Innovative Lactic Acid Production Techniques Driving Advances in Silage Fermentation

Abstract

Lactic acid (LA) plays a crucial role in the silage process, which occurs through LA fermentation. Consequently, there is a strong correlation between lactic acid production and the efficiency of the silage. However, traditional methods face challenges like long fermentation times, low acid production, and unstable quality, limiting agricultural preservation. This paper aims to explore innovations in lactic acid production technologies and show how these technologies have driven the development of silage fermentation for agricultural conservation. First, the important role of LA in agricultural preservation and the limitations of traditional silage techniques are presented. Next, advancements in LA production methods are thoroughly examined, covering the selection of microbial strains and the substitution of fermentation substrates. Following this, new technologies for silage fermentation are explored, drawing from innovations in LA production. These include the selection of LA strains, optimization of fermentation conditions, and improvements in fermentation techniques. These innovations have proven effective in increasing LA production, improving feed quality, extending shelf life, and providing new solutions to enhance agricultural production and sustainability.

1. Introduction

The importance of silage fermentation in agriculture, particularly for animal feed, has been increasingly recognized as the global demand for livestock products rises [1]. However, traditional silage fermentation methods face significant challenges, such as long fermentation periods, low acid production, and inconsistent quality, limiting their effectiveness in modern agricultural practice [2]. This article aims to address these limitations by reviewing advancements in lactic acid production and innovative silage fermentation technologies. By exploring these innovations, this article seeks to provide new approaches that enhance feed quality, reduce environmental impact, and support sustainable agricultural development.

It has been reported that global silage production continued to grow at a rate of about 2–4% over the period 2000–2020. Global population growth and rising living standards have led to increased demand for livestock products, which, in turn, has fueled the demand for silage [3]. Through silage technology, farmers can efficiently preserve and utilize herbages and crop residues [4]. According to the Food and Agriculture Organization of the United Nations (FAO) and related studies, it is estimated that the amount of crop residues produced globally is about 3–4 billion tonnes per year. This mainly includes stover from crops such as maize, rice, wheat, soybeans, and sorghum. During the silage process, these herbages and crop residues are fermented by lactic acid bacteria, producing lactic acid and other beneficial substances. This fermentation lowers the pH value and transforms the plant materials into stable products [5]. Consequently, the nutritional value of the feed is improved, making the feed more digestible [5]. Additionally, agricultural producers can effectively reduce the wastage of crop residues, thus reducing the environmental impact of incineration or decay.

Lactic acid is crucial in silage fermentation, serving multiple key roles: (1) it lowers the pH, preventing the growth of harmful microorganisms like mold and spoilage bacteria [5]; (2) it inhibits enzymes that cause feed degradation, thus preserving feed quality [6]; (3) it contributes to the production of fragrant compounds that enhance animals’ appetite [7]; (4) it acts as an energy source for animals, boosting the feed’s energy value [8]; and (5) it promotes digestive health by maintaining the microbial balance in the gastrointestinal tract, improving the feed’s digestibility [9].

With the development of agriculture and the progress of science and technology, traditional silage fermentation methods have become inadequate for modern agricultural needs [2]. Therefore, it is necessary to innovate the silage fermentation technology. For instance, technological innovations can lead to more effective control of the silage fermentation process, enhancing both the yield and quality of feed to meet the demands of large-scale feeding operations. Implementing new silage equipment and techniques can reduce production costs and improve economic returns [10]. Given the growing focus on sustainable agriculture, it is essential to develop environmentally friendly silage technologies that minimize environmental impact. Tailored silage technologies need to be developed for different regions and crop types to address local needs [11]. In the face of global challenges such as climate change and resource constraints, innovations in silage technology can help agricultural producers to adapt better and achieve sustainable development.

2. Traditional Silage Fermentation

The traditional silage method typically involves harvesting fresh crops (such as corn, sorghum, etc.) and storing them in closed cellars or towers. These crops eventually form silage with a strong sour taste, strong fragrance, and dark color through a natural fermentation process under hypoxic conditions [12].

However, this approach has some limitations: (1) Over-reliance on weather conditions: Especially during the harvest period of crops, adverse weather conditions, such as continuous rain or frost, can affect the quality of the silage. (2) Limitations of storage facilities: Storage facilities for traditional silage methods are usually fixed cellars or towers, making large-scale movement and transportation difficult, and limiting their application in different regions. In addition, the construction and maintenance of these fixed facilities require substantial capital investment and can be a burden for small farms [13]. (3) Uncontrollable fermentation process: Traditional silage methods depend on natural fermentation, making it challenging to control the fermentation time, temperature, and humidity. This lack of control can negatively impact the quality and yield of the silage [14]. (4) Low efficiency: Traditional silage methods often require a long time to complete the fermentation process, leading to low production efficiency. Low-efficiency fermentation means that it takes longer to fully ferment silage into a feed that can be preserved for a longer period of time and has a higher nutritional value. There may also be problems with incomplete fermentation or poor-quality fermentation during the fermentation process, which can lead to insufficient nutrient content or shortened shelf life of the final silage. These limitations bring several challenges and problems. For example, the quality of silage is unstable due to the uncontrollable fermentation process of traditional methods. Issues such as feed deterioration and mildew may occur, affecting animal health [6].

However, it is worth noting that, in recent years, silage bales have been gaining attention as an innovative storage method [15]. Silage bales, usually made of plastic film or other waterproof materials, can hold a certain amount of silage and maintain an anaerobic environment inside them by means of specific sealing techniques. This method not only solves the limitations of fixed storage facilities in terms of transportation and movement, it also opens up new possibilities for the control of the fermentation process [16,17]. By adjusting the degree of sealing and the internal environment (e.g., temperature, humidity) of the silage bale, the fermentation process can be controlled more effectively, improving the quality and stability of the silage. Silage bales offer a new perspective on the challenges posed by conventional storage facilities. In addition, the construction and maintenance costs of silage bales are relatively low, making this flexible storage option more attractive, especially for small farms [18,19,20]. It not only reduces the initial investment cost but also improves the availability and utilization rate of silage, which helps to promote sustainable agricultural development.

Given the limitations of traditional ensiling methods and the associated challenges and problems, combined with the emergence of new technologies such as silage bales, there is a strong need to improve silage techniques. Enhancing the quality and yield of silage while reducing production costs aligns better with the demands of modern agriculture and the goals of sustainable development. Therefore, it is crucial to research and develop novel silage technologies to achieve advantages in efficiency, cost reduction, quality improvement, and environmental protection.

3. Approaches to Lactic Acid Production

At present, new methods to increase the lactate content in the silage process mainly focus on innovative fermentation strains and the substitution of fermentation substrates. For example, efficient strains for producing LA have been developed for silage fermentation through genetic modification or strain screening. Alternatively, renewable resources such as agricultural waste can be utilized as fermentation substrates to reduce silage costs and make the process more environmentally friendly [21]. These new methods are more efficient and sustainable than traditional methods. Through biotechnology, the ensiling process has become more environmentally friendly, and improved fermentation strains and precise control have also increased the yield and quality of lactic acid. These innovations have greatly improved the efficiency of silage fermentation, as well as the quality and sustainability of silage feed [8].

For farmers, using renewable resources such as agricultural waste as fermentation base materials offers a wide range of raw material sources and low costs, reducing the direct cost of feed production. It not only enables the recycling of waste to resources but also significantly improves the quality of silage feed. In addition, this microbial fermentation method effectively locks in the nutrients in the raw materials, reducing the loss of nutrients during natural decay, thus producing silage feed that is rich in nutrients and stable in storage. This not only extends the shelf life of the feed but also ensures the quality of the feed, providing a more balanced and efficient source of nutrition for livestock, promoting their healthy growth and improving production performance. More importantly, by reducing waste emissions and minimizing feed waste, farmers can significantly reduce their overall production costs and improve their economic efficiency [12].

3.1. Lactic Acid-Producing Fermentation Strains

3.1.1. Lactic Acid Bacteria (LAB)

Lactic acid is an organic compound produced by fermentation by different microorganisms capable of utilizing various carbohydrate sources. Lactic acid bacteria (LAB) are widely distributed Gram-positive bacteria. As common fermentation strains for producing lactic acid, they offer high yield and productivity. Based on fermentation types, LAB can be categorized into homofermentative, facultative heterofermentative, and obligate heterofermentative lactic acid bacteria. Homofermentative LAB mainly include Lactococcus lactis, Pediococcus pentosaceus, and Enterococcus faecium. They primarily reduce pH by producing high concentrations of lactic acid, without producing ethanol, acetic acid, butyric acid, or other substances. Facultative heterofermentative LAB mainly include Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus rhamnosus. Compared to homofermentative LAB, facultative heterofermentative strains possess phosphoketolase, enabling them to ferment pentose to produce lactic acid, acetic acid, and ethanol. The most common obligate heterofermentative LAB are Lactobacillus buchneri and Lactobacillus reuteri.

