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Citation: Wu, Z.; Zhang, X.; Li, R.;
Hui, J.; Deng, L.; Kim, I.; Wei, J.; Yao, J.;
Lei, X. Effects of Cellulase and
Lactiplantibacillus plantarum on
Chemical Composition, Fermentation
Characteristics, and Bacterial
Community of Pennisetum giganteum
z.x.lin Silage. Agriculture 2025,15, 97.
https://doi.org/10.3390/
agriculture15010097
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licenses/by/4.0/).
Article
Effects of Cellulase and Lactiplantibacillus plantarum on
Chemical Composition, Fermentation Characteristics, and
Bacterial Community of Pennisetum giganteum z.x.lin Silage
Zhili Wu 1,2,† , Xiongfei Zhang 1 ,2 , †, Rongnuo Li 1,2, Jingtao Hui 1,2, Lu Deng 1,2 , Inho Kim 3, Jie Wei 4,
Junhu Yao 1,2 and Xinjian Lei 1,2,*
1College of Animal Science and Technology, Northwest A&F University, Xianyang 712100, China;
wzl1295354914@163.com (Z.W.); xiongfeizhang2024@nwafu.edu.cn (X.Z.); lrn000310@163.com (R.L.);
19939369481@163.com (J.H.); denglu128128@163.com (L.D.); yaojunhu2008@nwafu.edu.cn (J.Y.)
2Key Laboratory of Livestock Biology, Northwest A&F University, Xianyang 712100, China
3Department of Animal Resource and Science, Dankook University, Cheonan 31116, Republic of Korea;
inhokim@dankook.ac.kr
4Xi’an Architectural Design and Research Institute Co., Ltd., Xi’an 710000, China; 18829075888@163.com
*Correspondence: leixinjian@nwafu.edu.cn
†These authors contributed equally to this work.
Abstract: In order to explore the effects of additives on the chemical composition,
fermentation characteristics, and bacterial community of Pennisetum giganteum z.x.lin
silage, Pennisetum giganteum z.x.lin was ensiled with no additives (CON), cellulase (CE),
Lactiplantibacillus plantarum
(LP), or the combination of cellulase and Lactiplantibacillus plan-
tarum (LPCE) at room temperature for 60 days, respectively. The results indicated that
LPCE had the highest dry matter (DM) content. Compared with CON, LP exhibited higher
(p< 0.05) levels of water-soluble carbohydrate (WSC), crude protein (CP), and lactic acid
(LA), along with a higher (p< 0.05) ratio of LA/acetic acid (AA). Meanwhile, silage inoc-
ulated with cellulase (CE and LPCE) showed lower (p< 0.05) contents of acid detergent
fiber (ADF) and neutral detergent fiber (NDF) than CON. Furthermore, additive treatments
improved the bacterial community composition of silage, and Lactobacillus was abundant in
LPCE (LDA score > 4.0). Compared with CE and LP, LPCE more effectively promoted the
transformation of microbial functions, resulting in an upregulated (p< 0.05) carbohydrate
metabolism and a downregulated (p< 0.05) membrane transport. In conclusion, cellulase or
Lactiplantibacillus plantarum improved the silage quality of Pennisetum giganteum z.x.lin by
reducing the fiber content or enhancing LA fermentation, and their combination exhibited
a powerful ability to establish a bacterial community dominated by Lactobacillus, which
facilitated the production of high-quality silage.
Keywords: bio-additives; ensiling; enzyme-bacteria synergy; microbial diversity;
Pennisetum giganteum z.x.lin
1. Introduction
As people’s living standards improve and the population size continuous to grow,
people’s demand for high-quality meat and dairy products is also on the rise. However, the
efficient production of livestock products depends on a continuous supply of high-quality
feed, which is not available in many regions worldwide. For instance, in northwest China
and southwest Botswana, land degradation has hampered forage production, making local
Agriculture 2025,15, 97 https://doi.org/10.3390/agriculture15010097
Agriculture 2025,15, 97 2 of 16
high-quality forage scarce [
1
,
2
]. In addition, drought, a key environmental factor threaten-
ing plant growth, has become increasingly frequent and severe due to climate warming [
3
].
Therefore, it is urgent to develop novel feed resources to address the current and fu-
ture challenges of feed shortages caused by environmental factors.
Pennisetum giganteum
z.x.lin is a typical C
4
plant, characterized by a strong environmental stress resistance, well-
developed root system, and high biomass [
4
]. Currently, Pennisetum giganteum z.x.lin is
extensively utilized for improving soil quality [
5
] and producing biomass fuel [
6
]. Mean-
while,
Pennisetum giganteum
z.x.lin is also used as livestock feed due to its high yielding
ability and strong environmental adaptability [
4
]. However, the high fiber content, unstable
supply, and low water-soluble carbohydrate (WSC) content of Pennisetum giganteum z.x.lin
deeply limit its application in animal husbandry.
Ensiling is an effective approach to preserving green forage. The application of various
additives has further diversified feed resources and improved silage quality. Lactic acid
bacteria (LAB) inoculants are extensively employed to enhance lactic acid (LA) fermenta-
tion and prevent silage spoilage. For example, Lactiplantibacillus plantarum, which belongs
to the group of homofermentative LAB, has been proven to benefit the silage quality of
various forage crops, including alfalfa [
7
]. Additionally, Lactiplantibacillus plantarum have
been shown to enhance the microbial community of whole-plant corn silage, reducing the
levels of certain mycotoxins and the relative abundance of harmful bacteria [
8
]. However,
LAB inoculants have little effect on the nutrient digestibility of silage with a high fiber
content. Cellulase can break down structural carbohydrates into oligosaccharides and
monosaccharides, which can be used by LAB for generating LA and reducing the pH of
silage [
9
]. Similar to Pennisetum giganteum z.x.lin, Caragana korshinskii has the characteristics
of a well-developed root system, rapid growth, and high fiber content [
10
]. A previous
study showed that inoculation with cellulase and Lactiplantibacillus plantarum could effec-
tively enhance the fermentation quality of Caragana korshinskii silage [
11
]. However, the
palatability and digestibility of Caragana korshinskii silage are still poor due to the presence
of thorns and other factors [
12
]. Pennisetum giganteum z.x.lin is widely planted and applied
in northwest China. Surprisingly, in areas affected by soil erosion, the fresh weight yield
of Pennisetum giganteum z.x.lin can reach up to 178 t per hectare [
6
]. Although certain
characteristics of Pennisetum giganteum z.x.lin are not conducive to silage fermentation,
with the application of appropriate additives, it has the potential to become a practical
silage material, particularly in regions with poor soil and arid climates. But few stud-
ies have evaluated the effects of cellulase and Lactiplantibacillus on the silage quality of
Pennisetum giganteum z.x.lin.
