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Article

Microbial Additive Isolated from Exotic Semi-Arid Cactus and Cottonseed Byproduct in Sustainable Sorghum Silage Production

by
Raiane Barbosa Mendes
1,
Gabriel Rodrigues Silva Oliveira
2,
Maria Leonor Garcia Melo Lopes de Araújo
1,
Henry Daniel Ruiz Alba
1,
Cláudia Loianny Souza Lima
1,
Douglas dos Santos Pina
1,
Edson Mauro Santos
3,
Rodrigo Brito Saldanha
4,
Stefanie Alvarenga Santos
1 and
Gleidson Giordano Pinto de Carvalho
1,*
1
Department of Animal Science, Universidade Federal da Bahia, Av. Milton Santos, 500, Salvador 40170110, Brazil
2
Department of Animal Science, Universidade Estadual do Sudoeste da Bahia, Itapetinga 45700000, Brazil
3
Department of Animal Science, Universidade Federal da Paraíba, Areia 58397000, Brazil
4
Instituto Federal de Educação, Ciência e Tecnologia Baiano, Campus Alagoinhas, Alagoinhas 48007656, Brazil
*
Author to whom correspondence should be addressed.
Sustainability 2025, 17(10), 4595; https://doi.org/10.3390/su17104595 (registering DOI)
Submission received: 11 March 2025 / Revised: 3 May 2025 / Accepted: 14 May 2025 / Published: 17 May 2025
(This article belongs to the Special Issue Sustainable Agriculture with Innovative Technology and Equipment: Towards a Low-Carbon Era)
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Versions Notes

Abstract

:
Climate change and socio-economic challenges require greater production efficiency in the agricultural sector. Using microbial additives and biodiesel byproducts in silage production improves quality, reduces losses, and adds value to agro-industrial byproducts, thereby reducing environmental impacts. This study aimed to evaluate the potential of including cottonseed cake (CSC) and microbial inoculant isolated from forage cactus on the fermentation profile and quality of forage sorghum silage. The experimental design used was a completely randomized design, with four treatments: Control: sorghum (SS); sorghum + 10% CSC (% natural matter) (SSCSC); sorghum + Weissella cibaria (SSWC); and sorghum + 10% CSC + W. cibaria (SSCSCWC). There were increases of 15.1% in lactic acid bacteria, 11.4% in dry matter, and 62.9% in crude protein for SSCSC than SS (p = 0.001). There was a decrease of 96.4% in effluent losses (p = 0.002) and 21.6% in acid detergent fiber content (p = 0.005) in SSCSCWC compared to SS. Including 10% CSC and Weissella cibaria in sorghum silage was effective in improving nutritional composition with increased protein content and reduced fermentation losses. The cottonseed cake inclusion promotes greater efficiency in sorghum silage production, which can result in higher profitability and sustainability.

1. Introduction

Globally, efficient animal production is crucial in reducing environmental and economic challenges while enhancing sustainability [1,2]. In extensive systems, ruminant diets rely heavily—about 85 to 90%—on forage resources [3]. Nevertheless, forage availability and quality often decline due to seasonal variations and unfavorable weather conditions in these areas [4].
Pereira et al. [5] reported that forage production can decrease by as much as 63% during the dry season compared to the rainy season. Therefore, implementing sustainable conservation strategies is essential to maintain feed availability during droughts [6] or when conventional feed costs rise.
In this context, silage serves as a forage preservation method that relies on anaerobic fermentation to retain the nutritional value of the feed, making it a viable option during periods of feed scarcity [7]. For optimal fermentation, the forage should have a moisture content between 28% and 35% of its natural matter (NM), and the resulting silage should maintain a pH between 3.8 and 4.2 [8].
Sorghum (Sorghum bicolor (L.) Moench.) is a key forage crop in tropical and subtropical areas, valued for its nutritional quality and resilience in challenging environmental conditions, making it crucial for food security [9]. It is commonly used for silage due to its high forage productivity—ranging from 10 to 30 tons per hectare [10]—along with its broad adaptability, good palatability, and seasonal growth, which help maintain a steady feed supply. Additionally, sorghum is highly water-efficient, positioning it as a strong alternative to corn for ruminant feed, especially in regions with limited or irregular rainfall [11].
Despite its advantages, sorghum silage is vulnerable to aerobic deterioration due to its dry matter (DM) and soluble carbohydrate contents, typically around 25–30% and 30%, respectively [12,13]. This composition makes it more prone to alcoholic fermentation by acid-tolerant yeasts, which compete with lactic acid bacteria for nutrients. As a result, the silage becomes more susceptible to spoilage and increased effluent losses [14]. Enhancing the quality and yield of sorghum silage while lowering production costs is, therefore, essential in modern agriculture and supports sustainable development goals [15]. To address the limitations of monoculture sorghum silage, it is recommended to ensile it in combination with other feed sources [16].
In light of this, incorporating byproducts from biodiesel production offers a promising alternative to enhance livestock systems’ profitability and sustainability. This approach also addresses environmental concerns by promoting the proper use of biodiesel byproducts, reducing the risk of their inappropriate disposal. One such byproduct, cottonseed cake (CSC)—a residue from cottonseed oil extraction—has been studied extensively as a substitute for soybean meal in ruminant diets. With a crude protein (CP) content ranging from 23.0% to 35.7% DM, CSC presents a viable alternative protein source [17,18,19].
Cottonseed cake has also been studied as a promising additive in the ensiling of various forages, including millet [20], sorghum [14], and cactus pear [21]. Its inclusion has been shown to reduce fermentative losses and enhance the chemical composition of these silages. In addition to feed-based additives, recent studies have explored the use of novel microbial strains to stimulate the activity of lactic acid bacteria (LAB), to improve the overall fermentation process. This strategy helps minimize nutrient losses and enhances the efficiency and sustainability of silage production systems [17,18,19].
Among these microbial inoculants, strains from the genus Weissella, such as W. cibaria—an obligate heterofermentative LAB isolated from exotic cactus—have been evaluated for their potential to improve the aerobic stability of silages. These bacteria contribute to silage preservation by producing organic acids with potent antifungal properties, including acetic and propionic acids [22]. The primary pathway of these bacteria for organic acid production leads predominantly to the formation of lactic acid [23].
Accordingly, W. cibaria emerges as a promising microbial additive to enhance the fermentation process, resulting in improved DM recovery and better nutritional quality of silage. These improvements contribute to more sustainable forage conservation practices and help minimize environmental impacts.
Based on this, it was hypothesized that including CSC, with or without a microbial inoculant, could be an effective additive to reduce fermentative losses in sorghum silage by increasing its DM content. This approach is expected to improve both the silage’s nutritional value and aerobic stability compared to untreated silages. Therefore, this study aimed to evaluate the fermentation characteristics and overall quality of sorghum silage with the addition of cottonseed cake and the microbial inoculant (W. cibaria).