Different types of LAB exhibit varying capacities for lactic acid production. Currently, various species, including Lactobacillus (L-LA, D-LA, or DL-LA), Enterococcus (L-LA), Lactococcus (L-LA), Clostridium (D-LA), Pediococcus (L-LA or DL-LA), Leuconostoc (D-LA), and Streptococcus (L-LA), have emerged as strong producers of lactic acid [22]. For example, Tian et al. produced lactic acid from sweet sorghum juice using Lactobacillus thermophilus A69, which produced 114.6 g/L L-lactic acid in a 5 L bioreactor, with a productivity of 2.61 g/L/h. The lactic acid content was significantly increased, and the cost was greatly reduced by using cheap raw materials [23]. Silage is mainly fed to ruminants; therefore, lactic acid in silage is metabolized by rumen microorganisms. According to research, D-LA and L-LA are two optical isomers of lactic acid that differ significantly in their metabolism in the rumen. The metabolism of D-LA in the rumen is relatively slow, and although some rumen microorganisms can convert it into propionic acid or acetic acid, the conversion efficiency is limited. If the D-LA content in silage feed is too high, it may lead to an imbalance in the rumen environment and even pose a risk of ruminal acidosis. In contrast, L-LA is more easily rapidly metabolized into propionic acid by microorganisms in the rumen, which is one of the main pathways of ruminal microbial metabolism. Therefore, the accumulation of L-LA in the rumen is less, which helps maintain the stability of the ruminal environment [24].

In view of this, optimizing the inoculation strategy of lactic acid bacteria, promoting the production of L-LA while reducing the accumulation of D-LA, is of great significance for improving the fermentation quality of silage feed, ensuring the health and production performance of ruminants. During the ensiling process, it is necessary to select and mix lactic acid bacteria strains reasonably to regulate the type and proportion of lactic acid production, thereby optimizing the nutritional value and safety of the silage feed.

3.1.2. Bacillus

In recent years, Bacillus strains have become the fourth generation of inoculants for silage fermentation [25]. Firstly, the low nutrient requirements of Bacillus strains and their ability to grow in relatively poor media mean that cheap feedstocks can be used, reducing production costs. Additionally, Bacillus strains exhibit high carbon conversion efficiency in lactic acid production, enabling the conversion of more carbon sources into lactic acid, thereby enhancing the overall yield [22]. These advantages make Bacillus one of the dominant strains for lactic acid production. For example, Wang et al. explored the potential of Heyndrickxia coagulans as an inoculant for alfalfa silage and showed that the addition of Heyndrickxia coagulans increased the lactic acid content of the forage, while also increasing the abundance of Lactobacillus and decreasing the abundance of Enterococcus. The results showed that the lactic acid content of alfalfa silage supplemented with Heyndrickxia coagulans was 82.12 g/kg after 60 days, which was about 7 g/kg higher than that of the control group. Therefore, Heyndrickxia coagulans can be considered to be a viable microbial strain for improving the quality of silage fermentation feeds [26]. Bai et al. evaluated the effects of Bacillus amyloliquefaciens (BA) and Bacillus subtilis (BS) on the fermentation of wholemaize silage and found that inoculation with BA and BS increased the lactic acid concentration and the metabolism of cofactors and vitamins in whole-maize silage. After 60 days of silage, the lactic acid concentration under BA inoculation was 47.8 g/kg, and that under BS inoculation was 42.8 g/kg, both of which were significantly higher than that of the control group (36.9 g/kg, p < 0.001). This suggests that if BA and BS are inoculated into silage, not only can they improve the fermentation characteristics, they can also deliver live cells of BA or BS to ruminants, thus exerting their probiotic effects [25].

3.1.3. Escherichia coli

In recent years, Escherichia coli (E. coli) has opened up new avenues for the fermentation and quality improvement of silage feed as an alternative strain for lactic acid production.

E. coli has several advantages over traditional LAB in lactate production. Firstly, E. coli has a wide adaptability to sugar sources, including the ability to utilize complex sugars such as xylose, which enables it to more effectively utilize the polysaccharide components in plant cell walls during ensiling, thereby improving the conversion rate of lactic acid and the fermentation efficiency of silage feed. Secondly, E. coli can rapidly grow in nutrient-deficient media (similar to natural ingredients in silage feed), simplifying the conditions for silage fermentation. According to reports, wild-type E. coli has efficiently produced lactic acid using hexose and pentose sugars [27,28]. The modified E. coli optimized through modern biotechnology has higher lactic acid fermentation efficiency than the wild type, providing strong support for the rapid and efficient acidification of silage feed. Chang et al. reported the successful production of D-LA using recombinant E. coli. The recombinant strain JP201 produced approximately six times more D-LA than the parent strain RR1, with a lactate content of 60 g/L [29]. These modified strains can produce lactic acid from several disaccharides, including sucrose [30,31], monosaccharides (hexose [28], pentose [32], glucose [28,32,33,34,35,36], and xylose [32]), and glycerol [37,38]. For example, Dien et al. transformed three strains of E. coli, namely, strains FBR9, FBR10, and FBR11, using plasmids to convert glucose and pentose into lactic acid. Research has found that the improved strain FBR11 exhibits excellent fermentation performance, consuming 10% (w/v) concentrations of glucose and 7.32% (w/v) concentrations of lactic acid within 30 h [32].

At present, there is research on inoculating recombinant E. coli strains carrying antibiotic resistance marker plasmids and expressing green fluorescent protein into wheat silage and corn silage. The results show that adding E. coli during ensiling leads to a faster decrease in pH values. When the pH value drops below 5.0, E. coli disappears from the silage feed [39]. This result indicates that, during the ensiling process, the recombinant E. coli strain can rapidly exert its lactic acid fermentation ability, leading to a rapid decrease in the pH value of the silage feed. Therefore, in the future, genetic engineering technology could be used to further modify the lactate metabolism pathway and stress resistance genes of E. coli, making it more suitable for the fermentation environment of silage feed, and improving lactate production and stability.

3.1.4. Filamentous Fungi

Filamentous fungi are a group of microorganisms capable of producing lactic acid. Currently, several wild-type fungi, notably Rhizopus oryzae [40] and Rhizopus microsporus [41], are known as potent L-LA producers. The use of filamentous fungi for lactate production offers several benefits. These fungi possess starch-catabolizing enzyme activity, enabling them to convert various forms of starch-based biomass directly into L-LA [42]. For example, Oda et al. used Rhizopus oryzae IFO 4707 to produce lactic acid from agricultural waste potato pulp. The highest amount of L-LA (10 mg/g fresh matter) was observed in the pulp fermented for six days by Rhizopus oryzae IFO 4707. Ingredient analysis showed that enzymes secreted by fungal cells can effectively hydrolyze starch and partially degrade the cell wall. Therefore, Rhizopus oryzae can be used as an effective inoculant for other agricultural byproducts containing starch in silage [43]. However, it should be noted that not all filamentous fungi are beneficial for silage. Some filamentous fungi may produce harmful substances or toxins that pose a threat to the quality and safety of silage. Therefore, the species and quantity of microorganisms should be strictly controlled during silage to ensure the quality and safety of the silage [44].

3.1.5. Yeast

Currently, through genetic modification and other means, some yeasts can also produce lactic acid, including Saccharomyces cerevisiae [45], Candida species [46], Kluyveromyces lactis [47], Trichioron delbarii [33], Pichia pastoris [48], and Bacillus yeasts [49]. Because lactic acid is spontaneously produced by lactic acid-producing yeasts, the need for additional lactic acid bacteria or other additives in the silage process may be reduced, thus simplifying the silage process. Moreover, the addition of yeast to silage is also expected to provide a good source of protein for feeds. For example, Somayeh et al. obtained 0.078 g of protein/g of substrate by fermentation from agricultural waste sugarcane bagasse using Saccharomyces cerevisiae (PTCC 2486); therefore, this initiative is expected to be applied in silage fermentation as a good source of animal feed, providing better feed options for agricultural livestock production [50].

3.2. Alternative Fermentation Substrates for Lactic Acid Production

For lactic acid production, the fermentation medium requires substrates in addition to starter cultures. Various carbohydrate sources can be utilized, including starch materials, lignocellulose, organic wastes, glycerol, microalgae, and cyanobacteria, which have become viable alternative substrates for lactic acid production [51].