Silage quality is closely linked to its bacterial community. In order to investigate
the role of silage additives in the fermentation process of Pennisetum giganteum z.x.lin,
analyzing the bacterial community is essential. With the advancement of next-generation
sequencing technology, 16s rRNA gene sequencing has been extensively and deeply ap-
plied to the analysis of bacterial community [
13
]. At present, there are insufficient data
on the effects of cellulase and Lactiplantibacillus plantarum on the bacterial community of
Pennisetum giganteum
z.x.lin silage. Therefore, this experiment was conducted to explore
the effects of cellulase and Lactiplantibacillus plantarum on the chemical composition, fer-
mentation characteristics, and bacterial community of Pennisetum giganteum z.x.lin silage,
with the hypothesis that additive treatments could improve the silage quality.
2. Materials and Methods
2.1. Silage Preparation
Pennisetum giganteum z.x.lin was cultivated at a plot (about 1000 m
2
) of the “JunCao”
planting base of Shaanxi Fengqing Ecological Development Co., Ltd. (Xi’an, China) (a
Agriculture 2025,15, 97 3 of 16
warm, temperate continental climate, N 33
◦
44
′
50
′′
–35
◦
10
′
30
′′
, E 109
◦
20
′
17
′′
–109
◦
54
′
48
′′
,
altitude 554 m, Pucheng, China), with no fertilizers, irrigation, or herbicides. Pennisetum
giganteum z.x.lin cultivated at the base was about 2.5 m in height (elongation stage) and was
harvested using a silage harvester (4QZ-18B; Henan Changjun Agricultural Machinery Co.,
Ltd., Xinxiang, China), leaving a stubble of about 15 cm. During the harvesting process,
the Pennisetum giganteum z.x.lin was chopped into 2–3 cm pieces by the silage harvester
for making silage. The initial characteristics of pre-ensiled Pennisetum giganteum z.x.lin are
presented in Table 1.
Table 1. Characteristics (of g/kg DM) of the pre-ensiled Pennisetum giganteum z.x.lin.
DM CP NDF ADF
Hemicellulose
WSC
of g/kg FM
245.2 99.7 608.4 343.9 264.5 38.0
ADF, acid detergent fiber; CP, crude protein; DM, dry matter; FM, fresh matter; NDF, neutral detergent fiber; and
WSC, water-soluble carbohydrate.
Four treatments were established, as follows: (i) control (CON), treated with 5 mL
of double-distilled water/kg fresh material (FM); (ii) cellulase (CE, with an application
dose of 100 IU/g FM) [
14
]; (iii) Lactiplantibacillus plantarum (LP, with an application dose
of 1.0
×
10
6
colony-forming units (CFU)/g FM) [
13
]; and (iv) cellulase + Lactiplantibacillus
plantarum (LPCE, with application doses of cellulase and Lactiplantibacillus plantarum of
100 IU/g FM and 1.0
×
10
6
CFU/g FM, respectively). The cellulase powder (
5×105IU/g
)
and Lactiplantibacillus plantarum (Lp194, a native strain isolated from rumen contents;
1×1011 CFU/g)
were obtained from Guangdong VTR Bio-Tech Co., Ltd. (Zhuhai, China)
and Jiangsu Wecare Biotechnology Co., Ltd. (Suzhou, China), respectively. The additives
were mixed in 15 mL of double-distilled water and uniformly applied to a pile of ensiling
material weighing 3 kg. Then, each pile of forage was thoroughly blended and placed
into 30
×
40 cm polyethylene plastic bags with 1 kg of forage per bag. After that, each
polyethylene plastic bag was drained of air and sealed using a vacuum laminator (DLYS891;
Deli Group Co., Ltd., Ningbo, China). Then, 12 bags (4 treatments
×
3 replicates) were
kept at room temperature for 60 days. Upon fermentation’s completion, each bag of
silage samples was split into three equal portions, as follows: the first portion served for
nutritional value analysis, the second for fermentation parameter analysis, and the third
for microbial diversity analysis.
2.2. Chemical Analysis
The silage samples from the first portion were dried by an oven (DHG-9030A, Shanghai
Yiheng Scientific Instruments Co., Ltd., Shanghai, China) at 65
◦
C for 48 h. Then, the dried
silage samples were ground via a mill (HK-08B; Guangzhou Xulang Machinery Equipment
Co., Ltd., Guangzhou, China) to pass over a 1 mm sieve. The dry matter (DM; method
930.15) and crude protein (CP; method 990.03) of the silage samples were assessed following
the guidelines established by the Association of Official Analytical Chemists [
15
]. The CP
content was converted by total nitrogen (TN) with a conversion factor of 6.25. As described
by Van Soest et al. [
16
], neutral detergent fiber (NDF) was measured using a heat-stable
α
amylase, and then acid detergent fiber (ADF) was determined with acid detergent. The
contents of NDF and ADF were inclusive of the residual ash. The hemicellulose content
was derived by subtracting ADF from NDF. According to the manufacturer’ instructions,
the WSC content was measured through a plant-soluble sugar content test kit (A145-1-1;
Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
The second portions of the silage samples (10 g) were homogenized in 90 mL of double-
distilled water and stored at 4
◦
C for 24 h [
17
]. Once fully mixed, the liquid extracts were
Agriculture 2025,15, 97 4 of 16
filtered through a quantitative filtered paper. The pH value of each filtrate was assessed
with a pH meter (HI 90s24C; HANNA Instruments, Woonsocket, RI, USA). Subsequently,
the filtrate was passed through a 0.45
µ
m membrane, and then the concentrations of acetic
acid (AA), propionic acid (PA), and butyric acid (BA) were analyzed via gas chromatogra-
phy (GC7890A; Agilent, Santa Clara, CA, USA), as detailed by Wang et al. [
18
]. The LA
concentration was measured by an LA assay kit (A019-2-1; Nanjing Jiancheng Bioengi-
neering Institute, Nanjing, China). The ammonia nitrogen (NH
3
-N) content was assessed
with reference to the guidelines of Broderick and Kang [
19
]. The contents of WSC, LA, and
NH
3
-N were all measured by colorimetric methods with a microplate reader (UV-2300;
Shimadzu, Kyoto, Japan).
2.3. Bacterial Community Analysis
The total bacterial DNA from the third portion was extracted through a MagAtrract
PowerSoil Pro DNA Kit (Qiagen, Hilden, Germany) in accordance with the manufac-
turer’s protocol. Next, the quality of DNA was assessed by 1% agarose gel electrophoresis.