2. Materials and Methods

2.1. Experimental Site and Plant Material

The experiment was conducted at the Experimental Farm of São Gonçalo dos Campos, affiliated with the School of Veterinary Medicine and Animal Science at the Universidade Federal da Bahia. The farm is located along BR-101, kilometer 174, in the District of Mercês, Municipality of São Gonçalo dos Campos, Bahia (coordinates: 12°23′57.51″ S, 38°52′44.66″ W, at an altitude of 234 m).
According to the Köppen climate classification, the region has a tropical climate, with average monthly temperatures above 18 °C, a dry season during the period of greatest solar incidence, and longer daylight hours.
Forage sorghum was planted following soil correction to meet the crop’s nutritional requirements. Harvesting occurred when the grains reached the dough stage (approximately 33% DM). The sorghum was then chopped into approximately 3.0–5.0 cm2 particles using a stationary forage chopper (MC1001N, Laboremus, Campina Grande, Brazil).

2.2. Treatments, Experimental Design, and Sorghum Silage Production

The experiment was set up as a completely randomized design, consisting of four treatments and four replications in each treatment, totaling 16 experimental silos. The following four treatments were evaluated: Control: sorghum (SS); sorghum + 10% CSC (% natural matter) (SSCSC); sorghum + Weissella cibaria (SSWC); and sorghum + 10% CSC + W. cibaria (SSCSCWC).
The microbial inoculant used in the current study was W. cibaria isolated from the spineless cactus (Nopalea cochenillifera Salm-Dyck cv. Miúda) according to the methodology proposed by Leite et al. [22] and Pereira et al. [24]. The microorganism was incubated and maintained in MRS Agar (Man, Rogosa, and Sharpe agar) (KASVI, São José dos Pinhais, Brazil) at −20 °C and the activation process follows the method described in Santana et al. [20]. Thus, the inoculant was prepared according to procedures described by Ávila et al. [25]. The CSC used in the current study was purchased from COMFREITAS LTDA (Feira de Santana, Brazil).
Sorghum and CSC were individually weighed using a digital scale. For treatments involving the addition of CSC, the materials were manually mixed in 10 L containers at a ratio of 90% sorghum to 10% CSC on a NM basis, until a homogeneous mixture was achieved. In the W. cibaria treatments, 1.9 kg of the homogeneously mixed material was evenly distributed into vats, forming a layer approximately 2 cm thick. The inoculant was subsequently applied via spraying; specifically, 1.9 μL of the inoculant, previously diluted in 10 mL of distilled water to a concentration of −6 log CFU g−1, was used to ensure uniform distribution. The material was then immediately remixed to achieve a homogeneous preparation for ensiling. In the SS treatment, distilled water was applied at the same volume as the inoculant solution. After this process, the material was transferred to the mini-silos and compacted manually with cement sockets until reaching a specific density of approximately 600 kg m−3 NM in each experimental mini-silo, totaling approximately 1.9 kg NM for each mini-silo.
Each mini-silo included 1.5 kg of fine sand (previously dried in a forced-air oven for 48 h) at its bottom, separated from the chopped sorghum by a thin layer of non-woven fabric, preventing contamination of the ensiled material.
The ensiling process was performed using experimental polyvinyl chloride (PVC) mini-silos (15 cm diameter × 52 cm length) equipped with Bunsen’s valve, which allowed the gases produced in the fermentation process to escape.
All mini-silos were weighed prior to filling, immediately after sealing, and again after 60 days of the ensiling process in order to quantify effluent production and dry matter (DM) recovery. The mini-silos were sealed with PVC lids, with adhesive tape, weighed, and stored in a covered environment at room temperature, avoiding exposure to light until they were opened.

