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Article

Ensiling Cyanide Residue and In Vitro Rumen Fermentation of Cassava Root Silage Treated with Cyanide-Utilizing Bacteria and Cellulase

by
Waroon Khota
1,2,
Chatchai Kaewpila
1,
Rattikan Suwannasing
1,
Nikom Srikacha
1,
Julasinee Maensathit
1,
Kessara Ampaporn
1,
Pairote Patarapreecha
1,
Suwit Thip-uten
3,
Pakpoom Sawnongbue
3,
Sayan Subepang
4,
Kriwit Khanbu
5 and
Anusorn Cherdthong
2,*
1
Faculty of Natural Resources, Rajamangala University of Technology Isan, Sakon Nakhon 47160, Thailand
2
Tropical Feed Resources Research and Development Center (TROFREC), Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen 40002, Thailand
3
Faculty of Agricultural Technology, Sakon Nakhon Rajabhat University, Sakon Nakhon 47000, Thailand
4
Faculty of Liberal Arts and Sciences, Sisaket Rajabhat University, Sisaket 33000, Thailand
5
Lanpanyafarm Co., Ltd., Amnat Charoen 37110, Thailand
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(2), 151; https://doi.org/10.3390/fermentation9020151
Submission received: 16 December 2022 / Revised: 27 January 2023 / Accepted: 31 January 2023 / Published: 3 February 2023
(This article belongs to the Special Issue Recent Advances in Rumen Fermentation Efficiency)
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Abstract

:
Cyanide is a strong toxin in many tropical forage plants that can negatively affect ruminants. The aim of this study is to determine the cyanide removal efficiency, silage quality, and in vitro rumen fermentation of fresh cassava roots ensiled without an additive (control) and with Acremonium cellulase (AC), two cyanide-utilizing bacterial inoculants (Enterococcus feacium KKU-BF7 (BF7) and E. gallinarum KKU-BC10 (BC10)), and their combinations (BF7 + BC10, AC + BF7, AC + BC10 and AC + BF7 + BC10). A completely randomized design was used with eight treatments × four small-scale silo replicates. Additionally, extra silage samples (seven silos/treatment for individually opening after 0, 1, 3, 5, 7, 15, and 30 days of ensiling) were added to observe the changes in the total cyanide concentration and pH value. The fresh cassava root contained an optimal number of lactic acid bacteria (105 colony forming units/g fresh matter), and the contents of dry matter (DM) and total cyanides were 30.1% and 1304 mg/kg DM, respectively. After 30 days of ensiling, all silages demonstrated a low pH (<3.95; p < 0.01). Cyanide content ranged from 638 to 790 mg/kg DM and was highest in the control (p < 0.01). The addition of BF7 + BC10 increased the crude protein (CP) content (p < 0.01). The addition of AC decreased the fibrous contents (p < 0.01). The control had less acetic acid and propionic acid contents (p < 0.01) and a greater butyric acid content (p < 0.01). However, the degrees of in vitro DM digestibility (IVDMD) and gas production were similar among treatments. Methane production ranged between 29.2 and 33.3 L/kg IVDMD (p < 0.05), which were observed in the AC + BC10 and BF7 + BC10 treatments, respectively. Overall, our results suggested that the cyanide removal efficiency after 30 days of ensiling with good-quality cassava-root silage was approximately 39% of the initial value. The enterococci inoculants and/or AC could improve the ensiling process and cyanide removal efficiency (increasing it to between 47 and 51% of the initial value). The novel enterococci inoculants (BF7 + BC10) were associated with a decreased cyanide content and an increased CP content. They appeared to promote the methanogenesis potential of the cassava root silage. More research is required to validate the use of cyanide-utilizing bacterial inoculants in cyanogenetic plants, bioenergy fermentation, and livestock.