Following that, the concentration and purity of DNA were assessed by NanoDrop2000
(Thermo Fisher Scientific, Waltham, MA, USA). Then, polymerase chain reaction (PCR)
was conducted to amplify the 16S rRNA gene (V5–V7) with a pair of specific primers, 799F
(5
′
-AACMGGATTAGATACCCKG-3
′
) and 1993R (5
′
-ACGTCATCCCCACCTTCC-3
′
), using
a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA) [
20
]. The products of PCR were
analyzed through 2% agarose gel electrophoresis and then processed with a PCR Clean-Up
Kit (Shanghai Meiji Yuhua Biomedical Technology Co., Ltd., Shanghai, China). Next, the
amplified fragments were quantified by Qubit 4.0 (Thermo Fisher Scientific, Waltham,
MA, USA) for following sequencing on the NovaSeq platform (Illumina, San Diego, CA,
USA). Based on QIIME2 [
21
], the original 16S rRNA gene reads were filtered, merged,
screened, and denoised using DADA2 to obtain the representing sequence and abundance
information of amplicon sequence variants (ASVs). The ASVs were analyzed for taxonomic
annotation by RDP Classifier (v11.5) and the SILVA 16S rRNA database (v138) with a confi-
dence coefficient of 0.7. Five commonly used alpha-diversity-related indices, consisting
of Chao, Ace, Shannon, Simpson, and Coverage, were derived using Mothur (v1.30.2) to
assess the diversity and richness of the bacterial community. Based on the weighted Unifrac
distance, the beta diversity was assessed with principal coordinate analysis (PCoA) to
identity the differences between the bacterial community structures among treatments with
the ANOSIM test. According to the results of taxonomic analysis, bar charts of the bacterial
community composition at the phylum and genus were constructed using R (v3.3.1). The
bacterial differences among treatments were identified via linear discriminant analysis
effect size (LEfSe). Using PICRUSt2 (v2.2.0) and the Kyoto Encyclopedia of Genes and
Genomes (KEGG) database, functional genes were predicted and then matched to their
corresponding metabolic pathways.
2.4. Statistical Analysis
The basic data on chemical composition, fermentation characteristics, and bacterial
community were organized by Excel 2019 and assessed through the one-way analysis of
variance procedure of SPSS 27.0 (IBM Corp, Armonk, NY, USA), with each fermentation
bag considered as an experimental unit (n= 3 per treatment). The statistical model was
as follows:
Yi=µ+αi+εi(1)
where Y
i
is the is the observed value,
µ
is the overall mean,
αi
is the treatment effect (i= CON,
CE, LP, and LPCE), and
εi
is the experimental error. Tukey’s test was employed to compare
the mean values. The data on the bacterial community were processed through the resources
Agriculture 2025,15, 97 5 of 16
available on the Majorbio Cloud Platform (https://cloud.majorbio.com/page/tools/, accessed
on 10 May 2024). Statistical significance was declared when the pvalue < 0.05.
3. Results
3.1. Effects of Additives on Chemical Composition and Fermentation Characteristics of Pennisetum
giganteum z.x.lin Silage
The chemical composition of Pennisetum giganteum z.x.lin silage is given in Table 2. LP
showed a higher (p< 0.05) CP content than CON, but a lower (p< 0.05) DM content than
LPCE. Both CE and LPCE showed a lower (p< 0.05) NDF content compared with CON and
LP. In addition, CE, LP, and LPCE all exhibited a lower (p< 0.05) ADF content compared
with CON. Meanwhile, LPCE showed a lower (p< 0.05) ADF content compared with LP.
However, no significant difference (p> 0.05) was observed in hemicellulose among the four
treatments. Moreover, LP exhibited a higher (p< 0.05) WSC content than CON.
Table 2. Effects of additives on chemical composition of Pennisetum giganteum z.x.lin silage.
Item Treatments SEM p-Value
CON CE LP LPCE
DM (g/kg FM) 223.8 ab 226.9 ab 217.1 b229.5 a1.73 0.031
CP (g/kg DM) 95.2 b101.5 ab 104.8 a103.5 ab 1.38 0.032
NDF (g/kg DM) 607.2 a537.4 b588.1 a548.1 b9.34 0.001
ADF (g/kg DM) 390.7 a334.7 bc 344.6 b306.1 c9.81 <0.001
Hemicellulose (g/kg DM)
216.5 202.7 243.5 242.0 7.43 0.125
WSC (g/kg DM) 23.6 b29.8 ab 34.2 a24.2 ab 1.613 0.030
a–c
Values with different letters in the same row are significantly different (p< 0.05). CON, no additives; CE,
added cellulase (100 IU/g FM); LP, added Lactiplantibacillus plantarum (1
×
10
6
CFU/g FM); LPCE, added cellulase
(100 IU/g FM) and Lactiplantibacillus plantarum (1
×
10
6
CFU/g FM). ADF, acid detergent fiber; CP, crude
protein; DM, dry matter; FM, fresh matter; NDF, neutral detergent fiber; SEM, standard error of mean; and WSC,
water-soluble carbohydrate.
The fermentation characteristics of Pennisetum giganteum z.x.lin silage are presented in
Table 3. Interestingly, there was no significant difference (p> 0.05) in pH among the four
treatments. Additionally, LP had the highest LA content (p< 0.05) and a higher (p< 0.05)
ratio of LA/AA than CON. However, the additive treatments had little impact (p> 0.05) on
the AA content. LPCE exhibited a higher (p< 0.05) NH
3
-N content compared with CON.
Meanwhile, the contents of PA and BA were low and even undetectable in all treatments.
Table 3. Effects of additives on fermentation characteristics of Pennisetum giganteum z.x.lin silage.
Item
Treatments
SEM p-Value
CON CE LP LPCE
pH 3.75 3.69 3.73 3.79 0.03 0.672
LA (g/kg DM) 9.10 b10.98 b22.56 a11.59 b1.69 <0.001
AA (g/kg DM) 21.55 15.26 20.21 12.12 1.56 0.089
PA (g/kg DM) 0.23 0.25 0.20 ND - -
BA (g/kg DM) 0.08 ND ND ND - -
Ratio of LA/AA 0.42 b0.80 ab 1.16 a0.98 ab 0.10 0.037
NH3-N (g/kg TN) 81.3 b114.0 ab 114.0 ab 165.3 a11.31 0.034
a,b
Values with different letters in the same row are significantly different (p< 0.05). CON, no additives; CE, added
cellulase (100 IU/g FM); LP, added Lactiplantibacillus plantarum (1
×
10
6
CFU/g FM); LPCE, added cellulase
(100 IU/g FM) and Lactiplantibacillus plantarum (1
×
10
6
CFU/g FM). AA, acetic acid; BA, butyric acid; DM, dry
matter; LA, lactic acid; ND, no detected; NH
3
-N, ammonia nitrogen; PA, propionic acid; SEM, standard error of
mean; and TN, total nitrogen.
Agriculture 2025,15, 97 6 of 16
3.2. Effects of Additives on Microbial Diversity of Pennisetum giganteum z.x.lin Silage
The bacterial alpha-diversity-related indices of the Pennisetum giganteum z.x.lin silage
are presented in Table 4. The coverage values of all silage samples were more than 99.9%.
Meanwhile, there was no significant difference (p> 0.05) in Chao and Ace values among
the four treatments. Both CE and LPCE showed a lower (p< 0.05) Shannon value compared
with CON, while LPCE also exhibited a lower (p< 0.05) Shannon value compared with
LP. In addition, both CE and LPCE exhibited a higher (p< 0.05) Simpson value than CON.
The PCoA plot showed that PCoA1 and PCoA2 explained the changes in the bacterial
community structure by 85.63% and 6.71%, respectively (Figure 1). In the ANOSIM test,
p= 0.005
and R = 0.6543, indicating that the bacterial community structure of the silage was
significantly changed after the addition of additives. Meanwhile, the bacterial community
structure of CON was apparently separated from that of other treatments through PCoA1.
Table 4. The alpha diversity of bacterial community in Pennisetum giganteum z.x.lin silage.
