is different from the conventional cellulose-degrading bacteria that have been reported previously, such as Bacillus, Cellulomonas, Clostridium, Ruminococus, etc., as the application of such strains in environmental treatments is very rare. Experiments have shown that the cellulose-degrading bacterial strain D3-1 can not only efficiently produce cellulase but can also adapt to a wide pH range, as well as withstanding moderately high temperatures, thereby expanding its application in the industrial field. In addition, S. caprae D3-1 can be used to assist the denitrifying bacterial strain WH-01 in nitrogen removal, as they demonstrate synergistic effects.

First, the cellulase-producing strain D3-1 was identified as S. caprae. Subsequently, culture conditions, including incubation time, temperature, pH, inoculum size, carbon source, and nitrogen source, were optimized for cellulase production. Based on these results, RSM was used to further optimize the fermentation conditions to produce cellulase. The RSM optimization results demonstrated that the optimal conditions were a culture time of 91.7 h, a temperature of 41.8 °C, and a pH of 4.9; under these conditions, cellulase activity reached 16.67 U/mL, representing an approximately 165% increase compared to pre-optimization levels. Additionally, GC-MS analysis of the degradation liquid from maize straw degraded by strain D3-1 revealed that the primary components of the cellulose degradation liquid were monosaccharides. Key products identified included D-arabinose, xylitol, ribose, L-rhamnose, and D-sorbitol.

Carbon sources should be added to the municipal wastewater treatment process to ensure the normal growth of bacteria [25]. Strain D3-1 nitrogen removal efficiency in simulated sewage was approximately 50% of that achieved by the denitrifying strain WH-01. However, when strain D3-1 was combined with the denitrifying strain WH-01, the TN removal rate of the two strains together increased by 43% compared to that of WH-01 alone. Therefore, it is speculated that strain D3-1 has some denitrification ability. In future applications, it will be also possible to explore the direct addition of its degradation liquid to the denitrification system without filtration to supplement the carbon source, potentially decreasing the number of operation steps, reducing economic costs, and maximizing the utilization of this strain’s biological characteristics; therefore, the application scope of this strain may be even wider.

In municipal sewage experiments, the nitrogen removal capacity of the denitrification system with the addition of D3-1 degradation liquid as a carbon source was 70% of that achieved with the glucose carbon source, thus ranking second, indicating that, in real-world sewage application, the single-component glycogen may be utilized more rapidly by the bacteria in the system; there may also be other factors in the actual wastewater that affect its nitrogen reduction effect. Targeted exploration of how to better adapt cellulose degradation liquid as a carbon source is a direction for future research. Within 24 h, the reduction in TN approached 97% of that achieved with glucose, while the reduction in NO₃-N approached 80% of that achieved with glucose, results that are consistent with the experiment on simulated sewage; that is, after 24 h of denitrification, the cellulose degradation liquid plays the same carbon source role as glucose. It is speculated that, in a long-term anaerobic reaction environment, the denitrifying bacterial community in the activated sludge gradually adapts to the degradation liquid carbon source and gradually completes its normal metabolic process. After 72 h, TN was almost completely removed. These experimental results indicate that, in practical sewage treatment applications, MSFDL shows potential as a substitute for the glucose carbon source. However, within a reaction duration of 4 h, its nitrogen removal efficiency is slightly inferior to that of the glucose carbon source.