1. Introduction

Cassava (Manicot esculenta Crantz) is an annual root crop that is widely grown in tropic and sub-tropic regions. Cassava roots can be grown in soil with poor fertility, including low levels of available phosphorus (P), a low exchangeable base content, and a high aluminum content [1,2]. Cassava roots in the form of cassava chips are widely used as a carbohydrate source in animal feed. Cassava chips are produced from the chopped, dried roots, which are sun-dried until the moisture content is reduced to less than 14% [3]. During the rainy season, it is difficult to sun-dry cassava to produce cassava chips, so the use of fresh cassava roots could be a key alternative method. However, it is well known that fresh cassava roots are rich in cyanide content.
The natural presence of two cyanogenic glycosides, linamarine and lotaustraline, is an important factor that restricts the use of cassava as feed or food. When cyanogenic plants are damaged, they defend themselves by biologically releasing HCN gas from cyanogenic glycosides. All available cyanides are highly toxic to the cellular metabolism of humans and animals [4,5]. They combine with blood hemoglobin and restrict the activity of respiratory enzymes, eventually causing death [6]. Gensa [7] suggested that the lethal dosage of cyanogenic glycoside inhalation in ruminant species is approximately 4 mg of HCN equivalent/kg of body weight.
Silage is an efficient option to preserve the nutrient value and improve the shelf life of plant materials [8]. An ideal ensiling process is associated with employing an epiphytic lactic acid bacteria (LAB)-converted water-soluble carbohydrate (WSC) to lactic acids, resulting in a low pH and inhibiting the growth of harmful microorganisms [9]. Silage additives are widely known to improve ensiling characteristics [10,11,12,13,14,15,16]. There is an increasing interest in applying cellulase enzymes and LAB inoculants in ensiling because they can promote acidic conditions in the ensiling process [10,11]. Fibrolytic enzymes work by hydrolyzing structural carbohydrates to increase the WSC content for LAB utilization and improve silage digestibility [10,11,17].
In general, the ensiling process also significantly decreases the concentration of total cyanides [18,19]. Gomez and Valdivieso [20] reported that ensiling cassava root for 26 weeks reduced the cyanide content to 36% of the initial value. The reduction of residual cyanides or cyanide removal efficiency in silage might be associated with the acidic conditions that expel cyanogenic glycosides (HCN emission) and ensiling microbial activity that can use cyanogenic glycosides as C and/or N sources [21].
Presently, there is limited information available on the use of silage additives to efficiently prepare the silage of cyanogenic plants. In this study, we hypothesized that cellulase might help to reduce cyanides in silage through plant cell-wall hydrolysis and HCN release. Furthermore, we also hypothesized that cyanide-utilizing bacteria isolated from rumen fluids might use cyanogenic glycosides in the silo. Thus, the objective was to determine the effects of Acremonium cellulase (AC) and two strains of novel, cyanide-utilizing bacteria on the modification of the total cyanide concentration, ensiling characteristics, and in vitro rumen fermentation of cassava root silage. The bacteria examined were Enterococcus feacium KKU-BF7 (BF7) and E. gallinarum KKU-BC10 (BC10), which were recently isolated from bovine rumen.

2. Materials and Methods

2.1. Experimental Design, Ensiling Materials, and Silage Preparation

The experimental design was a completely randomized design (CRD). Cassava roots were treated with eight treatments: without additive (control), AC, BF7, BC10, BF7 + BC10, AC + BF7, AC + BC10, and AC + BF7 + BC10. The replicates were four silos per treatment.
Fresh cassava roots were collected on 4 June 2022, from farmers around Khon Kaen University, Khon Kaen, Thailand (16.4322° N, 102.8236° E), with a harvested age of 8 months. After harvesting, the sufficiently fresh cassava roots were immediately washed with water and left overnight. They were cut into pieces with an approximate length of 10 mm and sampled to mix them well with assigned additives. Each additive was supplemented into the ensiling material by spraying.
AC produced from Acremonium cellulolyticus (glucanase and pectinase 7350 U/g, Meiji Seika Pharma Co. Ltd., Tokyo, Japan [22]) was added at a level of 0.01% of fresh matter (FM) and diluted with distilled water (0.1 g per 1 mL). The novel, cyanide-utilizing bacteria BF7 and BC10 were previously isolated from the rumen fluids of swamp buffalo and beef cattle, respectively. These two enterococci strains were selected according to their rhodanese activities (unpublished data). The data of 16S rRNA gene sequences are uploaded to NCBI; the access numbers are MZ959830 and MZ959828 for strain BF7 and BC10, respectively. The strain was incubated in Lactobacilli de Man, Rogosa, Sharpe (MRS) broth (Difco Laboratories, Detroit, MI, USA) at 39 °C for 24 h. After incubation, the optical density at 600 nm of the suspension was adjusted to 1.0 (1 OD600) with 8.5 g/L of sterilized NaCl, using a spectrophotometer (UV/VIS Spectrometer, PG Instruments Ltd., London, UK). At 1 OD600, the bacterial cell density was approximately 108 colony forming units (cfu)/mL, and we added 1 mL/kg of FM chopped cassava roots.
Each experimental sample (200 g FM) was packed into a silo (laminated nylon and polyethylene, 200 × 300 mm, Hiryu KN, Asahikasei, Tokyo, Japan) and sealed using a vacuum sealer (SQ–303, Asahi Kasei Pax Corp., Tokyo, Japan). All silos were ensiled at ambient temperature (21 to 28 °C). After 30 days of ensiling, the silos were opened for the evaluation of ensiling loss, chemical composition, fermentation quality, microbial population, and in vitro rumen digestibility. In addition, seven extra silage samples/treatments were prepared for the estimation of the total cyanide concentration (Figure 1) and the pH value (Figure 2) after 0, 1, 3, 5, 7, 15, and 30 days of ensiling (one silo/treatment/day).