Items
Treatments
SEM p-Value
CON CE LP LPCE
Chao 250.3 184.4 287.0 193.4 17.82 0.097
Ace 251.4 186.1 291.4 196.5 17.48 0.102
Shannon 3.008 a2.226 bc 2.670 ab 2.137 c0.1149 0.001
Simpson 0.1155 b0.2803 a0.1966 ab 0.2609 a0.0223 0.008
Coverage 0.9998 0.9998 0.9993 0.9996 0.0001 -
a–c
Values with different letters in the same row are significantly different (p< 0.05). CON, no additives; CE, added
cellulase (100 IU/g FM); LP, added Lactiplantibacillus plantarum (1
×
10
6
CFU/g FM); and LPCE, added cellulase
(100 IU/g FM) and Lactiplantibacillus plantarum (1 ×106CFU/g FM). SEM, standard error of mean.
Agriculture 2025, 15, x FOR PEER REVIEW 6 of 17
Table 3. Effects of additives on fermentation characteristics of Pennisetum giganteum z.x.lin silage.
Item Treatments SEM p-Value
CON CE LP LPCE
pH 3.75 3.69 3.73 3.79 0.03 0.672
LA (g/kg DM) 9.10
b
10.98
b
22.56
a
11.59
b
1.69 <0.001
AA (g/kg DM) 21.55 15.26 20.21 12.12 1.56 0.089
PA (g/kg DM) 0.23 0.25 0.20 ND - -
BA (g/kg DM) 0.08 ND ND ND - -
Ratio of LA/AA 0.42
b
0.80
ab
1.16
a
0.98
ab
0.10 0.037
NH
3
-N (g/kg TN) 81.3
b
114.0
ab
114.0
ab
165.3
a
11.31 0.034
a,b
Values with different leers in the same row are significantly different (p < 0.05). CON, no addi-
tives; CE, added cellulase (100 IU/g FM); LP, added Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM);
LPCE, added cellulase (100 IU/g FM) and Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM). AA, acetic
acid; BA, butyric acid; DM, dry maer; LA, lactic acid; ND, no detected; NH
3
-N, ammonia nitrogen;
PA, propionic acid; SEM, standard error of mean; and TN, total nitrogen.
3.2. Effects of Additives on Microbial Diversity of Pennisetum giganteum z.x.lin Silage
The bacterial alpha-diversity-related indices of the Pennisetum giganteum z.x.lin silage
are presented in Table 4. The coverage values of all silage samples were more than 99.9%.
Meanwhile, there was no significant difference (p > 0.05) in Chao and Ace values among
the four treatments. Both CE and LPCE showed a lower (p < 0.05) Shannon value com-
pared with CON, while LPCE also exhibited a lower (p < 0.05) Shannon value compared
with LP. In addition, both CE and LPCE exhibited a higher (p < 0.05) Simpson value than
CON. The PCoA plot showed that PCoA1 and PCoA2 explained the changes in the bacte-
rial community structure by 85.63% and 6.71%, respectively (Figure 1). In the ANOSIM
test, p = 0.005 and R = 0.6543, indicating that the bacterial community structure of the silage
was significantly changed after the addition of additives. Meanwhile, the bacterial com-
munity structure of CON was apparently separated from that of other treatments through
PCoA1.
Figure 1. Principal coordinates analysis (PCoA) plot conducted based on weighted Unifrac distance
of bacterial community. ANOSIM value, R = 0.6543, p= 0.005. CON, no additives; CE, added
cellulase (100 IU/g FM); LP, added Lactiplantibacillus plantarum (1
×
10
6
CFU/g FM); and LPCE,
added cellulase (100 IU/g FM) and Lactiplantibacillus plantarum (1 ×106CFU/g FM).
The bacterial community composition of Pennisetum giganteum z.x.lin silage at the
phylum and genus levels is shown in Figure 2. In the silage, Firmicutes (CON, 57.6%; CE,
78.6%; LP, 73.6%; and LPCE, 88.1%) and Proteobacteria (CON, 46.4%; CE, 72.4%; CE, 63.1%;
and LPCE, 79.3%) were the major phyla, with a total relative abundance of >98.8% in all
Agriculture 2025,15, 97 7 of 16
treatments (Figure 2a). As expected, Lactobacillus (CON, 3.86%; CE, 5.08%; LP, 1.78%; and
LPCE, 85.16%) became the dominant genus after ensiling (Figure 2b). Compared with CON,
CE, LP, and LPCE all exhibited a decreased relative abundance of Serratia (CON, 20.9%; CE,
7.8%; LP, 9.9%; and LPCE, 4.8%), Pantoea (CON, 5.7%; CE, 2.7%; LP, 3.9%; and LPCE, 1.1%),
Klebsiella (CON, 3.9%; CE, 2.8%; LP, 2.9%; and LPCE, 2.0%), and Pediococcus (CON, 5.2%;
CE, 0.9%; LP, 2.6%; and LPCE, 1.4%), while exhibiting an increased relative abundance
of Lactococcus (CON, 2.4%; CE, 2.4%; LP, 4.3%; and LPCE, 3.3%). In addition, LPCE had
the highest abundance of Weissella (CON, 1.7%; CE, 1.5%; LP, 1.9%; and LPCE, 2.3%) and
Leuconostoc (CON, 0.9%; CE, 1.0%; LP, 0.9%; and LPCE, 1.2%), as well as the lowest relative
abundance of unclassified_f__Enterobacteriaceae (CON, 2.3%; CE, 2.2%; LP, 2.3%; and LPCE,
0.7%) and Enterobacter (CON, 2.1%; CE, 2.3%; LP, 1.7%; and LPCE, 0.7%).
Agriculture 2025, 15, x FOR PEER REVIEW 7 of 17
Figure 1. Principal coordinates analysis (PCoA) plot conducted based on weighted Unifrac distance
of bacterial community. ANOSIM value, R = 0.6543, p = 0.005. CON, no additives; CE, added cellu-
lase (100 IU/g FM); LP, added Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM); and LPCE, added
cellulase (100 IU/g FM) and Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM).
Table 4. The alpha diversity of bacterial community in Pennisetum giganteum z.x.lin silage.
Items Treatments SEM p-Value
CON CE LP LPCE
Chao 250.3 184.4 287.0 193.4 17.82 0.097
Ace 251.4 186.1 291.4 196.5 17.48 0.102
Shannon 3.008
a
2.226
bc
2.670
ab
2.137
c
0.1149 0.001
Simpson 0.1155
b
0.2803
a
0.1966
ab
0.2609
a
0.0223 0.008
Coverage 0.9998 0.9998 0.9993 0.9996 0.0001 -
a–c
Values with different leers in the same row are significantly different (p < 0.05). CON, no addi-
tives; CE, added cellulase (100 IU/g FM); LP, added Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM);
and LPCE, added cellulase (100 IU/g FM) and Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM). SEM,
standard error of mean.