3.2.1. Starch-Based Materials

In the field of silage feed, starch-based substances are increasingly becoming an important substrate choice for silage lactic acid production due to their abundant resources, low cost, and renewability [52]. Amylolytic lactic acid bacteria (ALAB), such as Lactobacillus manihotivorans, Lactobacillus amylophilus, and Lactobacillus amylovorus, can efficiently convert starch components in silage raw materials into lactic acid (LA) due to their α-amylase activity [33,34,53]. Various ALAB strains have been isolated from different starch compounds, including corn and fermented corn products [33,34,43], potato [33,34,43,54,55], cassava and fermented cassava products [33,34], rice and fermented rice products [33,34,53,56], sweet sorghum [33,34], wheat [33,34,53,57], rye [33,34], oats [33,34,53], barley [33,34,53], and other amyloid substrates [58,59]. For example, Okano et al. used Lactobacillus plantarum to produce L-LA from raw maize starch, and the strain produced 50.3 g/L L-LA with 98.6% optical purity [60]. The application of these strains and these starch-based materials provides abundant microbial and silage substrate resources for the production of silage feed, making the silage process more efficient and stable.

3.2.2. Lignocellulose Materials

There are abundant and inexpensive lignocellulosic materials in the world. These include agricultural residues (corn stover, sugarcane bagasse, and rice husks), forestry residues (sawdust), herbaceous plants, switchgrass and shrubs (switchgrass and thorn grass), woody plants (poplar), wheat, rice, barley, corn, sorghum stems, straw, leaves, stems, shells, and fruit peels [61,62]. For example, Azaizeh et al. used Heyndrickxia coagulans and yeast extracts to produce lactic acid from lignocellulosic biomass of banana peel stems, yielding 26.6 g LA/L. The fermentation of sugarcane with yeast extract resulted in 46.5 g of LA·L and yielded 0.88 g of LA/g of sugars. Carob showed that the addition of yeast extract resulted in higher productivity of 3.2 g of LA·L⁻¹·h⁻¹ compared to without yeast extract, where 1.95 g of LA·L⁻¹·h⁻¹ was obtained [63].

These cellulose materials are mainly composed of cellulose, xylan, arabinose, galactose, and lignin [33,34,64]. Studies have shown that adding pectinase and cellulase to fermentation media can increase the yield of LA [65]. For example, Hu et al. used Lactobacillus pentosus to produce lactic acid by fermentation from maize stover, in which cellulase was added at a concentration of 30 filter paper units (FPU)/g of stover, and the lactic acid production was 1.92 g/L/h for 30 h of fermentation [66]. Due to lignin being a complex polymer, it wraps around cellulose and hemicellulose and hinders their enzymatic hydrolysis. At present, some studies have shown that lignocellulose-degrading bacteria can degrade the structural carbohydrates of corn stover into non-fiber carbohydrates, which may increase the fiber degradation rate. For example, Guo et al. inoculated lignocellulose-degrading bacteria, including Lactobacillus plantarum, Bacillus subtilis Y-1d, and Bacillus subtilis MZS-3-6, into corn stover silage. The results showed that inoculation with lignocellulose-degrading bacteria increased the dry matter intake and growth performance of sheep, and it improved their digestion [67]. A study has also shown that adding Acremonium cellulase and Lactobacillus plantarum to corn stover silage results in high lactate content and good degradation of lignocellulose in the treated feed [68].

Therefore, with the in-depth study of the degradation mechanism of lignocellulose, more efficient and specialized enzyme systems or microbial strains can be developed in the future to break down lignin encapsulation more effectively, thereby improving the enzymatic hydrolysis efficiency of cellulose and hemicellulose. This will not only increase the proportion of digestible nutrients in the silage but also shorten the fermentation period of the silage and improve the overall feed quality.

3.2.3. Organic Wastes

Some organic wastes have the potential to be converted to lactic acid due to their richness in carbohydrates and other nutrients (Table 1). Agricultural residues are potential substrates for LA production. This category includes alfalfa (Medicago, Lotus corniculatus L.) fiber [69], wheat bran and straw [70,71], non-fat rice bran [72,73], food residue [74], corn straw and corn cobs [75,76], barley bran husks [75], sugarcane and cassava bagasse [77,78,79], pruned vine sprouts [75], pruned wine waste [75], artichokes [80], broccoli [80], olive mill waste [81], date palm leaves [82], deproteinised feta cheese waste [83], apple pomace [84], banana waste [85], mussel processing waste [33,34], kitchen waste [86], fish meal waste [87], and bagasse waste [88]. For example, Kalinowska et al. used Lactobacillus plantarum to produce lactic acid from apple pomace and extracted some bioactives [89]. Besma et al. demonstrated that Lactobacillus plantarum has the ability to produce lactic acid from Opuntia ficus-indica wastes [90]. Paula et al. used artichoke and broccoli byproducts for silage, which could provide feed with a shelf life of up to 200 days to ruminants, greatly reducing feed costs, and the fermentation process significantly enhanced the quality of silage through the production of lactic acid [80].

Food waste is also high in carbohydrates, and numerous studies have indicated that food waste is suitable for lactic acid production and can be applied as a substrate for lactic acid fermentation. Examples include food waste containing water, vegetables, fruits, meat/fish, and grains [91]. Specific examples include carrot peels, cabbage, potato peels, fruit peels such as banana peels, apple peels, and orange peels, baked fish, rice, and soaked tea [92,93]. For example, MA et al. used Lactobacillus delbrueckii inoculation to produce 28 g/L of highly concentrated lactic acid from banana peels, and the sequential saccharification and fermentation processes increased lactic acid production by about 2.8-fold [94].

In addition, the waste generated by the dairy industry, particularly expired or damaged dairy products like whey, represents a valuable resource. Whey is particularly well suited for lactic acid production due to its composition, which includes lactose, proteins, fats, water-soluble vitamins, minerals, and other nutrients that support microbial growth. When lactose is cleaved into 1 mol of glucose and 1 mol of galactose, homogeneous fermentation theoretically produces 4 mol of lactic acid from 1 mol of lactose [33]. Additionally, some dairy products, such as yogurt, often have added sugars like sucrose and glucose, leading to higher lactate production compared to cheese whey, which contains less sugar. For example, Alonso et al. achieved bioconversion of approximately 44% of total sugars to 25.9 g/L lactic acid in batch fermentation using yogurt whey [95].

These organic wastes are widely available but are often neglected or used only as low-value fuels. Through silage technology, with the inoculation of appropriate strains for fermentation, these wastes can be converted into nutrient-rich, palatable feed, providing a stable feed source for animal husbandry.

3.2.4. Glycerol

In recent years, a variety of microorganisms have shown the ability to efficiently convert glycerol to lactic acid, including Klebsiella [96], LAB [97], and genetically modified E. coli. In particular, the study of Tian et al. revealed that modified E. coli could efficiently produce L-lactic acid from glycerol under optimal conditions, with lactate productivity of 4.90 g/L/h and a yield of 93.7% in 27 h [98]. These findings highlight the potential of glycerol as an alternative substrate for lactate fermentation. Furthermore, Vaidyanathan et al. and Wang et al. also supported the widespread application of glycerol in microbial fermentation. For example, Vaidyanathan et al. studied the ability of genetically modified Lactobacillus reuteri strains to produce lactic acid and 1,3-propanediol in the presence of glycerol. Research has shown that glycerol can be used as an alternative fermentation substrate to efficiently produce lactic acid by adjusting fermentation conditions [99]. Wang et al. utilized a microbial consortium dominated by 57.97% Enterobacter and 39.25% Escherichia to convert crude glycerol to lactic acid (LA) and 1,3-propanediol. The microbial consortium showed better tolerance to crude glycerol and higher lactic acid production capacity [100]. In view of these advantages of glycerol in microbial fermentation, a study has found that glycerol can replace wheat starch as a substrate for silage feed to feed cows. Compared with wheat starch feed, the utilization of glycerol by ruminal microorganisms in cows is greater. In addition, dairy cows fed the glycerol diet benefited from de novo mammary gland synthesis and reduced adipose tissue breakdown, providing more energy for the cows [101].

Therefore, in future studies, the addition of glycerol to silage could be considered to provide higher energy density and a more optimized fatty acid composition of animal feed, which can help to improve the performance of dairy cows, including increasing their milk yield and improving their milk fat quality. This research is expected to provide a more efficient and sustainable feed solution.

3.2.5. Microalgae and Cyanobacteria

As photosynthetic autotrophic microorganisms, microalgae and cyanobacteria can grow in a wide variety of environments, have short harvest cycles, and produce lactic acid without the cost of carbohydrate feed. In recent years, microalgae and cyanobacteria have become substitutes for silage fermentation substrates due to their advantages. Some reports have evaluated the amounts of lactic acid produced by microalgae fermentation. For example, using microalgae, Scenedesmus obliquus strain D3 can produce D-LA as the main fermentation product [33,34]. Nannochlorum sp. 26A4 produced D-LA at a concentration of 26 g/L under anaerobic conditions, achieving a 70% yield and an optical purity of 99.8% (40% content per dry weight) [102].