2.2. Total Cyanide Measurement and Ensiling Trial

The concentrations of total cyanides in the fresh cassava root and silage samples were calorimetrically assayed using a UV/VIS spectrometer according to the method of Lambert et al. [23]. Briefly, 1 g of fresh cassava root and silage samples were homogenized with 9 mL of distilled water and left for cell lysis at −20 °C for 12 h. The sample was defrosted by shaking at 120 rpm. The extracted sample was then centrifuged at 4200 rpm and 4 °C for 10 min.
Next, 1 mL of the supernatant was added into a 15 mL conical tube (Eppendorf 15 mL, Eppendorf AG, Hamburg, Germany), followed by 0.4 mL of n-chlorosuccinimide-succinimide oxidizing reagent, 0.4 mL of hydantoin-pyridine reagent, and 8.2 mL of distilled water. The test tubes were closed with a screwcap, mixed well using a vortex mixer, and incubated at 25 °C for 1 min. The samples’ absorbance was measured at a wavelength of 403 nm with a standardized KCN. The total cyanide content was reported as mg of HCN equivalent.
To determine the ensiling loss, the final weight of silage samples was recorded and calculated as the % of initial ensiling material. The dry matter (DM) content was analyzed after air drying in an oven (Memmert UF450, Memmert GmbH, Schwabach, Germany) for 24 h at 100 °C. The samples were also dried for 48 h at 60 °C in a hot air oven (Memmert UF450) and ground to pass through a 1 mm sieve size using a grinding mill (IKA MF10, IKA Werke GmbH & Co. KG, Staufen, Germany). The organic matter (OM) and ether extract (EE) contents were analyzed according to methods 942.05 and 920.39 of the AOAC, respectively [24].
The crude protein (CP) content was analyzed using the combustion technique (N-analyzer, 828 Series, LECO, St. Joseph, MI, U.S.A.) with standardized EDTA and a factor of 6.25 for CP conversion. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were determined using a fiber analyzer (ANKOM 200, ANKOM Technology, Macedon, NY, U.S.A.). For NDF analysis, α-amylase (2500 U/mg, Sigma-Aldrich, St Louis, MO, U.S.A.) and Na2SO3 were used to eliminate starch and protein residues, respectively. The acid detergent lignin (ADL) was analyzed by solubilization with a 72% H2SO4 solution.
The ensiling fermentation products were measured from cold water extracts following the method of Cai et al. [13]. Each silage sample (10 g FM) was blended with 90 mL of sterilized distilled water and kept at 4 °C for 12 h. The extracted sample was then warmed to ambient temperature, and the pH was measured using a pH meter (FiveGo, Mettler-Toledo GmbH, Greifensee, Switzerland). The concentrations of lactic acid and volatile fatty acids were measured according to the periodic acid assay of Darwin et al. [25] using a gas chromatography (Nexis GC-2030, Shimadzu Co., Kyoto, Japan). This gas analyzer was equipped with a split-mode injector, capillary column (DB-WAX 30 m, 0.25 mm, 0.25 μm, Agilent Technologies, Inc., Santa Clara, CA, U.S.A.), and flame ionization detector. N2 was used as the carrier gas. The ammonia nitrogen (NH3N) content was measured using spectrophotometry (UV/VIS Spectrometer), following the method of Fawcett and Scott [26].
The microbial populations of the fresh cassava root and silage samples were counted using a spread plate method [22,27]. Briefly, 10 g FM of the sample was shaken well by hand with 90 mL of sterilized distilled water and diluted in a series of 10−1 to 10−5 with 0.85% NaCl solution. From each dilution, 20 µL was spread onto agar plates. Lactic acid bacteria (LAB) were counted after incubation in an anaerobic box (Sugiyamagen Ltd., Tokyo, Japan) at 30 °C for 48 h on MRS agar (Difco). Coliform bacteria and aerobic bacteria were counted after incubation at 30 °C in an incubator (Memmert IN260, Memmert GmbH, Schwabach, Germany) for 48 h on blue light broth agar (Nissui-seiyaku Ltd., Tokyo, Japan) and nutrient agar (Difco), respectively. Yeast and mold were counted on potato dextrose agar (Nissui-seiyaku) after incubation at 30 °C for 2 and 3 days, respectively, by colony appearance and cell morphology observation.