The bacterial community composition of Pennisetum giganteum z.x.lin silage at the
phylum and genus levels is shown in Figure 2. In the silage, Firmicutes (CON, 57.6%; CE,
78.6%; LP, 73.6%; and LPCE, 88.1%) and Proteobacteria (CON, 46.4%; CE, 72.4%; CE,
63.1%; and LPCE, 79.3%) were the major phyla, with a total relative abundance of >98.8%
in all treatments (Figure 2a). As expected, Lactobacillus (CON, 3.86%; CE, 5.08%; LP, 1.78%;
and LPCE, 85.16%) became the dominant genus after ensiling (Figure 2b). Compared with
CON, CE, LP, and LPCE all exhibited a decreased relative abundance of Serratia (CON,
20.9%; CE, 7.8%; LP, 9.9%; and LPCE, 4.8%), Pantoea (CON, 5.7%; CE, 2.7%; LP, 3.9%; and
LPCE, 1.1%), Klebsiella (CON, 3.9%; CE, 2.8%; LP, 2.9%; and LPCE, 2.0%), and Pediococcus
(CON, 5.2%; CE, 0.9%; LP, 2.6%; and LPCE, 1.4%), while exhibiting an increased relative
abundance of Lactococcus (CON, 2.4%; CE, 2.4%; LP, 4.3%; and LPCE, 3.3%). In addition,
LPCE had the highest abundance of Weissella (CON, 1.7%; CE, 1.5%; LP, 1.9%; and LPCE,
2.3%) and Leuconostoc (CON, 0.9%; CE, 1.0%; LP, 0.9%; and LPCE, 1.2%), as well as the
lowest relative abundance of unclassified_f__Enterobacteriaceae (CON, 2.3%; CE, 2.2%; LP,
2.3%; and LPCE, 0.7%) and Enterobacter (CON, 2.1%; CE, 2.3%; LP, 1.7%; and LPCE, 0.7%).
Figure 2. Relative abundance of top 5 bacterial phyla (a) and top 10 bacterial genera (b) of Pennisetum
giganteum z.x.lin silage. CON, no additives; CE, added cellulase (100 IU/g FM); LP, added
Figure 2. Relative abundance of top 5 bacterial phyla (a) and top 10 bacterial genera (b) of
Pennisetum giganteum z.x.lin silage. CON, no additives; CE, added cellulase (100 IU/g FM); LP, added
Lactiplantibacillus plantarum (1
×
10
6
CFU/g FM); and LPCE, added cellulase (100 IU/g FM) and
Lactiplantibacillus plantarum (1 ×106CFU/g FM).
The LEfSe showed that multiple bacterial taxa were potential biomarkers (LDA
score od > 4.0, Figure 3). Enterobacterales, Proteobacteria, Gammaproteobacteria, Er-
winiaceae, Pantoea, and Pediococcu were abundant in CON. Rikenellaceae was abundant
in LP.
Lactobacillus
, Firmicutes, Lactobacillaceae, Bacilli, Lactobacillales, Deinococci, and
Deinococcus were abundant in LPCE.
Agriculture 2025, 15, x FOR PEER REVIEW 8 of 17
Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM); and LPCE, added cellulase (100 IU/g FM) and Lac-
tiplantibacillus plantarum (1 × 10
6
CFU/g FM).
The LEfSe showed that multiple bacterial taxa were potential biomarkers (LDA score
od > 4.0, Figure 3). Enterobacterales, Proteobacteria, Gammaproteobacteria, Erwiniaceae,
Pantoea, and Pediococcu were abundant in CON. Rikenellaceae was abundant in LP. Lacto-
bacillus, Firmicutes, Lactobacillaceae, Bacilli, Lactobacillales, Deinococci, and Deinococcus
were abundant in LPCE.
Figure 3. Comparison of the bacterial variations in Pennisetum giganteum z.x.lin silage using LEfSe.
CON, no additives; LP, added Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM); and LPCE, added
cellulase (100 IU/g FM) and Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM).
3.3. Correlation Analysis Between Silage Quality Characters and Bacterial Community
The correlation between silage quality characteristics and bacterial community at the
genus level is given in Figure 4. CP exhibited a negative correlation (p < 0.05) with
Klebsiella. NDF showed a negative correlation (p < 0.05) with Lactobacillus and positive as-
sociation (p < 0.05) with Serratia, Pantoea, Klebsiella, and Pediococcus. ADF exhibited a neg-
ative correlation (p < 0.05) with Lactobacillus and a positive association (p < 0.05) with Ser-
ratia, Pantoea, Klebsiella, Pediococcus, unclassified_f__Enterobacteriaceae, and Enterobacter.
WSC showed a positive correlation (p < 0.05) with Lactobacillus and a negative correlation
(p < 0.05) with Serratia, Pantoea, Klebsiella, Pediococcus, unclassified_f__Enterobacteriaceae, and
Enterobacter. AA was inversely related (p < 0.05) to Lactobacillus and positively correlated
with Pantoea and unclassified_f__Enterobacteriaceae. NH
3
-N was positively associated (p <
0.05) with Lactobacillus and inversely related (p < 0.05) to Serratia, Pantoea, and Klebsiella.
Figure 3. Comparison of the bacterial variations in Pennisetum giganteum z.x.lin silage using LEfSe.
CON, no additives; LP, added Lactiplantibacillus plantarum (1
×
10
6
CFU/g FM); and LPCE, added
cellulase (100 IU/g FM) and Lactiplantibacillus plantarum (1 ×106CFU/g FM).
Agriculture 2025,15, 97 8 of 16
3.3. Correlation Analysis Between Silage Quality Characters and Bacterial Community
The correlation between silage quality characteristics and bacterial community at the
genus level is given in Figure 4. CP exhibited a negative correlation (p< 0.05) with Klebsiella.
NDF showed a negative correlation (p< 0.05) with Lactobacillus and positive association
(
p< 0.05
) with Serratia,Pantoea,Klebsiella, and Pediococcus. ADF exhibited a negative corre-
lation (p< 0.05) with Lactobacillus and a positive association (p< 0.05) with Serratia,Pantoea,
Klebsiella,Pediococcus,unclassified_f__Enterobacteriaceae, and Enterobacter. WSC showed a
positive correlation (p< 0.05) with Lactobacillus and a negative correlation (
p< 0.05
) with
Serratia,Pantoea,Klebsiella,Pediococcus,unclassified_f__Enterobacteriaceae, and Enterobacter.
AA was inversely related (p< 0.05) to Lactobacillus and positively correlated with
Pantoea
and unclassified_f__Enterobacteriaceae. NH
3
-N was positively associated (
p< 0.05
) with
Lactobacillus and inversely related (p< 0.05) to Serratia,Pantoea, and Klebsiella.
Agriculture 2025, 15, x FOR PEER REVIEW 9 of 17
Figure 4. Heatmap of Spearman correlation analysis of bacterial community and silage quality char-
acters at genus level. *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; and ***, p < 0.001. AA, acetic acid; ADF,
acid detergent fiber; CP, crude protein; DM, dry maer; LA, lactic acid; NDF, neutral detergent fiber;
NH
3
-N, ammonia nitrogen; and WSC, water-soluble carbohydrate.