2.3. Fermentation Losses and Aerobic Stability Assessment

The experimental mini-silos were weighed on the day of ensiling and after the ensiling period to determine the material losses in the form of gases and effluents and to estimate DM recovery according to the equations of Jobim et al. [26].
The aerobic stability assessment was performed by returning 0.5 kg of the representative sample silage to its respective mini-silo and submitting them to air exposure for 120 h (five consecutive days). Silage temperatures were measured only one time, in each mini-silo every 2 h, with the aid of a digital immersion thermometer (skewer type) (KASVI, São José dos Pinhais, Brazil) positioned into the silage mass at the geometric center. Additionally, an infrared thermometer controlled the room temperature to 24.2 °C, which was also evaluated every 2 h.
Aerobic stability was as calculated as the time taken in hours (up to 120 h), during which the silage remained stable before reaching 2 °C above the room temperature [27]. The difference between the maximum and the minimum temperature calculated thermal amplitude. Silage weights were recorded during 120 h after aerobic exposure to calculate DM recovery and, consequently, forage losses after aerobic exposure.

2.4. Fermentation Profile

Silage samples were collected to assess pH values, buffering capacity, ammonia nitrogen (NH3-N) levels, and organic acid concentrations. Buffering capacity was measured using a benchtop pH meter (KASVI, São José dos Pinhais, Brazil) following the methodology described by Playne and McDonald [28]. Each variable was analyzed in duplicate. Ammonia nitrogen (NH3-N) levels were determined by distillation with 2N potassium hydroxide, as outlined by Fenner [29].
Organic acid concentrations (lactic acid, acetic acid, propionic acid, and butyric acid) were analyzed using high-performance liquid chromatography (HPLC, model SPD-M20A-UV, SHIMADZU, São Paulo, Brazil) system coupled with an ultraviolet detector. The analysis followed the methodology adapted from Canale et al. [30]. The HPLC system used a MINEX HP87-H (BIORAD, Hercules, CA, USA) column (30 cm × 4.5 mm), with a flow rate of 0.6 mL min−1, column pressure of 6.0 MPa, and a temperature of 39 °C for 30 min. The detection was carried out at wavelengths between 203 and 209 nm, with an injection volume of 20 μL.
Although all organic acids were analyzed, the concentrations of propionic and butyric acids were below the detectable limits in all silage samples. Therefore, the results for these acids are not presented in the respective table.

2.5. Microbial Population Counts

The populations of LAB, molds, and yeasts were quantified in forage sorghum before ensiling the material, as in the silages using selective culture media for each microbial group, according to the recommendations of González and Rodríguez [31]. Ten grams of each treatment was weighed and added to 90 mL of distilled water, with manual shaking followed by serial dilutions (10−2 to 10−6).
All microorganisms were cultured in sterile Petri dishes following the pour-plate plating technique. For cultivation of the lactic acid bacteria (LAB) population, the MRS agar (KASVI, São José dos Pinhais, Brazil) with the addition of 0.1% acetic acid was used. The potato dextrose agar (KASVI, São José dos Pinhais, Brazil), supplemented with 1% tartaric acid at 10% (weight volume−1), was used for molds and yeasts cultivation. Accordingly, each experimental replicate was plated in duplicate for each culture medium.
The Petri dishes were incubated aerobically in a Biochemical Oxygen Demand incubator with the temperature and period determined for each group of microorganisms as follows: LAB, at 37 °C for 48 h; and molds and yeasts, 37 °C for 72 h. After the respective incubation period, the Petri dishes with colony-forming units (CFU) ranging from 30 to 300 were counted. The results were converted to a logarithmic scale (log10 CFU) for data evaluation and interpretation. Molds and yeasts were also counted, differentiating their colonies by morphological characteristics.

2.6. Chemical Composition

Samples of the mixtures were collected before sorghum ensiling (Table 1) and, at the opening, in each experimental mini-silo and kept at −20 °C. Before analyzing chemical composition, all samples were thawed at room temperature and homogenized. Then, they were pre-dried in a forced ventilation oven (55 °C—72 h) and ground in a Willey-type knife mill with a 1.0 mm sieve.
The dry matter (method 934.01), ash (method 930.05), ether extract (EE; method 920.39), CP (method 961.10), and lignin (Method 973.18) concentrations were analyzed in duplicate following the methodologies of the AOAC [32]. The difference between DM and ash content calculated organic matter (OM).
The methodology outlined by Van Soest et al. [33], with adaptations proposed by Mertens [34], was used to determine the contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF), with the addition of thermostable alpha-amylase. Similarly to the other chemical composition analysis, the NDF and ADF contents were assayed in duplicate. The NDF content was corrected for ash and protein to obtain NDFap. Hemicellulose and cellulose were calculated as the difference between NDFap and ADF and ADF and lignin, respectively.
Non-fibrous carbohydrates (NFC) were calculated from the equation proposed by Weiss [35], where NFC = 100 − (%CP + %EE + %Ash + %NDF); and total carbohydrates (TC) were calculated according to Sniffen et al. [36]: TC = 100 − (%CP + %EE + %Ash).