2.3. In Vitro Rumen Test

An in vitro gas production technique [28] was conducted to evaluate in vitro DM digestibility (IVDMD), gas production, and methane production after 24 h of incubation. The substrates of cassava root silage samples were prepared as total mixed rations (TMR). The ingredients of TMR on a dry basis were rice straw (30%), silage sample (50%), soybean meal (14%), rice bran (4.3%), urea (0.7%), vitamin premix (0.5%), and minerals (0.5%). However, the fresh cassava root sample was used alone. The substrates were weighed into 50 mL serum bottles (three bottles/replicate). Bottles were closed with a rubber stopper and aluminum seal cap and warmed at 39 °C in a water bath.
The buffers were prepared [28] and warmed at 39 °C with CO2 flushing. Two heads of Thai native beef cattle with an average body weight of 250 ± 16 kg were used as rumen fluid donors. The cattle were housed in individual pens and fed concentrated feed consisting of 16% CP and 11.0 MJ metabolizable energy/kg DM at 0.5% body weight. Rice straw was given daily ad libitum, and the animals had free access to fresh water. The feed was fed in two equal meals at 08:00 and 16:00 h.
Rumen fluid was collected using a stomach tube sucker before morning feeding. The first 200 mL of collected rumen fluid was discarded to avoid saliva contamination, according to Muizelaar et al. [29]. The collected rumen fluid was immediately transferred to the laboratory, filtered with four sheet-cloth layers, and mixed with the buffer. The rumen fluid medium (40 mL) was injected into the bottles. Three bottles without substrate were added as blanks. All bottles were incubated in an incubator shaker (Innova 40, Hamburg, Germany) at 39 °C with shaking at 120 rpm.
The gas produced was measured every 2 h using a glass syringe. After measuring, the gas content was transferred into a gas bag to analyze the methane production [15]. After 24 h of incubation, the methane concentration was measured using gas chromatography (Nexis GC-2030 equipped with SH-Rt Q-BOND Column, 30 m, 0.53-mm ID, 20 um, Shimadzu Co., Kyoto, Japan), according to the method described by Kaewpila et al. [30]. The residual samples in bottles were filtered through a glass filter crucible (ROBU, GmbH, Hattert, Germany), washed using a pepsin solution and distilled water, dried at 100 °C in an airdry oven for 24 h, and weighed for IVDMD determination.

2.4. Statistical Analysis

The data were analyzed by analysis of variance (ANOVA) in SAS (V. 6.12, SAS Institute Inc., Cary, NC, USA) with the following models:
Yij = μ + τi + εij
where Yij is the observation, μ is the overall mean, τi is the treatment effect (i = 1 to 8), and εij is the error. Duncan’s test was used to determine the differences among treatment means, and the significance level was considered to be p ≤ 0.05 [31].

3. Results

3.1. Cassava Root Materials

The populations of LAB, coliform bacteria, aerobic bacteria, yeast, and mold from fresh cassava root ranged from 105 to 108 cfu/g FM (Table 1). The pH value and DM content were 5.1 and 38.1%, respectively. The OM content was high (98.3% DM). The other chemical contents ranged from 0.5 to 10.8% DM. The total cyanide content was 1304 mg/kg DM. The IVDMD, gas production, and methane production were 81.7% DM, 194.0 L/kg DM, 16.62 L/kg DM, and 22.25 L/kg IVDMD, respectively.

3.2. Changes of Total Cyanides and pH Values in Cassava Root Silage during Ensiling

The total cyanides and pH values of the cassava root silage treated with additives are shown in Figure 1 and Figure 2, respectively. The total cyanides after day 1 in all treatments decreased slowly and then decreased sharply after day 3. From day 3 to day 30, the cyanide content slowly decreased, and the final value was less than 800 mg/kg DM. The pH value sharply decreased until day 3, and a stable pH seemed to appear among treatments from day 7 onwards.

3.3. Ensiling Loss, Chemical Composition, and Total Cyanides Content of Silage

The ensiling loss, chemical compositions, and residue cyanide of the cassava root silages after 30 days of ensiling are shown in Table 2. The ensiling loss and ADL content were not different between the treatments (p > 0.12). Compared to the control, the silage treated with AC, AC + BF7, AC + BC10, and AC + BF7 + BC10 clearly showed a greater OM content (p < 0.01) and a lower NDF content (p < 0.01). The CP contents ranged from 1.90 to 2.31% DM.
The use of BF7 + BC10 resulted in a higher CP content than other treatments (p < 0.01) except for BF7, which had a medium value. Compared to the control, only BC10 improved the EE content of the cassava root silage. The ADF content of the AC, AC + BC10, and AC + BF7 + BC10 treatments was lower than that of the control (p < 0.05). The total cyanide content in the cassava root silage after 30 days of ensiling ranged from 638 to 790 mg/kg DM. The silage treated with additives had significantly fewer residual cyanides than the control (p < 0.01).

3.4. Silage Fermentation

The DM content, pH value, and fermentation products of the cassava root silage are presented in Table 3. The DM, lactic acid, and NH3-N contents were not different among treatments (p ≥ 0.09). Although the pH value was low (in the range of 3.85 to 3.95), the pH of the AC + BF7 + BC10 treatment was significantly lower than that of the control (p < 0.01). When compared to the control, the silages prepared with additives had significantly greater acetic acid and propionic acid contents (p < 0.01) and a lower butyric acid content (p < 0.01).