In addition, microalgae have extremely high protein contents, with spirulina having a protein content of over 60%, containing all 18 amino acids required by animals, especially lysine and methionine, which are often lacking in leguminous foods. For silage feed substrates, this means that microalgae can provide a more comprehensive and balanced amino acid profile, which is beneficial for animals’ growth and development. Microalgae are also rich in various vitamins (such as vitamin A, vitamin E, folate) and minerals (such as iron, calcium, etc.), as well as a large amount of bioactive substances such as phycobiliprotein and phycocyanin, which have multiple biological functions, including antioxidant, anti-inflammatory, and antiviral properties [103]. For animals, these bioactive substances help to enhance immunity, resist diseases, and promote health. More importantly, the growth cycle of microalgae is very short, usually taking only a few days to complete one growth cycle. Compared to traditional crops, microalgae have higher production efficiency and can produce a large amount of feed materials in a short period of time. At present, there is a study using spirulina platensis microalgae as a substitute for rapeseed meal and broad beans as a substrate for silage feed. Studies have shown that the addition of spirulina platensis microalgae improves the milk production performance of cows and greatly increases their milk yield [104]. However, microalgae have poor palatability as silage feed. In order to overcome the palatability issue of microalgae feed, it is necessary to add other substrates to the silage to further improve the feeding strategy and processing methods of microalgae [105].

Table 1. Sources of substrates for lactic acid production from organic wastes.

Type	Substrate Name	Strengths	Weaknesses	Potential Recommendations for LA Production	Reference
Agricultural wastes	Alfalfa fiber	Rich in cellulose, can serve as a carbon source for lactic acid production	Low starch content, lower fermentation efficiency	Suitable for pre-treatment before fermentation to increase LA yield	[69]
	Bran and straw	Rich in cellulose and hemicellulose, suitable for microbial fermentation	Requires pre-treatment, may produce inhibitors affecting fermentation	Enzymatic pre-treatment before LA fermentation to improve efficiency	[70,71]
	Non-fat rice bran	Contains residual sugars and cellulose, supports lactic acid bacteria growth	Fermentation efficiency may be limited	Can be directly used for lactic acid fermentation or mixed with other carbon sources	[72,73]
	Food residue	High organic content, rich in carbon sources	Variable composition, inconsistent fermentation efficiency	Optimize processing methods to increase LA production	[74]
	Corn straw and corn cob	Rich in cellulose and hemicellulose, potential for lactic acid production	Requires complex pre-treatment to release fermentable sugars	Used as a cellulose source after pre-treatment for LA fermentation	[75,76]
	Barley bran husk	Contains fermentable cellulose and hemicellulose	Low starch content, may have lower fermentation efficiency	Can be used as auxiliary material in lactic acid fermentation	[75]
	Sugarcane and cassava bagasse	Byproducts of the sugar and starch industries, rich in cellulose	Needs processing to remove components unfavorable to fermentation	Suitable as a fermentation substrate for lactic acid production	[77,78,79]
	Pruned vine sprouts	Rich in cellulose, can be used for anaerobic fermentation to produce LA	Low starch content, requires pre-treatment	Used for LA fermentation after pre-treatment	[75]
	Wine pruned waste	Rich in organic matter, suitable for fermentation	High phenolic content may inhibit fermentation	Optimize fermentation conditions to reduce inhibition and increase LA yield	[75]
	Artichoke	Rich in carbohydrates and cellulose, suitable for fermentation	High moisture content may affect fermentation	Detailed chemical composition analysis is required to determine its feasibility as a substrate for lactic acid production	[80]
	Broccoli	Rich in carbohydrates and cellulose, suitable as a substrate for lactic acid fermentation	Compared to other agricultural byproducts, broccoli may have less waste volume and higher pre-treatment and processing costs	It can be mixed with other carbon sources for lactic acid production	[80]
	Olive mill waste	Rich in large amounts of organic material, including carbohydrates and oils, and is a good substrate for lactic acid fermentation	High levels of moisture and impurities may affect fermentation, and harmful substances may be produced during fermentation	Strengthen the pre-treatment process to remove water and impurities, optimize fermentation conditions, and reduce the production of odor and harmful substances	[81]
	Date palm leaves	Rich in lactose and other fermentable sugars, and is a good substrate for lactic acid fermentation	Low conversion efficiency of cellulose and hemicellulose	Methods to improve the efficiency of cellulose and hemicellulose conversion, or mixed with other carbon sources	[82]
	Deproteinised feta cheese waste	Rich in lactose and other fermentable sugars, and is a good substrate for lactic acid fermentation	The fermentation process may be disturbed by the original microorganisms in the dairy products	Optimize fermentation conditions to inhibit the growth of original microorganisms in dairy products, and strengthen the pre-treatment process	[83]
	Apple pomace	Rich in sugars and pectin, suitable for lactic acid fermentation	LA production efficiency may be limited	Mainly used to ferment pectin and sugars for LA production	[84]
	Banana waste	Rich in sugars, suitable for efficient lactic acid fermentation	High moisture content, prone to spoilage	Dried and then used for efficient lactic acid fermentation	[85]
	Mussel processing waste	High in organic matter, rich in proteins	No starch, unsuitable for lactic acid fermentation	Not suitable for lactic acid production	[33,34]
	Cellulosic biological sludge	Rich in cellulose, can serve as a substrate for LA fermentation	Variable composition, affects fermentation efficiency	Suitable for the conversion of cellulose into lactic acid	[106]
	Kitchen waste	High in organic matter and potential sugars	Complex composition, may produce inhibitors during fermentation	Sorting and optimizing processing methods to increase LA production	[86]
	Fish meal waste	Rich in proteins and oils, unsuitable for lactic acid fermentation	No starch or sugars, very low fermentation efficiency	Not suitable for lactic acid fermentation	[87]
	Paperboard waste	Rich in cellulose, recyclable for fermentation	No starch, low fermentation efficiency	Suitable for the conversion of cellulose into lactic acid	[107]
	Bagasse waste	Rich in cellulose, suitable for lactic acid production	Low starch content, byproduct of sugar extraction	Suitable as a fermentation substrate for lactic acid production	[88]
Food waste	Vegetables (carrot peels, cabbage, potato peels)	Contains some residual starch and sugars, easy to process	Varied types, potential contaminants	Suitable for efficient lactic acid fermentation from vegetable waste	[92,93]
	Fruits (banana peels, apple peels, and orange peels)	Rich in sugars, suitable for lactic acid fermentation	High moisture content, prone to spoilage	Dried and enzymatically hydrolyzed before lactic acid fermentation	[92,93]
	Baked fish	Rich in proteins and oils, unsuitable for lactic acid fermentation	No starch or sugars, unsuitable for lactic acid production	Not suitable for lactic acid production	[92,93]
	Rice	High in starch, suitable for lactic acid fermentation	Requires processing to improve purity	Directly used for lactic acid fermentation	[92,93]
	Soaked tea	Contains polyphenols, unsuitable for lactic acid fermentation	No starch or sugars, unsuitable for lactic acid production	Not suitable for lactic acid production	[92,93]
Dairy products	Yogurt whey	Rich in proteins and lactose, suitable as a medium	No starch or sugars, may limit acid production efficiency	Can be used as a medium for lactic acid bacteria	[95]

Table 1. Sources of substrates for lactic acid production from organic wastes.