2.7. Statistical Analysis

The results obtained were evaluated through analysis of variance (ANOVA) in a completely randomized design. Means were compared by Tukey’s test, with a critical probability level of 0.05 for type-I error using PROC MIXED of SAS 9.4 software (SAS Institute, Cary, NC, USA). Data were analyzed using the following model:
Yij = μ + Ti + ɛij,
where Yij is the observed value of the dependent variable; μ is overall mean; Ti is the fixed effect of the cottonseed cake and/or microbial inoculant (W. cibaria); and ɛij is residual error, assumed to be independently and normally distributed with mean zero and variance NID ~ (0, σ2).

3. Results

3.1. Fermentation Profile and the Microbial Population Counts

The observed results showed that the pH values were significantly higher (p < 0.001) in the SSCSCWC treatment, with an increase of 15.5% compared to the SS treatment. Similarly, buffering capacity was significantly greater (p < 0.001) in silages with CSC and W. cibaria inclusion, showing a 21.9% increase compared to the control silages.
Ammonia nitrogen concentrations were also significantly influenced (p < 0.001) by the inclusion of CSC. Silages with CSC, including the SSCSC and SSCSCWC treatments, had higher NH3-N concentrations (5.32% and 7.41% of total nitrogen, respectively); while SS and SSWC treatments had lower NH3-N levels (2.03% and 2.62% of total nitrogen, respectively).
Acetic acid levels were significantly affected (p < 0.001), with the lowest concentration observed in the SSWC treatment (0.83 mg mL−1) and the highest in the SSCSC and SSCSCWC treatments (1.47 and 1.52 mg mL−1, respectively) (Table 2).
Lactic acid concentrations also showed a significant increase (p < 0.001) in silages without inoculant, with higher levels in SS and SSCSC (2.77 and 2.63 mg mL−1, respectively) and lower levels in SSWC and SSCSCWC (2.08 and 1.81 mg mL−1, respectively).
Microbial counts of molds and yeasts were not significantly affected by the inclusion of CSC or W. cibaria (p > 0.05) (Table 2).
The concentrations of lactic acid and acetic acid were significantly influenced (p = 0.001 and p < 0.001, respectively). Lactic acid concentrations were higher in SS and SSCSC treatments (1.47 and 1.52 mg mL−1), while acetic acid levels were higher in the SSCSC and SSCSCWC treatments (2.77 and 2.63 mg mL−1, respectively).
Including CSC and/or W. cibaria significantly affected LAB populations (p = 0.001). Silages with W. cibaria had a higher number of CFU, with a 17.3% increase in SSCSCWC compared to SSCSC (Figure 1).
Mold and yeast populations were not sufficiently high for quantification, with counts less than 1 × 10−2 log10 CFU g−1 of silage in all treatments.

3.2. Fermentative Losses and Aerobic Stability

There was no significant effect (p > 0.05) of the inclusion of CSC and/or W. cibaria on gas losses (Table 3). However, including CSC and W. cibaria significantly affected effluent production (p = 0.001) and dry matter recovery (p = 0.001). Specifically, the inclusion of CSC resulted in a 96.4% reduction in effluent losses and an 11.5% increase in DM recovery compared to the control silage.
Minimum temperature, thermal amplitude, forage losses, and aerobic stability were not significantly influenced by the inclusion of CSC or W. cibaria in the silages (p > 0.05) (Table 4). However, significant effects were observed for the average and maximum temperatures (p = 0.035 and p = 0.029, respectively). The highest average and maximum temperatures were recorded in sorghum silage without adding CSC or W. cibaria, compared to those including W. cibaria and CSC.

3.3. Chemical Composition

The contents of hemicellulose, lignin, and NFC were not significantly affected by the inclusion of CSC and/or W. cibaria in the silages (p = 0.130, p = 0.241, and p = 0.635, respectively) (Table 5).
In contrast, the inclusion of CSC significantly increased the DM, ash, CP, and EE contents (Table 5). Additionally, the NDFap, cellulose, total carbohydrates, and ADF contents decreased with the inclusion of CSC and/or W. cibaria compared to the control treatment.