3.4. 16S rRNA Gene-Predicted Functional Profiles of Pennisetum giganteum z.x.lin Silage
The major KEGG metabolic pathways in the silage are shown in Figure 5. The domi-
nant microbial metabolic pathways consisted of global and overview maps, carbohydrate
metabolism, amino acid metabolism, and membrane transport, with their proportions all
> 5%. Compared with CON, the other treatments significantly upregulated (p < 0.05) the
global and overview maps. Meanwhile, CON was less abundant (p < 0.05) than CE and
LPCE in the carbohydrate metabolism and more abundant (p < 0.05) than LP and LPCE in
membrane transport. Additionally, the addition of additives also decreased (p < 0.05) the
abundance of the energy metabolism and the metabolism of cofactors and vitamins, while
simultaneously increasing (p < 0.05) the abundance of the nucleotide metabolism, transla-
tion, signal transduction, and replication and repair to varying degrees.
Figure 4. Heatmap of Spearman correlation analysis of bacterial community and silage quality
characters at genus level. *, 0.01
≤
p< 0.05; **, 0.001
≤
p< 0.01; and ***, p< 0.001. AA, acetic acid;
ADF, acid detergent fiber; CP, crude protein; DM, dry matter; LA, lactic acid; NDF, neutral detergent
fiber; NH3-N, ammonia nitrogen; and WSC, water-soluble carbohydrate.
3.4. 16S rRNA Gene-Predicted Functional Profiles of Pennisetum giganteum z.x.lin Silage
The major KEGG metabolic pathways in the silage are shown in Figure 5. The domi-
nant microbial metabolic pathways consisted of global and overview maps, carbohydrate
metabolism, amino acid metabolism, and membrane transport, with their proportions
Agriculture 2025,15, 97 9 of 16
all > 5%.
Compared with CON, the other treatments significantly upregulated (p< 0.05)
the global and overview maps. Meanwhile, CON was less abundant (p< 0.05) than CE and
LPCE in the carbohydrate metabolism and more abundant (p< 0.05) than LP and LPCE
in membrane transport. Additionally, the addition of additives also decreased (p< 0.05)
the abundance of the energy metabolism and the metabolism of cofactors and vitamins,
while simultaneously increasing (p< 0.05) the abundance of the nucleotide metabolism,
translation, signal transduction, and replication and repair to varying degrees.
Agriculture 2025, 15, x FOR PEER REVIEW 10 of 17
Figure 5. Level 2 Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog functional predic-
tions of the top 10 metabolic functions using PICRUSt2 (v2.2.0).
a–c
Different leers indicate signifi-
cantly difference (p < 0.05) between values for a given metabolic category. CON, no additives; CE,
added cellulase (100 IU/g FM); LP, added Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM); and LPCE,
added cellulase (100 IU/g FM) and Lactiplantibacillus plantarum (1 × 10
6
CFU/g FM). *, 0.01 ≤ p < 0.05.
4. Discussion
4.1. Effects of Additives on Silage Quality of Pennisetum giganteum z.x.lin
Due to its excellent biological characteristics, Pennisetum giganteum z.x.lin is expected
to alleviate the forage shortage in northwest China as a potential feed resource for rumi-
nants, such as goats [22]. With a good harvest speed and low weather damage, ensiling
may be a practical strategy for preserving Pennisetum giganteum z.x.lin [23]. For a suitable
anaerobic fermentation condition, silage raw material should contain WSC content of
>6% [9]. Therefore, the low WSC content (38.0 g/kg DM) of Pennisetum giganteum z.x.lin
may be insufficient to ensure successful LA fermentation. Generally, ruminants prefer di-
ets with low fiber [9]. Meanwhile, fiber is one of the most challenging nutrients to digest
in feed. A previous study reported that ADF content impacts feed digestibility and has a
negative correlation with DM digestibility [24]. In this study, Pennisetum giganteum z.x.lin
contained high levels of NDF (608.4 g/kg DM) and ADF (343.9 g/kg DM), which may have
a negative impact on its digestibility. Therefore, adding cellulase and Lactiplantibacillus
plantarum to Pennisetum giganteum z.x.lin may help to improve its silage quality and feed-
ing value by degrading fiber into WSC.
Previous studies have showed that cellulase could reduce the DM content of silage
due to its function of degrading structural carbohydrates [13,25]. However, LP had the
lowest DM content in the present study, which is consistent with the study of Xiao et al.
[26], who reported that Lactiplantibacillus plantarum could decrease the DM content of
forage oat silage because of increased LA fermentation. Indeed, LP showed a higher LA
content than CON, but the reason for its reduced DM content needs further investigation.
In this study, a lower CP content was found in CON, which may be aributed to the deg-
radation of proteins by microorganisms during the fermentation process. Interestingly,
the CP content in the additive treatment groups increased compared to the pre-ensiled
forage. This was similar to the study of Zhang et al. [12], who reported that the LAB and
cellulase inoculations resulted in a higher CP content in the silage compared to the fresh
Figure 5. Level 2 Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog functional predictions of
the top 10 metabolic functions using PICRUSt2 (v2.2.0).
a–c
Different letters indicate significantly difference
(p< 0.05) between values for a given metabolic category. CON, no additives; CE, added cellulase (100 IU/g
FM); LP, added Lactiplantibacillus plantarum (1
×
10
6
CFU/g FM); and LPCE, added cellulase (100 IU/g
FM) and Lactiplantibacillus plantarum (1 ×106CFU/g FM). *, 0.01 ≤p< 0.05.
4. Discussion
4.1. Effects of Additives on Silage Quality of Pennisetum giganteum z.x.lin
Due to its excellent biological characteristics, Pennisetum giganteum z.x.lin is expected to
alleviate the forage shortage in northwest China as a potential feed resource for ruminants,
such as goats [
22
]. With a good harvest speed and low weather damage, ensiling may
be a practical strategy for preserving Pennisetum giganteum z.x.lin [
23
]. For a suitable
anaerobic fermentation condition, silage raw material should contain WSC content of
>6% [
9
]. Therefore, the low WSC content (38.0 g/kg DM) of Pennisetum giganteum z.x.lin
may be insufficient to ensure successful LA fermentation. Generally, ruminants prefer diets
with low fiber [
9
]. Meanwhile, fiber is one of the most challenging nutrients to digest in feed.
A previous study reported that ADF content impacts feed digestibility and has a negative
correlation with DM digestibility [
24
]. In this study, Pennisetum giganteum z.x.lin contained
high levels of NDF (608.4 g/kg DM) and ADF (343.9 g/kg DM), which may have a negative
impact on its digestibility. Therefore, adding cellulase and
Lactiplantibacillus plantarum
to
Pennisetum giganteum z.x.lin may help to improve its silage quality and feeding value by
degrading fiber into WSC.