3.5. Microbial Population

The counts of the microbial populations of the cassava root silage are shown in Table 4. The LAB numbers were not significantly different among treatments (p = 0.23) and ranged from 5.70 to 6.93 Log10 cfu/g FM. The additions of BF7 + BC10 and AC + BF7 + BC10 resulted in significantly lower aerobic bacteria populations compared with the control (p < 0.05). Coliform bacteria, yeast, and mold populations were below the detectable level (<102 cfu/g FM).

3.6. In Vitro Rumen Fermentation

The in vitro rumen test results after 24 h of incubation of the cassava root silage in TMR (50:50 w/w) are shown in Table 5. The IVDMD (g/kg) and gas productions (L/kg DM and L/kg IVDMD) were not significantly different among treatments (p > 0.05). Although the methane productions (L/kg DM and L/kg IVDMD) of the TMR prepared with the control were similar to those of the other treatments, the degrees in the AC, BC10, and AC + BC10 treatments were less than in BF7 + BC10 (p < 0.05).

4. Discussion

4.1. Cassava Root Materials

In the fermentation process, the microorganism population in ensiled material plays an important role and influences the silage quality [32,33]. A high population of epiphytic LAB can improve the ensiling process by increasing lactic acid production and decreasing the pH value, resulting in the inhibition of harmful bacteria growth [16,34]. A suitable number of LAB in tropical materials for good-quality silage is at least 105 cfu/g FM [14]. In this study, the LAB number of a fresh cassava root was 3.3 × 105 cfu/g FM (Table 1), which might be sufficient for fermentation and the production of lactic acid.
The numbers of coliform bacteria, aerobic bacteria, yeasts, and molds were higher (106 to 108 cfu/g FM) and dominated over the LAB. These strains influence the fermentation quality and the preservation of nutrients in silage [16,22]. Thus, to inhibit the undesirable microflora and preserve nutrients, external factors such as silage additives were required.
The factors affecting the chemical compositions of a plant include the varieties, harvest stage, water availability, and soil fertility [35]. The DM, OM, CP, and EE of cassava root in this study (Table 1) were consistent with those reported by Pitirini et al. [36]. The NDF, ADF, and ADL contents were lower than those reported by Chumpawadee et al. (12.3, 4.9, and 2.5% DM, respectively) [37]. It is well known that fresh cassava root is rich in cyanogenic glycosides. The total cyanide content of fresh cassava root in other studies ranged from 1 to 2000 mg/kg FM [19,38,39]. Therefore, the level of total cyanides in the fresh cassava root sample used in this study was within the typical concentration range (496.43 mg/kg FM) (Table 1). Gensa [7] revealed that the concentration of cyanogenic glycosides can vary extremely with plant varieties, planting managements (frost, drought, and herbicide application), growing stages, and animal feeding methods (grazing, cut and carry, haymaking, and ensiling).

4.2. Effect of Additive on Total Cyanides Removal Efficiency in Silage

Our results demonstrated that 30 days of an ensiling process can partially decrease the total cyanides of cassava root silage, which remained at 61% of the initial level, although the pH was less than 4.0 (Figure 1 and Figure 2, and Table 2). These findings generally suggest that an effective preparation of cassava root silage requires a specific additive for expelling or utilizing cyanides in alternative ways to employ a prolonged fermentation period.
The ensiling disappearance of cyanides in cassava root silage agreed with previous findings [19,20,37]. Iwuoha et al. [19] reported the highest and fastest total cyanide removal efficiency of 94.7% of the initial value at 6 days of fermentation. In contrast, Gomez and Valdivieso [20] found that the ensiling efficiency in un-chopped cassava root silage after 22 weeks of fermentation ranged from 25 to 36% of the initial HCN equivalent level. Ngwa et al. [6] reported that the concentration of cyanides in silage of another cyanogenetic plant, Acacia sieberiana, dropped by 24% after 28 days of ensiling.
In the present study, the use of additives improved the total cyanide removal efficiency of cassava root silage (39 to 51% of initial value remaining; p < 0.05; Table 2). These results are in agreement with those of Kyawt and Lukkananukool [21], who found that cassava tubers ensiled with the fermented juice of epiphytic LAB had significantly greater HCN reduction than the control silage. However, Man and Wiktorsson [40] reported that the addition of molasses at 0, 30, 60, and 90 kg/t FM had no effect on cyanide reduction of cassava top silage.
The reduction of cyanide contents during the ensiling process could be attributable to the leaching of glycosides and free cyanide, which produced the large quantity of gas effluent. Based on the hydrolysis effects of AC, the lower amount of retained cyanide contents should be attributable to the sharply decreased pH within 3 days of ensiling (<4.2; Figure 2). The pH value decreased because the epiphytic LAB transformed WSC in the cassava root material into organic acids during the fermentation process [9,14]. In contrast, the beneficial effects on cyanide of the novel enterococci inoculants could also relate to their rhodanese activities in cyanide detoxification. Therefore, they can degrade the glycosides as a nutrient source [41]. However, when compared with BF7 or BC10 alone, only the addition of BF7 + BC10 clearly increased the CP content, which might result from the use of cyanide.