4. Discussion

Climate change can affect forage production and nutritional quality [37]. As a result, silage conservation offers a valuable strategy to mitigate the impact of seasonal fluctuations in forage availability, helping to preserve nutrients and allow for long-term storage.
Effluent losses, which lead to nutrient leaching, contribute to nutritional losses in the silage and reduce sustainability in production, primarily due to the environmental risks associated with effluent pollution. Typically, DM losses from effluents range from 10 to 58 kg per ton of NM, while gas losses can vary from 22% to 144% of DM during fermentation [38]. However, these losses can be minimized by using forage mixtures that help balance the moisture content in silage. For instance, Santana et al. [20] demonstrated that adding 10% CSC to pearl millet silage resulted in a 10% reduction in losses compared to silage made from millet alone.
In the current study, the silages with 10% CSC, either alone or combined with W. cibaria, demonstrated approximately 90% lower effluent losses. This effect is attributed to the increased DM content provided by the CSC, which is 90% DM, and its ability to absorb excess moisture from the silage. These findings align with those of Santana et al. [20], who observed similar reductions in effluent losses when ensiling millet with CSC, helping to achieve the ideal DM range of 28% to 32%, as recommended by McDonald et al. [8].
Moreover, the DM content in the silages in this study increased from 30.35% to 33.81%, a result attributed to CSC’s higher DM content than sorghum. This reinforces the efficiency of CSC as an additive. These findings are consistent with Justino et al. [14], who evaluated CSC inclusion in sorghum silage. Although the silage DM content exceeded the range recommended by McDonald [8], it did not surpass 40%, an important threshold for reducing water activity and limiting the growth of undesirable microorganisms [8].
Gas losses are influenced by the specific fermentation pathways that occur within the silo environment [39]. Minimal gas losses typically indicate limited activity of enterobacteria and Clostridium species during the ensiling process [40]. In the current study, neither W. cibaria nor CSC influenced gas losses in sorghum silages, nor did their combination. However, all the silages evaluated exhibited low gas losses, with values under 12% [41].
Additionally, higher DM recoveries were observed in sorghum silages with W. cibaria compared to those with CSC alone or combined with W. cibaria. This result aligns with expectations, as higher DM recovery values are typically observed in silages containing CSC. This finding further supports the idea that CSC helps mitigate effluent losses and reduces the occurrence of secondary fermentation.
The fermentation profile of the studied silages was altered by the inclusion of CSC and the addition of W. cibaria. As expected, an increase in pH was observed in sorghum silage with CSC inclusion, which exceeded the recommended pH range of 3.8 to 4.2 by McDonald et al. [8]. However, according to Vieira et al. [42], pH values up to 4.6 are associated with silages of satisfactory quality when coupled with a source of buffering capacity, such as the high protein content found in CSC.
Lactic acid is considered the most desirable chemical component in silage production, as it plays a crucial role in nutrient preservation during fermentation [8,41]. This is consistent with earlier findings, and as emphasized by Zeng et al. [43], lactic acid, a product of silage fermentation, rapidly lowers the pH of silage, preventing the growth of undesirable microorganisms like fungi and yeasts.
Furthermore, as heterofermentative bacteria, W. cibaria also produces acetic acid. This has proven to be effective in preserving silage by increasing its aerobic stability to minimize aerobic deterioration after opening the silo.
Although there was a decrease in lactic acid content with the addition of W. cibaria in association with CSC, a desirable increase in acetic acid content was observed. According to Pereira et al. [24], W. cibaria promotes lactic acid production and stimulates acetic acid production, likely due to its heterofermentative capacity. Kung Jr. et al. [41] recommend that acetic acid concentrations for corn silage should be between 1 and 3%. In this study, all the evaluated silages exhibited adequate acetic acid concentrations, remaining below 2%, indicating they were aerobically stable [44].
In contrast, the lactic acid levels were below the recommended range of 3 to 6% for silages, as suggested by Kung Jr. et al. [41]. Nevertheless, there was a noteworthy increase in the LAB population, from 4.51 to 5.29 log10 CFU g−1 NM, which indicates suitable fermentation in silages with W. cibaria inclusion.
Additionally, no growth of molds and yeasts was observed, likely due to the inhibition of their metabolism during the silage fermentation process [45]. This result is likely associated with the acetic acid content in the sorghum silages, which contributed to a significant enhancement in aerobic stability. Molds and yeasts are key microorganisms involved in aerobic spoilage, and their growth is linked to the presence of oxygen [41]. The antifungal properties of acetic acid are responsible for inhibiting the development of lactate-utilizing yeasts and fungi, which are known to initiate aerobic deterioration [46]. Therefore, appropriate levels of acetic acid can help minimize silage losses once the silo is opened.
The results confirmed that all silages remained stable in aerobic conditions for up to 120 h, suggesting that no aerobic deterioration of DM occurred during this period. This indicates a delay in the reactions that would typically lead to the heating of the ensiled mass. Justino et al. [14] observed a similar pattern, associating this stability with low populations of molds and yeasts and a low count of colony-forming units.
Yeasts are primarily responsible for aerobic deterioration in silages [47]. These microorganisms can remain dormant inside the silo, but once exposed to air upon opening, they reactivate and begin to cause spoilage. The results from the current study corroborate the high aerobic stability observed in the silages, confirming that the inclusion of CSC and W. cibaria contributed to minimizing the risk of deterioration upon air exposure.