Previous studies have showed that cellulase could reduce the DM content of silage due
to its function of degrading structural carbohydrates [
13
,
25
]. However, LP had the lowest
DM content in the present study, which is consistent with the study of Xiao et al. [
26
], who
reported that Lactiplantibacillus plantarum could decrease the DM content of forage oat silage
Agriculture 2025,15, 97 10 of 16
because of increased LA fermentation. Indeed, LP showed a higher LA content than CON,
but the reason for its reduced DM content needs further investigation. In this study, a lower
CP content was found in CON, which may be attributed to the degradation of proteins
by microorganisms during the fermentation process. Interestingly, the CP content in the
additive treatment groups increased compared to the pre-ensiled forage. This was similar
to the study of Zhang et al. [
12
], who reported that the LAB and cellulase inoculations
resulted in a higher CP content in the silage compared to the fresh Caragana korshinskii,
but the underlying reasons for this require further investigation. As expected, the silage
inoculated with cellulase (CE and LPCE) showed lower contents of NDF and ADF than
CON, indicating that cellulase promoted the decomposition of the fiber components of
Pennisetum giganteum z.x.lin [
9
]. This may also help to improve the ruminal digestibility of
Pennisetum giganteum z.x.lin silage [
11
]. However, the effect of cellulase on decomposing
hemicellulose was limited in this study, which is inconsistent with the findings from
Si and Iannaccone et al. [
13
,
27
], who reported that cellulase could effectively degrade
the hemicellulose of mixed alfalfa and Leymus chinensis silage or industrial feeds. This
may be due to the different degradation abilities of cellulases from different sources on
hemicellulose. A suitable WSC content is important for LA fermentation. Compared to
CON, the WSC content of CE, LP, and LPCE grew by 26.3%, 44.9%, and 2.5%, respectively.
Therefore, cellulase and Lactiplantibacillus plantarum may help to preserve more WSC
during ensiling, which agrees with the findings from Guo et al. [
28
], who reported that the
addition these two additives increased the WSC content in mixed silage of corn and hulless
barely straw compared with the control silage. On the one hand, cellulase could degrade
some structural carbohydrates into WSC; on the other hand, Lactiplantibacillus plantarum
could promote LA fermentation and suppress the utilization of WSC by undesirable
microorganisms [13,28].
pH is a key parameter for assessing silage quality, and a pH of < 4.2 is suitable for
conventional silage [
1
]. In the fermentation process, LA is a key beneficial organic acid,
while AA is the product of hetero-fermentation [
29
]. In this study. LP exhibited the
highest LA content and ratio of LA/AA, suggesting that Lactiplantibacillus plantarum could
promote homo-fermentation and increase LA production during ensiling [
30
]. Previous
studies have showed that cellulase can also inhibit hetero-fermentation and reduce AA
production by increasing the WSC content of silage [
13
,
31
]. In this study, no significant
difference was observed in the LA content or ratio of LA/AA among CON, CE, and
LPCE. Nevertheless, compared to CON, the ratio of LA/AA in CE and LPCE climbed by
90.48% and 133.33%, respectively, suggesting that cellulase could enhance the fermentation
quality of silage to some extent. PA is generated from the secondary fermentation of LA by
clostridium [
32
]. Previous studies have showed that cellulase and LAB can reduce the PA
content of silage [
12
,
33
]. In this study, all treatments had a low PA content, and even the
PA content in LPCE was undetected, suggesting that the secondary fermentation of LA by
clostridium was inhibited in the silage. Generally, the levels of NH
3
-N and BA in high-quality
silage are <100 g/kg TN and 2.0 g/kg DM, respectively [
34
]. Interesting, all treatments had
a BA content below 2.0 g/kg DM, but only CON had an NH
3
-N content below 100 g/kg
TN. NH
3
-N is generally considered as an important indication of the protein breakdown
caused by microorganisms [
35
]. In this study, although there was no significant difference
in the CP content between LPCE and CON, and the NH
3
-N level in LPCE was significantly
higher than that in CON, suggesting that the combination of cellulase and Lactiplantibacillus
plantarum may promote NH
3
-N production in a way independent of protein breakdown.
Considering that LPCE exhibited the highest abundance in nucleotide metabolism, NH
3
-
N may be generated through nucleotide decomposition, but this hypothesis requires
further investigation.
Agriculture 2025,15, 97 11 of 16
4.2. Effects of Additives on Bacterial Community of Pennisetum giganteum z.x.lin Silage
The bacterial community of silage plays a critical role in the anaerobic fermentation
process. In the present study, all treatments had a coverage of > 99.9%, indicating that the
sequencing was adequate to accurately represent the status of the bacterial community.
Chao and Ace were employed to measure the bacterial community richness, while Sannon
and Simpson to were used to assess the bacterial community diversity. Previous studies
have indicated that cellulase and Lactiplantibacillus plantarum can reduce the richness and
diversity of bacterial communities [
9
,
36
]. In the present study, the addition of additives did
not significantly change the bacterial community richness of the silage, but both CE and
LPCE had a lower bacterial community diversity than CON, suggesting that the addition
of additives may promote LA fermentation and then suppress the growth of undesirable
microorganisms during ensiling. In addition, PCoA showed that the bacterial community
structures of LP, CE, and LPCE were obviously separated from those of CON through
PCoA1. Therefore, cellulase and Lactiplantibacillus plantarum can decrease the bacterial
community diversity and change the bacterial community structure of silage.
In terms of bacterial community composition, Firmicutes was the most abundant phy-
lum, and its relative abundance was upregulated to varying degrees due to the inoculation
of additives, which agrees with the study reported by Si et al. [
13
], who found that cellulase,
Lactiplantibacillus plantarum, and their combination could all increase the relative abundance
of Firmicutes in mixed silage of alfalfa and Leymus chinensis. Firmicutes is an important
acid-producing hydrolytic phylum that is capable of growing rapidly under acidic condi-
tions [
37
,
38
]. In the course of ensiling, LAB use WSC to generate LA, reduce the pH, and
prevent the proliferation of undesirable microorganisms, so that the nutrients of silage can
be preserved for an extended period [
39
]. Moreover, LAB are an important component of
Firmicutes. Therefore, the addition of additives may promote the proliferation of LAB and
increase the relative abundance of Firmicutes.
As an acid-tolerant LAB, Lactobacillus plays an essential role in enhancing LA ac-
cumulation and improving silage quality. Previous studies have showed that cellulase
and Lactiplantibacillus plantarum can increase the relative abundance of Lactobacillus in
silage [
13
,
25
,
40
]. Expectedly, Lactobacillus became the major genus in all four treatments,
with its relative abundance in CE, LP, and LPCE being higher than that in CON. Members
of Enterobacteriaceae,Serratia,Pantoea,Klebsiella,unclassified_f__Enterobacteriaceae, and En-
terobacter were found in the silage, which was harmful to LA fermentation and nutrient
preservation. Interestingly, most of them were more abundant in CON than the other treat-
ments, suggesting that the additive treatments promoted LA fermentation and inhibited
the activity of these harmful bacteria. Meanwhile, Serratia was the main harmful bacteria,
and its relative abundance was surpassed only by that of Lactobacillus, which was similar to
the findings from Li et al. [
35
], who reported that the relative abundance of Serratia was
high in mixed silage of faba bean with forage wheat. It is noteworthy that LPCE had the
highest relative abundance of Lactobacillus, but the lowest relative abundances of Serratia,
Pantoea,Klebsiella,unclassified_f__Enterobacteriaceae, and Enterobacter, suggesting that the
combination of cellulase and Lactiplantibacillus plantarum could more effectively improving
the bacterial community composition of Pennisetum giganteum z.x.lin silage. Other LAB,
including Lactococcus,Pediococcus,Weissella, and Leuconostoc, were also present in the silage.