Type	Substrate Name	Strengths	Weaknesses	Potential Recommendations for LA Production	Reference
Agricultural wastes	Alfalfa fiber	Rich in cellulose, can serve as a carbon source for lactic acid production	Low starch content, lower fermentation efficiency	Suitable for pre-treatment before fermentation to increase LA yield	[69]
	Bran and straw	Rich in cellulose and hemicellulose, suitable for microbial fermentation	Requires pre-treatment, may produce inhibitors affecting fermentation	Enzymatic pre-treatment before LA fermentation to improve efficiency	[70,71]
	Non-fat rice bran	Contains residual sugars and cellulose, supports lactic acid bacteria growth	Fermentation efficiency may be limited	Can be directly used for lactic acid fermentation or mixed with other carbon sources	[72,73]
	Food residue	High organic content, rich in carbon sources	Variable composition, inconsistent fermentation efficiency	Optimize processing methods to increase LA production	[74]
	Corn straw and corn cob	Rich in cellulose and hemicellulose, potential for lactic acid production	Requires complex pre-treatment to release fermentable sugars	Used as a cellulose source after pre-treatment for LA fermentation	[75,76]
	Barley bran husk	Contains fermentable cellulose and hemicellulose	Low starch content, may have lower fermentation efficiency	Can be used as auxiliary material in lactic acid fermentation	[75]
	Sugarcane and cassava bagasse	Byproducts of the sugar and starch industries, rich in cellulose	Needs processing to remove components unfavorable to fermentation	Suitable as a fermentation substrate for lactic acid production	[77,78,79]
	Pruned vine sprouts	Rich in cellulose, can be used for anaerobic fermentation to produce LA	Low starch content, requires pre-treatment	Used for LA fermentation after pre-treatment	[75]
	Wine pruned waste	Rich in organic matter, suitable for fermentation	High phenolic content may inhibit fermentation	Optimize fermentation conditions to reduce inhibition and increase LA yield	[75]
	Artichoke	Rich in carbohydrates and cellulose, suitable for fermentation	High moisture content may affect fermentation	Detailed chemical composition analysis is required to determine its feasibility as a substrate for lactic acid production	[80]
	Broccoli	Rich in carbohydrates and cellulose, suitable as a substrate for lactic acid fermentation	Compared to other agricultural byproducts, broccoli may have less waste volume and higher pre-treatment and processing costs	It can be mixed with other carbon sources for lactic acid production	[80]
	Olive mill waste	Rich in large amounts of organic material, including carbohydrates and oils, and is a good substrate for lactic acid fermentation	High levels of moisture and impurities may affect fermentation, and harmful substances may be produced during fermentation	Strengthen the pre-treatment process to remove water and impurities, optimize fermentation conditions, and reduce the production of odor and harmful substances	[81]
	Date palm leaves	Rich in lactose and other fermentable sugars, and is a good substrate for lactic acid fermentation	Low conversion efficiency of cellulose and hemicellulose	Methods to improve the efficiency of cellulose and hemicellulose conversion, or mixed with other carbon sources	[82]
	Deproteinised feta cheese waste	Rich in lactose and other fermentable sugars, and is a good substrate for lactic acid fermentation	The fermentation process may be disturbed by the original microorganisms in the dairy products	Optimize fermentation conditions to inhibit the growth of original microorganisms in dairy products, and strengthen the pre-treatment process	[83]
	Apple pomace	Rich in sugars and pectin, suitable for lactic acid fermentation	LA production efficiency may be limited	Mainly used to ferment pectin and sugars for LA production	[84]
	Banana waste	Rich in sugars, suitable for efficient lactic acid fermentation	High moisture content, prone to spoilage	Dried and then used for efficient lactic acid fermentation	[85]
	Mussel processing waste	High in organic matter, rich in proteins	No starch, unsuitable for lactic acid fermentation	Not suitable for lactic acid production	[33,34]
	Cellulosic biological sludge	Rich in cellulose, can serve as a substrate for LA fermentation	Variable composition, affects fermentation efficiency	Suitable for the conversion of cellulose into lactic acid	[106]
	Kitchen waste	High in organic matter and potential sugars	Complex composition, may produce inhibitors during fermentation	Sorting and optimizing processing methods to increase LA production	[86]
	Fish meal waste	Rich in proteins and oils, unsuitable for lactic acid fermentation	No starch or sugars, very low fermentation efficiency	Not suitable for lactic acid fermentation	[87]
	Paperboard waste	Rich in cellulose, recyclable for fermentation	No starch, low fermentation efficiency	Suitable for the conversion of cellulose into lactic acid	[107]
	Bagasse waste	Rich in cellulose, suitable for lactic acid production	Low starch content, byproduct of sugar extraction	Suitable as a fermentation substrate for lactic acid production	[88]
Food waste	Vegetables (carrot peels, cabbage, potato peels)	Contains some residual starch and sugars, easy to process	Varied types, potential contaminants	Suitable for efficient lactic acid fermentation from vegetable waste	[92,93]
	Fruits (banana peels, apple peels, and orange peels)	Rich in sugars, suitable for lactic acid fermentation	High moisture content, prone to spoilage	Dried and enzymatically hydrolyzed before lactic acid fermentation	[92,93]
	Baked fish	Rich in proteins and oils, unsuitable for lactic acid fermentation	No starch or sugars, unsuitable for lactic acid production	Not suitable for lactic acid production	[92,93]
	Rice	High in starch, suitable for lactic acid fermentation	Requires processing to improve purity	Directly used for lactic acid fermentation	[92,93]
	Soaked tea	Contains polyphenols, unsuitable for lactic acid fermentation	No starch or sugars, unsuitable for lactic acid production	Not suitable for lactic acid production	[92,93]
Dairy products	Yogurt whey	Rich in proteins and lactose, suitable as a medium	No starch or sugars, may limit acid production efficiency	Can be used as a medium for lactic acid bacteria	[95]

4. Enhancing Lactic Acid Production in Silage Fermentation

LAB are pivotal in silage fermentation due to their ability to produce lactic acid, which rapidly lowers pH, inhibiting the growth of unwanted microorganisms and ensuring dominance in the fermentation process. Ideal LAB should reproduce rapidly, produce acid efficiently, tolerate acidic environments, resist adverse conditions, and be safe. Numerous studies have delved into factors influencing LAB populations during silage fermentation. It has been found that LAB inoculants can significantly enhance LAB populations, thereby improving the silage quality. Genetic modification presents new avenues for boosting LAB’s fermentation potential. This section discusses strategies for optimizing LAB populations in silage fermentation, including inoculum selection, dosing, timing, and the fermentation environment. The importance of biotechnology—particularly genetic modification—in enhancing silage fermentation is emphasized.

4.1. Selecting the Appropriate Lactic Acid-Producing Microorganisms

The diversity of LAB in silage necessitates careful selection and application of inoculants to achieve high-quality silage. Selected strains undergo screening for organic acid production, protein breakdown, and growth rates at various pH values and temperatures, along with performance testing (Figure 1). Effective LAB strains must adapt well, ferment efficiently, and be compatible with raw silage material. Homofermentative lactic acid bacteria mainly reduce pH by producing high concentrations of lactic acid, and they hardly produce ethanol, acetic acid, and butyric acid. Compared with homofermentative lactic acid bacteria, facultative heterofermentative lactic acid bacteria strains have ketolase phosphate, which enables them to ferment pentose and produce lactic acid, acetic acid, and ethanol. Obligate heterofermentative LAB can decompose lactic acid into ethanol and 1,2-propanediol during ensiling fermentation to inhibit the growth of harmful microorganisms and prolong the aerobic stability time of the feed; at the same time, obligate heterofermentative LAB can also produce ferulic acid esterase, which is beneficial to cellulose degradation, promotes lactic acid production, and inhibits the activity of harmful bacteria [108].

Experiments help identify LAB strains that perform well under specific silage conditions. When identifying and screening strains formed during the silage process, their acid-producing capacity, acid tolerance, ability to inhibit harmful microorganisms, fermentation efficiency, survival and multiplication, storage tolerance, and nutrient value-added capacity should be taken into account. These parameters help to select the right strain to ensure the quality and nutritional value of the silage. For example, Ding et al. isolated two strains of lactic acid bacteria producing feruloyl esterase (FAE)—namely, Lactobacillus plantarum A1 (LP) and Lactobacillus breveus A3 (LBr)—from alfalfa on the Qinghai–Tibet Plateau [109]. After silage for 60 days, the lactic acid content was significantly increased. The lactic acid content was increased by 18.7 g/kg compared with the control group. Meanwhile, the combination of commercial cellulase and FAE-producing LAB synergistically improved the fermentation quality of the silage. The addition of cellulase increased the lactic acid content by nearly 2-fold compared to the inoculum.

In addition, Saarisalo et al. evaluated strains that could rapidly reduce the pH value of forage substrates and had low proteolytic activity in a laboratory-scale silo containing natural microbial flora, and they selected four of the most promising strains (two Pediococcus (E315 and E390) and two Lactobacillus (E76 and E98) strains), among which Lactobacillus plantarum strain E76 was superior to the other candidates [110]. It was also applied to a pure lactic acid fermentation of fescue silage in Timothy and Meadow hay, and the results were consistent with those of the screening program, correctly predicting the potential of Lactobacillus plantarum strain E76 as an inoculant for pasture silage.

In another study, Liu et al. characterized Pediococcus pentosaceus SC1 and Lactobacillus paraphysium SC2 strains isolated from silage grass, and they investigated their effectiveness in improving the fermentation quality of stem silage. The results showed that, compared with SC2, strain SC1 was most effective at improving the fermentation quality of silage at 20 °C for 45 days. The lactic acid content was increased by nearly 2-fold compared with the control group [111]. In another similar study, Xu et al. also screened Pediococcus valericus Q6 from the drapephalus growing on the Qinghai–Tibet Plateau, and the study showed that Q6 could grow at pH 3.0 and 4 °C for 30 days. Under these conditions, the lactic acid efficiency was increased by 50% [112]. At the same time, Q6 can ferment mannitol, sucrose, sorbitol, and rhamnose, which helps improve the quality of low-temperature silage, providing a candidate strain to produce high-quality silage in cold-climate regions. This strain has achieved good results in the local area, and it is planned to spread it to neighboring farmers in the future to promote the development of animal husbandry in the whole region.