Moreover, molds and yeasts had no growth due to inhibiting their metabolism during the silage fermentation [45]. This result is likely related to the acetic acid content found in the sorghum silages. Thus, this acid contributed to a marked enhancement in the aerobic stability of the silage, as the microorganisms involved are linked to aerobic spoilage processes [41]. This effect is attributed to the antifungal properties of acetic acid, which inhibit the development of lactate-utilizing yeasts and fungi responsible for initiating aerobic deterioration [46]. Therefore, appropriate levels of acetic acid may help minimize silage losses following silo opening.
The results corroborated that all silage remained stable in aerobiosis for up to 120 h, indicating that during this time, there was no aerobic DM deterioration. Thus, it is possible to infer that there was a delay in the reactions that heated the ensiled mass. According to Justino et al. [14], this behavior may be associated with a low population of molds and yeasts with a low count of colony-forming units.
Yeasts are responsible for silage aerobic deterioration [47]. It happens because yeasts can remain dormant inside the silo until it is opened, and contact with the air reactivates these microorganisms. Thus, the results corroborate with the high aerobic stability in the silages.
Furthermore, the evaluated doses, including CSC or W. cibaria, either separately or in combination, did not improve aerobic stability compared to silages without either of the additives. This suggests that sorghum silage maintained satisfactory fermentative quality regardless of the additives, likely due to the minimal growth of fungi and yeasts.
With the inclusion of CSC, the silages showed an increase in buffer capacity from 50.94 to 63.98 mEq NaOH 100 g−1 DM and protein content from 6.5% to 11.41%. This result aligns with expectations, as cottonseed cake is rich in protein and acts as a buffer, thereby slowing the rate of pH reduction during fermentation. These findings are consistent with those of Dos Santos et al. [48], who reported that as crude protein content in sorghum silage increased, so did its buffering capacity.
Ammoniacal nitrogen serves as an indicator of protein degradation (proteolysis) during silage fermentation, which is an undesirable characteristic. High NH3-N levels in silages result in nutrient losses due to the volatilization of one of the most expensive dietary nutrients. For quality silage, NH3-N content should ideally be less than 10% of total nitrogen [8].
Although silages with CSC exhibited increased buffering capacity, protein content, and higher proteolysis compared to sorghum silages without using CSC or W. cibaria, all silages showed low levels of proteolysis. This can be attributed to the rapid acidification typically observed in these silages [41]. Consequently, lower levels of NH3-N contribute to greater sustainability by reducing the loss of this costly nutrient. Despite the increased protein content in the CSC treatments, all silages displayed adequate fermentation, as evidenced by their high aerobic stability (>120 h), the absence of propionic and butyric acids, and low NH3-N levels.
Regarding the chemical composition of the silages, the addition of CSC proved beneficial, as it increased the DM, CP, and EE contents in the silage, making it a valuable source of nutrients. The CSC used in this study had a DM content of 93.2%, CP of 26.1%, and EE of 9.5%. Therefore, the observed increases in DM, CP, and EE levels were expected, as CSC is a byproduct known for its high CP and DM content and its residual EE content resulting from its extraction process.
Moreover, silages supplemented with CSC—both with and without adding W. cibaria—showed satisfactory CP levels that would support a weight gain of 200 g day−1 in woolly sheep. This is in line with the nutritional needs of these animals, which require a diet containing 10 to 15% CP [49].
The neutral detergent fiber and ADF levels are crucial factors influencing silage quality, fermentation, and the DM intake and digestibility of the diet [50]. Reductions in NDF and ADF levels contribute to improved silage digestibility. In the current study, CSC was auspicious, as it helped reduce NDF content, which is negatively correlated with DM intake [51,52].
Including W. cibaria in combination with CSC also led to significant reductions in ADF content, which is advantageous because ADF is negatively correlated with the apparent digestibility of the material and feed intake [33]. This reduction in ADF content was primarily due to a significant decrease in cellulose levels. According to Zhao et al. [53], this decrease can be attributed to acid hydrolysis at low pH, which makes cellulose more susceptible to breakdown. Additionally, the potential fibrolytic effects of W. cibaria likely contributed to this reduction.
Based on these results, sorghum silages with the inclusion of CSC, whether associated with W. cibaria or not, could be effectively used in ruminant diets. This combination enhances ruminant nutrient intake by improving the digestibility of fibers and addressing limitations associated with high energy concentrations and poorly digestible fibers like ADF.
Farm profitability and the sustainability of livestock production are significantly influenced by total dry matter losses and the nutritive value of silage from field harvest through to animal feeding. In this context, it is essential to assess the effects of incorporating additives or microbial inoculants—either individually or in combination—on silage quality. Alongside proper silage management practices, the use of silage additives plays a crucial role in counteracting the negative effects of undesirable microorganisms (e.g., yeasts and molds), which can compromise fermentation, reduce nutritional value, and diminish aerobic stability. These interventions not only enhance silage quality but also contribute to the sustainability of silage production by reducing its environmental footprint.
The findings of the present study, which investigated the effects of W. cibaria and CSC on sorghum silage, are particularly relevant in this regard. The results indicate that the application of these additives—whether alone or in combination—can substantially improve silage quality, thereby offering viable and sustainable solutions for ruminant production in tropical regions. Consequently, this study provides valuable insights that may inform future advancements in animal nutrition and farm management, ultimately supporting the development of more efficient and sustainable agricultural systems.