4.3. Effect on Ensiling Loss and Chemical Composition in Silage

The ensiling loss of silage fermentation in a closed silo system is a biological cost in production; therefore, minimizing this value significantly improves silage production [30]. After day 30 of ensiling, the results showed that the ensiling loss of all treatments was not significantly different (p > 0.05; Table 2). The present study is consistent with another study, which reported that the addition of Enterococcus spp. as a LAB inoculant could not improve the DM losses of forage crop silage [42].
The chemical composition of silage determines the nutritive value for ruminants [16]. The silage additives influence the chemical composition [14,22,43]. In the present study, the addition of AC and its combination with the inoculant improved the OM content and reduced the NDF and ADF contents of cassava root silage (Table 2). These results are in agreement with previous studies [12,16,24].

4.4. Effect on Silage Quality and Microbial Population of Silage

The results show that the cassava root silages were all well-preserved, with low pH values (3.85 to 3.95) and NH3-N contents (Table 3). The silage quality of the control silage was similar to that of treated silage because the physiological properties of epiphytic bacteria that ferment the soluble carbohydrate produce sufficient organic acid, reduce pH, and inhibit the growth of another harmful microorganisms [41]. However, the silage treated with AC + BF7 + BC10 had a lower pH than other treatments (p < 0.01), except AC + BC10. This could be attributed to a greater synergistic effect of the enzyme and bacterial inoculants.
The cassava root silage treated with additives had higher acetic acid and propionic contents (p < 0.01) and a lower butyric acid content (p < 0.01) when compared to the control. The main reason for this was that Enterococcus spp. is a type of heterofermentative LAB. Therefore, it could increase acetic acid production in the silage. Our finding is consistent with another study, which reported that alfalfa silage treated with Enterococcus spp. showed higher acetic acid and butyric acid contents than the control silage [42].
We found that cassava root silage ensiled for 30 days still had abundant LAB and aerobic bacteria, while yeast and mold were below the detectable level (Table 3). Silage treated with BF7 + BC10 and AC + BF7 + BC10 showed lower aerobic bacteria numbers than other treatments (p < 0.05). These findings are attributed to the additive improving the ensiling process [42].

4.5. Effect on In Vitro Rumen Fermentation of Dietary Silage

In vitro rumen fermentation is important for understanding the utilization of forage by ruminants [30,44]. In this study, the data demonstrated that the IVDMD and gas production of cassava root silage in TMR form did not differ among treatments (p > 0.05; Table 5). Enteric methane is a major source of dietary energy losses [45,46]. In previous studies, the effects of cellulase and LAB on methane production have not been consistent among additive prototypes and ensiling materials [14,17,30]. Therefore, the selection of additives based on decreasing methanogenesis, in combination with improved silage quality, is interesting for ruminant production.
Unfortunately, an additive to reduce methane production in cassava root silage was not found in this study. The results suggest that the silage treated with BF7 and AC + BC10 had lower degrees of methane production when compared to BF7 + BC10 (p < 0.05). The reason for this might be a stimulation of increased CP content in ensiling (Table 2). Our previous study found that sorghum silage treated with cellulase and LAB inoculant significantly decreased rumen methane production in vitro [17]. Recently, the same additives were used to treat green manure sunn hemp crop silage and increased methane production [30]. Cao et al. [14] showed that LAB-treated cabbage silage had decreased rumen methane production in vitro. Similarly, the addition of exogenous cellulase decreased the methane production of crop straws and grasses [43].

5. Conclusions

The present results demonstrate that an ensiling process could be employed to decrease the total cyanide content in cassava root silage by 39% of the initial value. The addition of AC and the two Enterococcus spp. as novel, cyanide-utilizing bacterial inoculants decreased the total cyanide content by 47 to 51% of its initial value. The addition of BF7 + BC10 was highlighted to increase the CP, acetic acid, and propionic acid contents of the silage. However, it did not improve IVDMD or gas production, and might stimulate the methanogenesis potential of the diet. To ensure the benefits to cyanide removal efficiency and improvement of silage quality, an in vivo evaluation will be needed.