Shah et al. isolated and identified LAB from King grass (Pennisetum, Pennisetum purpureum Rich × Pennisetum americanum cv. Reyan No.4) and applied them to improve the fermentation quality of sweet sorghum [113]. Three isolates, L. plantarum (HDASK), P. acidilactici (SK3907), and P. acidilactici (ASKDD), were added to sweet sorghum, and the lactate content was significantly increased. After 7 days of silage, the lactic acid content increased by approximately 40% compared with the control group. The screening criteria for potential lactobacilli inoculants for silage are shown in Figure 2. Therefore, it is crucial to carefully select new strains to enhance the silage quality. Additionally, these strains must possess characteristics suitable for industrial cultivation and must maintain stability during storage.

4.2. Optimizing the Dose and Timing of Inoculation

The dose and time of inoculation have an important effect on the amount and activity of LAB during silage fermentation. Through experiments, we can determine the optimal inoculation dose and inoculation time to ensure that lactic acid bacteria (LAB) maintain a dominant position during the silage fermentation process, thereby optimizing the quality of the silage. Ma et al. studied the effects of Enterococcus faecalis (E. faecalis) combined with protease on soybean meal (SBM) silage and used response surface methodology (RSM) to optimize the optimal growth conditions of protease-producing Enterobacter faecalis ZZUPF95 and fermented SBM under the optimal fermentation conditions [114]. The results showed that the optimal fermentation conditions of ZZUPF95 were 10% inoculum, a 1:1 solid–liquid ratio, and 36 h of fermentation time. After fermentation, the ZZUPF95 and ZZUPF95+ protease groups could reduce the pH value of the feed, increase the content of lactic acid in the fermentation system, increase the content of crude protein, and reduce the content of crude fiber. Among them, the addition of ZZUPF95+ protease increased the lactic acid content by approximately 40 mg/g compared with the control group after 18 days of silage. This has certain theoretical value for improving the fermentation and storage of SBM.

4.3. Regulation of the Fermentation Environment

The growth and fermentation activity of LAB are affected by environmental factors, including temperature, pH, and oxygen concentration. By regulating these environmental factors, the fermentation environment can be optimized to increase their number and activity during silage fermentation, e.g., appropriately lowering the fermentation temperature, adjusting the pH value, providing adequate oxygen, etc. Shan et al. developed a multi-sensor micro-bioreactor (MSMB) to monitor microbial fermentation in situ and proposed a mathematical model based on the Bolza equation for optimal evaluation of candidate inocula. This model uses data from three sensors (pH, CO₂, and ethanol) and includes four additional sensors (O₂, gas pressure, temperature, and atmospheric pressure) to control the fermentation environment [115]. These novel rapid data processing methods associated with MSMBs may accelerate the development of microbial amendments for silage additives.

Aerobic stability is also a critical factor during silage fermentation. Vigorous aerobic metabolism will reduce the nutritional quality and animal acceptance of silage [116]. Therefore, Shan et al. introduced an innovative interruption-free dual-sensor method to demonstrate that both in situ silage temperature (T_si) and pH are related to the preservation of lactic acid. Their study assessed aerobic deterioration using two sources of corn silage, one supplemented with biological additives, at 23 °C and 33 °C incubation temperatures. The results indicated a time delay between the rise in T_si and the rise in pH after aerobic exposure at both incubation temperatures. The T_si reached 2 °C above ambient temperature at a lactate loss of 11% to 25%. In contrast, when the silage pH increased by 0.5 units above its initial value, over 60% of the lactic acid had been metabolized. Although pH is typically used as the primary indicator of aerobic deterioration in maize silage, T_si proved to be more sensitive and timely under the same conditions, making temperature monitoring a more reliable early warning indicator [115].

4.4. Use of Combined Inoculum

Since different LAB species have different fermentation characteristics, single LAB species often cannot achieve optimal fermentation results. Composite inoculants leverage the synergistic effects between different LAB species, improving their number and activity, which enhances the fermentation quality of silage.

Okoye et al. analyzed the performance of homofermentative LAB (Lactobacillus plantarum (Lp) or Pentosaccharomyces pellucida (Pp)) and heterofermentative LAB (Lactobacillus buchneri (Lb) or combinations (LbLp or LbPp)) in silage alfalfa forage fermentation, and the results showed that LbLp inoculation resulted in higher contents of water-soluble carbohydrates (WSCs) and lactobacilli, as well as a significant improvement in alfalfa carbohydrate metabolism and significantly inhibiting the growth of Clostridia, molds, and yeasts [117]. The results showed that, after 30 days of silage, the WSC contents of silages inoculated with Lp, Lb, and LbLp increased to 10.46 g/kg DM, 10.33 g/kg DM, and 10.84 g/kg DM, respectively. The most prominent genus among all treatments after different silage days was Lactobacillus. After 60 days of silage, the highest abundance of lactic acid bacteria was detected in alfalfa silage inoculated with Lp, Lb, and LbLp, with values of 93.3%, 94.5%, and 96.02%, respectively.

In addition to complex Lactobacillus inoculants, Lactobacillus is commonly used in combination with other types of inoculants in silage fermentation. Fang et al. evaluated the fermentation quality of high-moisture whole-plant quinoa (WPQ) silage with different additives (molasses (M), LAB (L), and combinations of molasses and LAB (ML)); the results showed that the M and ML groups moved the fermentation pattern towards increasing the intensity of lactic acid fermentation. Under natural fermentation conditions, the lactic acid content was low (37.0 g/kg DM) and the pH was high (5.65). The addition of molasses, alone or in combination with lactic acid bacteria inoculum, reduced the pH value (<4.56) while increasing the lactic acid content (>60.5 g/kg DM) and the relative abundance of lactic acid bacteria (>83.0%). It greatly alleviated the problem of the lack of fermentable sugars in WPQ, and this is expected to be a useful strategy for the production of high-quality WPQ silage [118]. Similar studies, such as the work of Abbasi et al., added 5% molasses to LAB and amaranth silage fermented for 45 d. It was found that the addition of molasses resulted in an increase in feed ash and lactic acid concentration. The lactic acid content was increased by approximately 20% [119].

Creatively, in another study, Amado et al. identified a selective bacteriocin (Pediocin SA-1), and the co-inoculation of Lactobacillus and this bacteriocin into corn silage showed that the combination of the two reduced the Listeria monocytogenes content and significantly increased the concentrations of antimicrobial compounds (acetic acid, ethanol, and 1,2-propanediol). The abundance of lactic acid bacteria decreased by 2 × 10¹⁰ CFU/g [120].

In addition, enzyme addition is considered to be an effective strategy to enhance silage fermentation quality. Zhao et al. explored the effects of LAB and cellulase treatment (LAB + CE) on mixed silage fermentation of soybean residue and corn stover, and the results showed that the LAB + CE group had a significant increase in crude protein content and LAB content, along with a significant decrease in the contents of undesirable bacteria. The crude protein content was increased by 30%, and the number of lactic acid bacteria was increased by approximately 6 × 10¹⁰ CFU/g [121]. In another study, Liu et al. investigated the effect of cellulase addition on enhancing silage fermentation with Bacillus inoculants, and the results showed that the lactic acid accumulation in silage in the cellulase–Bacillus group was significantly higher than that of the Bacillus inoculant group [122]. Lactobacillus acted as the dominant bacteria with the highest abundance, and the addition of cellulase increased the bacterial communities, resulting in a significant increase in neutral detergent fiber (NDF) and acid detergent fiber (ADF) degradation. After 60 days of silage, NDF and ADF decreased by approximately 10% and 5%, respectively. This reveals the potential of cellulase to improve the nutritional quality of straw.

The effects of different additives on the fermentation of silage are shown in

Table 2. Effects of different additives on the fermentation effects of silage.