5. Conclusions

The inclusion of 10% cottonseed cake (% NM) and W. cibaria in forage sorghum silage improved the nutritional composition, increased protein content, and reduced fermentative losses compared to sorghum silages without microbial inoculant and/or cottonseed cake.
The cottonseed cake was particularly efficient in reducing nutrient losses through leaching (effluent losses) and enhancing the nutritional value of the silage, making it a sustainable alternative for sorghum silage production.
However, future studies are needed to assess the impact of these silages—containing cottonseed cake and/or W. cibaria—for a more comprehensive nutritional evaluation when included in feedlot ruminant diets. Such studies would provide insights into their effects on productive performance, health, carcass characteristics, and meat quality, all of which are crucial parameters for evaluating the overall benefits of this feeding strategy.

Author Contributions

Conceptualization, G.G.P.d.C., D.d.S.P., E.M.S. and S.A.S.; Methodology, G.G.P.d.C., R.B.S. and D.d.S.P.; Validation, G.G.P.d.C., D.d.S.P., E.M.S. and S.A.S.; Formal Analysis, G.G.P.d.C., D.d.S.P. and H.D.R.A.; Investigation, R.B.M., C.L.S.L. and G.R.S.O.; Resources, G.G.P.d.C.; Data Curation, G.G.P.d.C. and D.d.S.P.; Writing—Original Draft Preparation, R.B.M., C.L.S.L., R.B.S. and G.R.S.O.; Writing—Review and Editing, G.G.P.d.C., D.d.S.P., E.M.S., S.A.S., H.D.R.A. and M.L.G.M.L.d.A.; Visualization, G.G.P.d.C., D.d.S.P., H.D.R.A. and M.L.G.M.L.d.A.; Supervision, G.G.P.d.C., D.d.S.P., H.D.R.A. and M.L.G.M.L.d.A.; Project Administration, G.G.P.d.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created.

Acknowledgments

The authors thank the Bahia State Research Foundation (FAPESB), the “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq), and the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES-Brazil) for granting the students scholarships.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
ADFAcid detergent fiber
CFUColony-forming units
CPCrude protein
TCTotal carbohydrates
DMDry matter
EEEther extract
LABLactic acid bacteria
NDF apNeutral detergent fiber corrected for ash and protein
NMNatural matter
NH3-NAmmoniacal nitrogen
NFCNon-fibrous carbohydrates
PVCPolyvinyl chloride
SSSorghum silage
SSCSCSorghum silage + 10% cottonseed cake
SSCSCWCSorghum silage +10% cottonseed cake + Weissella cibaria
SSWCSorghum silage + Weissella cibaria