Author Contributions

Conceptualization, W.K., C.K. and A.C.; formal analysis, W.K., C.K., P.S. and S.T.-u.; investigation, W.K. and C.K.; resources, W.K., C.K. and A.C.; writing—original draft preparation, W.K. and C.K.; writing—review and editing, W.K., C.K., N.S., R.S., J.M., K.A., P.P., S.S. and A.C.; supervision, W.K., C.K. and A.C.; funding acquisition, A.C., K.K., C.K. and W.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research has received funding support from the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation [grant number B01F640043] under the Industrial Post-doctorate Development for Agriculture, Food, Energy and Bio-materials for Future Phase II, Khon Kaen University (KKU), Thailand [grant number (KKU-PMU-B) 64-014]. The authors express their most sincere gratitude to the National Research Council of Thailand (NRCT) (Grant No. NRCT5-RSA63003-01) for providing financial support, the Research Program on the Research and Development of Winged Bean Root Utilization as Ruminant Feed, and the Increased Production Efficiency and Meat Quality of Native Beef and Buffalo Research Group and Research and Graduate Studies, KKU. The APC was funded by Khon Kaen University.

Institutional Review Board Statement

The animal experimental protocols performed in this work were approved by the Animal Care and Use Committee of Khon Kaen University, Khon Kaen, Thailand, based on the Ethics of Animal Experimentation of the National Research Council, Thailand (Record No. IACUC-KKU-45/64).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors thank the Rajamangala University of Technology Isan, Khon Kaen University, and the Animal Nutrition Laboratory (Khon Kaen University) for the infrastructure and laboratory facilities. W.K. and C.K. would like to thank Yimin Cai from the Japan International Research Center for Agricultural Sciences (JIRCAS) for expert technical support.