Silage Crop	Additives	Ferment Effect	Comparison of Lactic Acid Content	Reference
Alfalfa	Lactobacillus brucei PC-C1 + Lactobacillus plantarum YC1-1-4B	It significantly improved carbohydrate metabolism and inhibited the growth of Clostridium, mold, and yeast in alfalfa	Treatment group: 5.35 g/kg CK group: 0.74 g/kg	[117]
Whole-plant quinoa with high moisture	Lactobacillus + molasses/Lactobacillus + cellulolytic enzymes	The fermentation mode was developed to increase the intensity of lactic acid fermentation, which greatly alleviated the limitation of lactic acid fermentation by the lack of fermentable sugars in WPQ	Treatment group: 60.5 g/kg CK group: 37.0 g/kg	[118]
Three-colored amaranth	Lactic acid bacteria + 5% molasses	The addition of molasses resulted in increased feed ash and lactic acid concentrations, and also improved the fermentation quality of silage	Treatment group: 69.2 g/kg CK group: 57.0 g/kg	[119]
Corn	Lactobacillus plantarum/Lactobacillus brucei/Enterococcus faecium + selective bacteriocin (Pediocin SA-1)	It also significantly increased the concentrations of antimicrobial compounds (acetic acid, ethanol, and 1,2-propylene glycol) and enhanced the aerobic stability of maize silage	Treatment group: 40.2 g/kg CK group: 34.9 g/kg	[120]
Soybean residue + corn stover	Lactic acid bacteria + cellulase	The content of crude protein was significantly higher than that in the control group, the content of lactic acid bacteria was significantly higher than that in other groups, and the contents of undesirable bacteria were significantly lower than in the other treatment groups	Treatment group: 5.45 g/kg CK group: 3.81 g/kg	[121]
Whole-crop corn	Cellulase + Bacillus sp.	Lactic acid accumulation was significantly higher in the Bacillus inoculant group and the control group; lactic acid bacteria were the most abundant dominant bacteria, and the addition of cellulase increased the bacterial community, leading to significant degradation of neutral detergent fiber (NDF) and acid detergent fiber (ADF)	Treatment group: 6.0 g/kg CK group: 8.3 g/kg	[122]

. All of the above studies showed that the fermentation effect of multiple inoculants on different silage materials was better than that of single inoculants, but some of the mechanisms remain to be studied. However, further development of these combinations to adapt to various silage conditions, such as different plant biomass types and climates, may be more beneficial in controlling the initial active fermentation period, inhibiting pathogenic microorganisms, and leading to a faster decline in pH and dry matter (DM) losses.

4.5. Genetic Modification Technology

Advances in genetic modification technology have provided new ways to improve LAB strains for silage fermentation. Techniques such as metagenomics, genomics, proteomics, and metabolomics offer deeper insights into LAB’s physiological characteristics and metabolic processes, aiding in strain selection and optimization (Figure 3) [8]. Gene editing technologies like CRISPR/Cas9 allow for the development of LAB with enhanced fermentation capacities and stress resistance. However, genetic modification must be carefully evaluated for safety and feasibility to prevent environmental and health risks [8].

4.5.1. Metagenomics

Metagenomics is a powerful tool for understanding the intricate changes in microbial communities during silage fermentation, including the interactions between bacterial and viral communities. Saenz et al. utilized metagenomics to analyze the composition of bacterial and viral communities over a 40-day silage preservation period [123]. Their findings revealed a decreasing trend in the diversity of dominant viral operational taxonomic units (vOTUs) throughout the process. The changes in bacterial communities mirrored those of the vOTUs predicted to be their hosts, with only 10% of recovered vOTUs clustering with the reference genome. The bacterial community rapidly shifted between days 0 and 2: Firmicutes increased from 8 to 75%, Proteobacteria decreased from 79% to 22%, and Actinobacteria decreased from 11% to 2%. After 40 days, Firmicutes dominated the community, reaching 84%, while Proteobacteria and Actinobacteria decreased to 15% and 0.5%, respectively. On the fresh grass (day 0), Pantoea, Pseudomonas, and Curtobacterium were the most abundant genera, while Weissella, Lentilactobacillus, Pantoea, and Lactiplantibacillus were the dominant ones at the end of the preservation. Notably, phage infection history was observed only in Lactobacillus lentil and Levilactobacillus, and vOTUs harbored potential accessory metabolic genes related to carbohydrate metabolism, organic nitrogen, stress resistance, and transport. This study suggests that vOTUs are enriched in silage and may influence bacterial community establishment, offering new perspectives for optimizing forage preservation strategies.

Xu et al. conducted a metagenomic analysis to explore the occurrence and transmission mechanisms of antibiotic resistance genes (ARGs) and their microbial hosts in whole-maize silage inoculated with homofermentative lactic acid bacteria (Lactobacillus plantarum) and heterofermentative lactic acid bacteria (Lactobacillus brucella) at different temperatures (20 °C and 30 °C) [124]. The study found that Lactobacillus, Leuconostoc, Lactobacillus platyphylla, and Lactobacillus were dominant in maize silage, and that both temperature and inoculation significantly altered the silage microbiota. The ARGs of L. brucella increased significantly regardless of temperature, while mobile genetic elements (MGEs) were higher at low temperatures. The corn silage microbial community exhibited diverse ARGs, primarily MacB, RanA, bcrA, msbA, TetA, and TetT, mainly associated with macrolides and tetracyclines. Plasmids were the most abundant MGEs, significantly linked with high-risk ARGs like tetM, TolC, mdtH, and NorA, whose abundance was reduced during the silage process. The study demonstrated that Lactobacillus brucei inoculation and higher storage temperatures could improve the biosafety of maize silage by reducing the abundance and transmission of high-risk ARGs. In a similar study, Zhang et al. used metagenomics to investigate the effects of Lactobacillus plantarum MTD/1 or Lactobacillus brucei 40788 inoculations on ARG distribution and transmission in alfalfa silage [125]. Their results showed that multidrug and bacitracin resistance genes were the dominant ARGs in Medicago sativa. The natural ensiling process increased the abundance of bacitracin, beta-lactams, and aminoglycosides in 30% DM alfalfa silage, as well as the abundance of vancomycin in 40% DM alfalfa silage. Prolonged wilting also increased ARG enrichment in fresh alfalfa. Notably, inoculation with Lactobacillus plantarum MTD/1 or Lactobacillus brucella 40788 reduced the abundance of total ARGs and of multidrug, MLS, vancomycin, aminoglycosides, tetracycline, and fosfomycin resistance genes by reducing host bacteria and enriching ARGs in plasmids. The study concluded that Lactobacillus plantarum inoculation was more effective in reducing the abundance of ARGs in green alfalfa, compared to L. brucella.

4.5.2. Genomics

Next-generation sequencing (NGS) has revolutionized the study of LAB’s performance in silage. Ni et al. used NGS to compare the microbiota of wilted Italian ryegrass (Lolium, Lolium multiflorum) and wilted alfalfa silage [126]. Their results revealed that LAB were prevalent in all silages after two months of storage, but the dominant groups varied: Leuconostoc and Pediococcus for wilted Italian ryegrass silages, and Enterococcus in wilted alfalfa silages.

Genetic engineering technology is also used to modify inoculants. Rossi et al. employed recombinant cellulolytic Lactobacillus plantarum as a silage inoculant, genetically modifying Lactobacillus plantarum B41 by integrating the celA gene of alkaline endo-1,4-β-glucanase from the Bacillus genus into its chromosome through vector-free cloning technology [127]. This modification significantly increased the acidifying capacity of the Lactobacillus plantarum B41 cellulolytic clone in silage samples incubated at 37 °C.

Several genetic engineering approaches have been developed to produce high-quality silage. For instance, enhancing the digestibility of forage maize is crucial for improving the feed intake, growth rate, and milk yield in animals. Genetic methods include using known mutants of the lignin pathway, genetic engineering of genes involved in the lignin, cellulose, and hemicellulose pathways, and breeding low-fiber and low-lignin concentrations using conventional methods or marker-assisted selection (MAS) [128]. Modern biotechnological tools like CRISPR/Cas9, RNA interference (RNAi), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) enable site-directed mutagenesis or base editing in maize silage applications [129]. Genetic engineering has produced maize plants overexpressing genes like ZmPLA1, ZmDof1, ZmGLK1, ZmEmBP-1, and Zmm28, which show increased biomass accumulation. Additionally, downregulating genes like GA20 oxidase and certain lignin synthesis genes can improve the quality and quantity of silage [128].

4.5.3. Transcriptomics

Transcriptomics allows for the investigation of gene expression processes in LAB, offering insights into their functional significance [128]. Eikmeyer et al. performed transcription analysis of Lactobacillus brucei strain CD034, known for enhancing silage quality, by sequencing transcriptomes isolated under anaerobic and aerobic conditions [130]. RNA sequencing data revealed that the most abundant transcribed genes were necessary for essential cellular functions such as protein biosynthesis, energy metabolism, and lactate fermentation, regardless of oxygen status. Under aerobic conditions, 283 genes were upregulated, while 198 genes were downregulated (p < 0.01). Upregulated genes were associated with oxygen consumption, oxidative stress response, and reactive oxygen species (ROS) breakdown, while genes related to pH homeostasis and redox potential balance were downregulated. The genes required for lactic acid fermentation were largely unaffected by growth conditions. Identifying differentially transcribed genes based on oxygen status can help pinpoint strains with favorable performance in silage formation [130].