Although their relative abundance was much lower than that of Lactobacillus, they may be
significant in the initiation of LA fermentation [
41
,
42
]. Due to their poor acid resistance,
these LAB were gradually replaced by Lactobacillus during ensiling [
41
,
43
]. LEfSe was
widely applied to further explore the differences in the bacterial communities of different
treatments [
12
]. In this study, some undesirable microorganisms such as Enterobacterales
were abundant in CON, while Lactobacillus was abundant in LPCE, suggesting that the
Agriculture 2025,15, 97 12 of 16
combination of cellulase and Lactiplantibacillus plantarum can synergistically enhance LA
fermentation and inhibit the growth of undesirable microorganisms. Interestingly, Pediococ-
cus was abundant in CON, which was likely due to the fact that the pH of CON decreased
at a lower rate compared with other treatments; thus, Pediococcus had a better growing
environment in CON.
4.3. Effect of Bacterial Community on Silage Quality of Pennisetum giganteum z.x.lin
The bacterial community is closely involved in the fermentation process and signif-
icantly affects silage quality [
29
]. As a major beneficial bacterium, Lactobacillus played
an indispensable role in suppressing the activity of harmful bacterial and improving the
silage quality of Pennisetum giganteum z.x.lin. In this study, cellulase promoted fiber de-
composition and WSC preservation, thus providing more fermentation substrates for LAB.
Meanwhile, the addition of Lactiplantibacillus plantarum promoted homo-fermentation and
reduced AA production. As expected, Lactobacillus was inversely related to NDF, ADF, and
AA and positively associated with WSC, which was similar to the study of Bao et al. [
41
],
who reported that Lactobacillus had a negative correlation with AA and a positive correla-
tion with WSC in alfalfa silage. Generally, the NH
3
-N content is low om high-quality silage,
but there are also reports that LAB can increase the NH
3
-N content [
44
–
46
]. Interestingly, a
similar situation was observed in this study, as Lactobacillus showed a significantly positive
correlation with NH
3
-N. As members of Enterobacteriaceae,Serratia,Pantoea,Klebsiella,un-
classified_f__Enterobacteriaceae, and Enterobacter had a similar effect on the silage quality, as
they accelerated WSC consumption and AA production. In this study, Klebsiella was nega-
tively correlated with CP, suggesting that it was probably the main bacteria that degraded
the proteins [
47
]. Additionally, the role of Pediococcus on the silage quality seemed to be
opposite to that of Lactobacillus, which was possibly due to the fact that the rapid growth of
Lactobacillus had an inhibitory effect on Pediococcus in the late stage of ensiling [43,46].
4.4. Effect of Additives on Function Shifts of Bacterial Community
During ensiling, metabolic gene clusters have a profound impact on the metabolism
and metabolites of microorganisms by participating in a variety of secondary metabolic
pathways, thereby affecting the silage quality [
48
]. In this study, global and overview
maps were the most common metabolic category and were upregulated by additive
treatments. This agreed with the study of Du et al. [
9
], who found that global and
overview maps were the main metabolic category of microorganisms in high-cellulose
silage, and their abundance could be upregulated by the combination of cellulase and
Lactiplantibacillus plantarum.
In the global and overview maps, there was a series of
metabolic pathways, such as the nucleotide metabolism and biosynthesis of cofactors [
49
],
but the reasons for the upregulation of global and overview maps caused by additive
treatments need to be further studied. In this study, carbohydrate metabolism, amino
acid metabolism, and membrane transport were also important metabolic categories. Bai
et al. [
50
] reported that the amino acid metabolism should be suppressed in high-quality
silage. Nevertheless, the amino acid metabolism did not differ significantly among the four
treatments. This could be attributed to the fact that all treatments had a pH of <4.2 [
51
],
which effectively inhibited the protein breakdown and amino acid utilization caused by
undesirable microorganisms. Previous studies have showed that the relative abundance of
total LAB is positively associated with the abundance of carbohydrate metabolism [
12
,
52
].
As expected, compared with CON, both LPCE and CE upregulated the carbohydrate
metabolism, which was likely due to the fact that the fiber degradation caused by cellulase
could provide LAB with more WSC for LA fermentation, thus enhancing their carbohydrate
metabolic activity. However, compared with CON, both LP and LPCE downregulated
Agriculture 2025,15, 97 13 of 16
membrane transport, which may have been due to the fact that the silage without additives
had more abundant transporters [
53
]. Interestingly, compared with CE and LP, LPCE
exerted a more profound impact on function shifts in the bacterial community, suggest-
ing that cellulase and Lactiplantibacillus plantarum probably exerted synergistic effects on
accelerating bacterial community succession and metabolic function transformation in
high-fiber silage.
5. Conclusions
This study was conducted to investigate the roles of cellulase and Lactiplantibacillus
plantarum in the silage fermentation process of Pennisetum giganteum z.x.lin. Cellulase inoc-
ulation significantly decreased the contents of NDF and ADF. Lactiplantibacillus plantarum
inoculation effectively increased the ratio of LA/AA and LA content, which benefited the
preservation of CP and WSC. The combination of cellulase and Lactiplantibacillus plantarum
showed the highest abundance of Lactobacillus and accelerated function shifts in the bacte-
rial community, leading to the lowest membrane transport and the highest carbohydrate
metabolism. In summary, the cellulase or Lactiplantibacillus plantarum inoculation could
improve the silage quality of Pennisetum giganteum z.x.lin and their combination showed a
greater ability to reshape bacterial community.
Author Contributions: Conceptualization, L.D., I.K., J.Y. and X.L.; methodology, X.Z., R.L. and J.H.;
software, R.L. and J.H.; validation, R.L. and J.H.; formal analysis, Z.W. and X.Z.; investigation, Z.W.
and X.Z.; resources, J.W., J.Y. and X.L.; data curation, Z.W., X.Z., R.L. and J.H.; writing—original draft
preparation, Z.W. and X.Z.; writing—review and editing, Z.W., X.Z., R.L., J.H., L.D., I.K., J.W., J.Y. and
X.L.; visualization, Z.W. and X.Z.; supervision, X.L.; project administration, X.L.; funding acquisition,
X.L. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Shaanxi Livestock and Poultry Breeding Double-chain Fusion
Key Project (2022GD-TSLD-46-0501), the Science & Technological Project of Shaanxi Province, China
(2022ZDLNY01-09; 2023KJXX-132; 2024NC-YBXM-125), and the Science & Technological Project of
Xianyang, Shaanxi Province, China (L2022-QCYZX-NY-004).
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Data are available upon request to the corresponding author.
Acknowledgments: The authors thank the Key Laboratory of Livestock Biology, Northwest A&F
University, Yangling, China for the technical assistance and use of the research facilities.
Conflicts of Interest: Author Jie Wei was employed by the company Xi’an Architectural Design and
Research Institute Co., Ltd. The remaining authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a potential conflict
of interest.
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