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Sustainability 17 04595 g001
Figure 1. Lactic acid bacteria counts in sorghum silage with or without cottonseed cake and/or microbial inoculant (W. cibaria; log10 CFU g−1 NM). Different lowercase letters indicate differences between lines by Tukey’s test (p < 0.05).
Figure 1. Lactic acid bacteria counts in sorghum silage with or without cottonseed cake and/or microbial inoculant (W. cibaria; log10 CFU g−1 NM). Different lowercase letters indicate differences between lines by Tukey’s test (p < 0.05).
Sustainability 17 04595 g001
Table 1. Chemical composition, pH, and lactic acid bacteria of mixtures pre-ensiling.
Table 1. Chemical composition, pH, and lactic acid bacteria of mixtures pre-ensiling.
Item, % DM Additive
(CSC) 		Treatments
SS SSCSC SSWC SSCSCWC
Dry matter, % natural matter (NM)89.332.940.233.741.6
Ash5.43.54.13.74.3
Organic matter94.696.595.996.395.7
Crude protein28.05.711.35.211.4
Ether extract9.51.93.92.33.7
Neutral detergent fiber ap55.137.535.241.734
Acid detergent fiber26.519.717.522.816.6
Cellulose18.516.414.419.113.3
Hemicellulose28.617.817.718.817.4
Lignin7.993.33.13.73.3
Non-fibrous carbohydrates2.051.545.547.246.5
Total carbohydrates57.188.880.488.680.2
pH-5.45.54.85.5
Lactic acid bacteria, log10 CFU g−1 NM-2.1302.622.25
CSC: cottonseed cake; SS: sorghum silage; SSCSC: sorghum silage + 10% cottonseed cake; SSWC: sorghum silage + W. cibaria; SSCSCWC: sorghum silage + 10% cottonseed cake + W. cibaria.
Table 2. Fermentative profile and microbial counts of sorghum silage with or without cottonseed cake and/or microbial inoculant.
Table 2. Fermentative profile and microbial counts of sorghum silage with or without cottonseed cake and/or microbial inoculant.
Item, % DMTreatments 1SEMp-Value
SSSSCSCSSWCSSCSCWC
pH4.18 b4.51 ab4.19 b4.83 a0.08<0.001
Ammoniacal nitrogen, % TN2.03 c5.32 b2.62 c7.41 a0.58<0.001
Buffer capacity, mEq NaOH 100 g−1 DM52.47 b61.85 a50.74 b63.98 a1.60<0.001
Organic acids
Acetic, % DM1.18 b1.27 a0.83 c1.52 a0.07<0.001
Lactic, % DM2.77 a2.63 a2.08 b1.81 b0.11<0.001
Molds and yeasts, log10 CFU g−1 NM<1 × 10−2<1 × 10−2<1 × 10−2<1 × 10−2--
1 Treatments: SS: sorghum silage; SSCSC: sorghum silage + 10% cottonseed cake; SSWC: sorghum silage + W. cibaria; SSCSCWC: sorghum silage + 10% cottonseed cake + W. cibaria. Different lowercase letters indicate differences between lines by Tukey’s test (p < 0.05).
Table 3. Fermentative losses of sorghum silage with or without cottonseed cake and/or microbial inoculant.
Table 3. Fermentative losses of sorghum silage with or without cottonseed cake and/or microbial inoculant.
Item, % DMTreatments 1SEMp-Value
SSSSCSCSSWCSSCSCWC
Gas Losses, % DM0.070.050.050.080.020.350
Effluent losses, kg ton−1 NM30.75 a4.44 b22.92 a1.12 b3.340.001
Dry matter recovery, %89.67 a79.37 b84.90 a78.49 b1.310.001
1 Treatments: SS: sorghum silage; SSCSC: sorghum silage + 10% cottonseed cake; SSWC: sorghum silage + W. cibaria; SSCSCWC: sorghum silage + 10% cottonseed cake + W. cibaria. Different lowercase letters indicate differences between lines by Tukey’s test (p < 0.05).
Table 4. Aerobic stability of sorghum silage with or without additive with cottonseed cake and/or microbial inoculant.
Table 4. Aerobic stability of sorghum silage with or without additive with cottonseed cake and/or microbial inoculant.
Item, % DMTreatments 1SEMp-Value
SSSSCSCSSWCSSCSCWC
Average temperature, °C24.60 a24.35 ab24.33 b24.4 ab0.050.035
Minimum temperature, °C23.0023.0523.1023.230.070.717
Maximum temperature, °C27.25 a26.75 b26.8 ab26.93 ab0.070.029
Thermal amplitude, °C4.253.703.703.700.090.061
Forage losses, % NM5.574.665.325.520.200.380
Aerobic stability, hours>120>120>120>120--
1 Treatments: SS: sorghum silage; SSCSC: sorghum silage + 10% cottonseed cake; SSWC: sorghum silage + W. cibaria; SSCSCWC: sorghum silage + 10% cottonseed cake + W. cibaria. Different lowercase letters indicate differences between lines by Tukey’s test (p < 0.05).
Table 5. Chemical composition of sorghum silage with or without cottonseed cake and/or microbial inoculant.
Table 5. Chemical composition of sorghum silage with or without cottonseed cake and/or microbial inoculant.
Item, % DMTreatments 1SEMp-Value
SSSSCSCSSWCSSCSCWC
Dry matter30.35 b33.81 a29.79 b33.59 a0.49<0.001
Mineral matter3.50 c4.06 ab3.70 bc4.31 a0.090.002
Organic matter96.50 a95.94 bc96.30 ab95.69 c0.090.002
Crude protein6.83 b11.13 a6.50 b11.41 a0.60<0.001
Ether extract2.55 b4.55 a2.93 b4.31 a0.22<0.001
NDFap 244.79 ab40.38 b47.23 a39.58 b0.990.003
Acid detergent fiber27.46 ab24.11 bc27.71 a21.61 c0.740.005
Cellulose22.94 a19.25 b23.12 a17.73 b0.710.001
Hemicellulose17.3316.2619.5217.980.500.130
Lignin4.524.864.593.880.170.241
Non-fibrous carbohydrates36.8534.4234.8535.70.670.635
Total carbohydrates87.12 a80.25 b86.88 a79.97 b0.89<0.001
1 Treatments: SS: sorghum silage; SSCSC: sorghum silage + 10% cottonseed cake; SSWC: sorghum silage + W. cibaria; SSCSCWC: sorghum silage + 10% cottonseed cake + W. cibaria. Different lowercase letters indicate differences between lines by Tukey’s test (p < 0.05); 2 NDFap: Neutral detergent fiber corrected for ash and protein.
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Mendes, R.B.; Oliveira, G.R.S.; Araújo, M.L.G.M.L.d.; Alba, H.D.R.; Lima, C.L.S.; Pina, D.d.S.; Santos, E.M.; Saldanha, R.B.; Santos, S.A.; Carvalho, G.G.P.d. Microbial Additive Isolated from Exotic Semi-Arid Cactus and Cottonseed Byproduct in Sustainable Sorghum Silage Production. Sustainability 2025, 17, 4595. https://doi.org/10.3390/su17104595

AMA Style

Mendes RB, Oliveira GRS, Araújo MLGMLd, Alba HDR, Lima CLS, Pina DdS, Santos EM, Saldanha RB, Santos SA, Carvalho GGPd. Microbial Additive Isolated from Exotic Semi-Arid Cactus and Cottonseed Byproduct in Sustainable Sorghum Silage Production. Sustainability. 2025; 17(10):4595. https://doi.org/10.3390/su17104595

Chicago/Turabian Style

Mendes, Raiane Barbosa, Gabriel Rodrigues Silva Oliveira, Maria Leonor Garcia Melo Lopes de Araújo, Henry Daniel Ruiz Alba, Cláudia Loianny Souza Lima, Douglas dos Santos Pina, Edson Mauro Santos, Rodrigo Brito Saldanha, Stefanie Alvarenga Santos, and Gleidson Giordano Pinto de Carvalho. 2025. "Microbial Additive Isolated from Exotic Semi-Arid Cactus and Cottonseed Byproduct in Sustainable Sorghum Silage Production" Sustainability 17, no. 10: 4595. https://doi.org/10.3390/su17104595

APA Style

Mendes, R. B., Oliveira, G. R. S., Araújo, M. L. G. M. L. d., Alba, H. D. R., Lima, C. L. S., Pina, D. d. S., Santos, E. M., Saldanha, R. B., Santos, S. A., & Carvalho, G. G. P. d. (2025). Microbial Additive Isolated from Exotic Semi-Arid Cactus and Cottonseed Byproduct in Sustainable Sorghum Silage Production. Sustainability, 17(10), 4595. https://doi.org/10.3390/su17104595

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