DNA microarrays have also been used to characterize LAB species and their transcriptomic analyses, providing insights into gene expression associated with metabolic pathways. This approach helps understand the expression of genes encoding enzymes involved in beneficial organic acid production, proteolysis, and mycotoxins during silage [131].

4.5.4. Proteomics

Proteomics involves studying the structure, function, interactions, and dynamic changes of proteins in organisms. It aims to reveal information about protein localization, quantitative distribution, isoforms, intermolecular interactions, and post-translational modifications. Proteomics can determine the type and number of proteins on LAB cell surfaces and their roles in LAB–host interactions. Additionally, it helps map the protein expression profiles of LAB under different fermentation conditions, enhancing the understanding of LAB’s adaptation mechanisms [8].

Techniques used in proteomics include mass spectrometry (MS), gel chromatography (such as SDS-PAGE and 2-DE), circular dichroism (CD), nuclear magnetic resonance (NMR), protein interaction research (such as affinity chromatography and co-immunoprecipitation), and some bioinformatics analysis techniques for protein sequence alignment, annotation, and functional prediction.

Amortegui et al. employed proteomic techniques to characterize bacteriocins produced by Lactobacillus plantarum LE5 and LE27 isolated from maize silage, purified by ammonium sulfate precipitation and dual dialysis using 12 kDa and 1 kDa membranes [132]. These bacteriocins exhibited activity against Listeria innoculus, Listeria monocytogenes, and Enterococcus faecalis. Molecular weight estimation by Tricine-SDS-PAGE, followed by Mueller–Hinton agar overlay with Listeria monocytogenes, revealed an inhibition zone in the 5–10 kDa range. Nano-LC-MS/MS analysis identified the UPF0291 protein (UniProtKB/Swiss-Prot Q88VI7), which has an α-helix-rich structure and a large positively charged region. The bacteriocin remained stable at 4–121 °C and pH 2–12 but was inhibited by SDS and protease. These findings suggest a potential novel class IIa bacteriocin produced by Lactobacillus plantarum, which could serve as an antimicrobial peptide with biopreservative properties for silage [132].

4.5.5. Metabolomics

LAB produce a wide range of metabolites during fermentation, including oligosaccharides, amino acids, fatty acids, vitamins, and aromatic compounds [133]. Metabolomics, a subfield of omics, focuses on the in-depth study of low-molecular-weight endogenous metabolites in organisms, which play crucial roles in various physiological and pathological processes. Metabolomics employs targeted and non-targeted approaches, each with unique characteristics, complementing each other to provide a comprehensive understanding of metabolic profiles. Analytical techniques like mass spectrometry (MS) and nuclear magnetic resonance (NMR) are commonly used, with MS providing quantitative analysis of metabolic profiles and NMR revealing structural and functional information.

Separation techniques such as gas chromatography (GC), high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC), and capillary electrophoresis (CE) are often combined with MS and NMR to accurately identify and quantify metabolites. GC and HPLC/UPLC efficiently separate complex mixtures, while CE is particularly suitable for chiral compounds and biological macromolecules [134].

In silage metabolomics, these combined techniques help elucidate microbial metabolic activities and their impact on feed quality and nutritional value. For example, Guo et al. used GC-MS to analyze the metabolome of alfalfa silage without Lactobacillus plantarum or Lactobacillus brucella, identifying 280 substances and 102 different metabolites [135]. Inoculation with L. brucella significantly increased the concentrations of 4-aminobutyric acid, certain free amino acids, and polyols in green alfalfa, while L. plantarum inoculation led to a significant subaccumulation of cadaverine and succinate. Kharazian et al. used metabolomics to identify 10 bioactive metabolites in sorghum silage with high dry matter content [136], including chrysin, isorhamnetin, petunidin 3-glucoside, apigenin, caffeic acid, gallic acid, p-coumaric acid, trans-cinnamic acid, colic, and 3, 4-dihydroxytrans-cinnamate. These studies demonstrate that metabolomics analysis provides insight into silage metabolites, facilitating target-based modulation approaches to achieve high-quality silage production.

5. Future Prospects and Challenges

As an environmentally friendly and efficient method of feed preservation, lactic acid silage fermentation has shown broad application prospects in animal husbandry. With the increasing attention to environmental protection and sustainable development, lactic acid silage fermentation will be more widely used. Its unique fermentation process effectively inhibits the growth of harmful microorganisms, prolongs the shelf life of feed, and improves the nutritional value of feed. This will help reduce the dependence of animal husbandry on chemical preservatives, reduce environmental pollution, and promote the healthy development of agricultural ecosystems.

In addition, with the continuous progress of biotechnology, the processes and technology of lactic acid silage fermentation will also be further optimized. By screening more efficient LAB strains, optimizing fermentation conditions, and improving fermentation efficiency, the quality and yield of lactic acid silage can be further improved. This will ensure a more stable and reliable feed source for animal husbandry, thereby promoting its sustainable development.

However, with the continuous development of technology and continuous changes in the market, lactic acid silage fermentation also faces some challenges and areas for further research. The processes and technology of lactic acid silage fermentation still need to be further improved and optimized. How to screen more efficient lactic acid bacteria strains according to different environments, as well as how to control factors such as temperature, humidity, and oxygen content in the fermentation process to achieve the best fermentation effect, still needs further research [137].

Future technological innovation in silage fermentation should highlight the development of new, efficient, and environmentally friendly additives [137]. For example, using bioengineering technology, microbial strains with specific functions can be screened and applied as biological additives in silage to improve fermentation efficiency and feed quality. At the same time, natural additives such as plant extracts can also be studied to explore their application potential in silage [138].

In addition, with the swift advancement of technologies like the Internet of Things (IoT), big data, and artificial intelligence (AI), the research and development of intelligent silage fermentation equipment will become an important direction in the future. Through intelligent equipment, the fermentation process of silage could be monitored in real time, including key parameters such as temperature, humidity, and oxygen content, to achieve precise control of the fermentation process. Based on the current literature, the application of AI technology in silage fermentation is rather scarce. Therefore, the focus of future research could be turned to the combination of AI and silage. By providing data analysis functions, this could help farmers better understand the state and effect of feed fermentation and provide data support for optimizing the fermentation process. Although some new technologies (such as the IoT, AI, etc.) applied to silage fermentation itself may have high initial investment costs, the long-term benefits they bring (e.g., improved productivity, reduced energy consumption, reduced waste, etc.) may more than make up for these costs or even achieve a reduction in overall costs. We are still of the positive attitude that these new technologies and methods will contribute to silage development in the future.

To meet the nutritional requirements of different animals at different growth stages, future silage fermentation technology should pay more attention to the precise nutritional regulation of feed. Through in-depth study of the nutritional composition of silage and its relationship with animal growth performance, it is possible to develop customized silage for different animals and growth stages. This can not only improve the utilization efficiency of feed and reduce the cost of breeding but also help to promote the healthy growth of animals and improve their production performance.

Finally, silage will produce some byproducts during fermentation, such as waste liquid and waste gas [139,140]. How to recycle these byproducts and reduce environmental pollution will also be an important direction of future technological innovation. For example, the waste liquid can be used as organic fertilizer after harmless treatment, or the waste gas can be used for energy recovery. The advancement and implementation of these resource utilization technologies will support the sustainable development of silage. By continuously strengthening technological innovation and R&D efforts, we can promote the sustainable development of silage fermentation technology and provide more environmentally friendly, efficient, and sustainable feed solutions for the livestock industry. Although it is unlikely that traditional producers’ silage practices can be easily changed at the regional or global level, we are taking a proactive approach to improve the quality of silage through a number of agricultural and biotechnological techniques.

At the same time, interdisciplinary cooperation and support can be sought. The knowledge and technology of many disciplines, such as biology, chemistry, agricultural engineering, and computer science, can provide strong support for the research and application of lactic acid silage fermentation. By strengthening interdisciplinary cooperation and communication, we can jointly solve the technical problems and market challenges faced by lactic acid silage fermentation, promote its rapid development in animal husbandry, and contribute to the sustainable development of animal husbandry.

6. Conclusions

Today’s agricultural sector is moving towards sustainability, prioritizing the optimal use of resources. Appropriate and efficient utilization of roughage, with an emphasis on enhancing farm productivity, is a key objective for the coming years. As a method of feed storage, silage technology is central to achieving this goal. Selecting or screening suitable strains, utilizing industrial byproducts, and diversifying additives can contribute to cost-effectiveness. Additionally, advancing biotechnology and applying multi-omics approaches have progressively increased lactic acid production in silage. These advancements are being integrated into agricultural practices to enhance productivity and sustainability, thereby helping to realize the promise of sustainable agriculture.