Conflicts of Interest

The authors declare that they have no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Fermentation 09 00151 g001 550
Figure 1. The changes in total cyanide content of cassava root silage during 30 days of ensiling. AC—Acremonium cellulase; BF7—E. feacium KKU-BF7 inoculant; BC10—E. gallinarum KKU-BC10 inoculant.
Figure 1. The changes in total cyanide content of cassava root silage during 30 days of ensiling. AC—Acremonium cellulase; BF7—E. feacium KKU-BF7 inoculant; BC10—E. gallinarum KKU-BC10 inoculant.
Fermentation 09 00151 g001
Fermentation 09 00151 g002 550
Figure 2. The changes in pH value of cassava root silage during 30 days of ensiling. AC—Acremonium cellulase; BF7—E. feacium KKU-BF7 inoculant; BC10—E. gallinarum KKU-BC10 inoculant.
Figure 2. The changes in pH value of cassava root silage during 30 days of ensiling. AC—Acremonium cellulase; BF7—E. feacium KKU-BF7 inoculant; BC10—E. gallinarum KKU-BC10 inoculant.
Fermentation 09 00151 g002
Table
Table 1. Microbial counts, pH, chemical compositions, and in vitro rumen parameters at 24 h incubation of fresh cassava root material used to prepare silage in this study.
Table 1. Microbial counts, pH, chemical compositions, and in vitro rumen parameters at 24 h incubation of fresh cassava root material used to prepare silage in this study.
Item Fresh Cassava Root
Microbial counts
(cfu/g FM)		LAB3.3 × 105
Coliform bacteria5.0 × 106
Aerobic bacteria3.7 × 107
Yeast1.8 × 108
Molds2.1 × 106
pH value 5.12
Chemical composition,
% DM		DM (%)38.07
OM98.27
CP2.10
EE0.50
NDF10.84
ADF3.60
ADL0.88
Total cyanides,
mg/kg DM		 1304
In vitro parametersIVDMD, %81.69
Gas production (L/kg DM)193.98
Methane production (L/kg DM)16.62
Methane production (L/kg IVDMD)22.25
cfu—colony forming unit; FM—fresh matter; DM—dry matter; LAB—lactic acid bacteria; OM— organic matter; CP—crude protein; EE—ether extract; NDF—neutral detergent fiber; ADF—acid detergent fiber; ADL—acid detergent lignin; IVDMD—in vitro DM digestibility.
Table
Table 2. Ensiling loss (%), chemical composition (% DM), and total cyanide content (mg/kg DM) of cassava root silage after 30 days of ensiling.
Table 2. Ensiling loss (%), chemical composition (% DM), and total cyanide content (mg/kg DM) of cassava root silage after 30 days of ensiling.
ItemEnsiling LossOMCPEENDFADFADLTotal Cyanides
Control1.5798.21 c2.08 bc0.55 b6.98 a3.54 a1.04790.06 a
AC1.7798.58 a1.91 c0.62 ab4.09 b2.59 b1.05657.69 b
BF71.6998.33 bc2.18 ab0.64 ab7.31 a3.50 a1.07695.69 b
BC101.6898.35 bc2.01 bc0.87 a7.63 a3.54 a1.14668.33 b
BF7 + BC101.8098.22 c2.31 a0.56 b6.93 a3.49 a1.07677.18 b
AC + BF71.8398.53 a2.03 bc0.34 b5.11 b2.93 ab1.22637.74 b
AC + BC101.9198.58 a1.90 c0.34 b4.27 b2.54 b1.10664.79 b
AC + BF7 + BC102.0698.38 b2.04 bc0.36 b4.35 b2.54 b0.91669.99 b
SEM0.1120.0050.0650.0920.4930.2600.14617.706
P0.127<0.0010.0030.005<0.0010.0110.912<0.001
a–c Means within columns with difference superscript letters differ at p < 0.05. Control—silage prepared without additive; AC—Acremonium cellulase; BF7—E. feacium KKU-BF7 inoculant; BC10—E. gallinarum KKU-BC10 inoculant; SEM—standard error of the means; OM—organic matter; CP—crude protein; EE—ether extract; NDF—neutral detergent fiber; ADF—acid detergent fiber; ADL—acid detergent lignin.
Table
Table 3. Silage quality of cassava root silage prepared with additives measured after 30 days of ensiling.
Table 3. Silage quality of cassava root silage prepared with additives measured after 30 days of ensiling.
ItemDMpHLactic AcidAcetic AcidPropionic AcidButyric AcidNH3-N
%g/kg DM
Control38.043.95 a1.885.72 b1.41 e8.86 a0.90
AC38.003.92 ab2.8111.46 a3.90 a4.03 b0.92
BF737.343.95 a2.6510.96 a3.60 a4.88 b0.90
BC1038.773.91 ab2.2211.25 a3.30 ab4.41 b0.92
BF7 + BC1038.063.91 ab2.4110.59 a3.22 ab3.49 b0.92
AC + BF739.483.91 ab2.4811.35 a2.58 bc2.21 bc0.89
AC + BC1039.633.88 bc2.2711.57 a1.60 de4.75 b0.90
AC + BF7 + BC1038.533.85 c2.6111.58 a2.23 cd0.41 c0.92
SEM0.8230.0130.2050.2580.1200.4480.009
P0.5140.0010.088<0.001<0.0010.0010.180
a–e Means within columns with difference superscript letters differ at p < 0.05. Control—silage prepared without additive; AC—Acremonium cellulase; BF7—E. feacium KKU-BF7 inoculant; BC10—E. gallinarum KKU-BC10 inoculant; SEM—standard error of the means; DM—dry matter; NH3-N—ammonia nitrogen.
Table
Table 4. Microbial population of cassava root silage prepared with additives measured after 30 days of ensiling.
Table 4. Microbial population of cassava root silage prepared with additives measured after 30 days of ensiling.
ItemLABColiform BacteriaAerobic BacteriaYeastMold
Log10 cfu/g FM
Control5.70ND5.62 aNDND
AC6.42ND5.27 abNDND
BF76.63ND5.36 aNDND
BC106.55ND5.24 abNDND
BF7 + BC106.47ND4.32 cNDND
AC + BF76.47ND5.51 aNDND
AC + BC105.96ND5.56 aNDND
AC + BF7 + BC106.93ND4.47 bcNDND
SEM0.320-0.279--
P0.228-0.016--
a–c Means within columns with difference superscript letters differ at p < 0.05. Control—silage prepared without additive; AC—Acremonium cellulase; BF7—E. feacium KKU-BF7 inoculant; BC10—E. gallinarum KKU-BC10 inoculant; SEM—standard error of the means; LAB—lactic acid bacteria; cfu—colony forming unit, FM—fresh matter; ND—not detected.
Table
Table 5. In vitro rumen fermentation after 24 h incubation of the dietary TMR prepared with cassava root silage (50:50 w/w).
Table 5. In vitro rumen fermentation after 24 h incubation of the dietary TMR prepared with cassava root silage (50:50 w/w).
ItemIVDMDGas ProductionMethane Production
(g/kg)(L/kg DM)(L/kg IVDMD)(L/kg DM)(L/kg IVDMD)
Control667.3184.70276.7321.00 abc31.49 abc
AC664.2186.40280.8720.11 bc30.28 bc
BF7676.8190.91282.0521.78 ab32.20 ab
BC10674.1185.10274.5920.19 bc29.96 bc
BF7 + BC10675.6197.72292.5922.47 a33.25 a
AC + BF7680.0183.56270.1620.20 bc29.70 bc
AC + BC10673.9190.48282.6619.67 c29.21 c
AC + BF7 + BC10685.5193.23281.8921.69 ab31.63 abc
SEM7.8803.3344.5550.5810.872
P0.6540.0730.0780.0220.040
a–c Means within columns with difference superscript letters differ at p < 0.05. TMR—total mixed ration; Control—TMR prepared from cassava root silage ensiled without additive; AC—Acremonium cellulase; BF7—E. feacium KKU-BF7 inoculant; BC10—E. gallinarum KKU-BC10 inoculant; SEM—standard error of the means; IVDMD—in vitro dry matter digestibility; DM—dry matter.
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Khota, W.; Kaewpila, C.; Suwannasing, R.; Srikacha, N.; Maensathit, J.; Ampaporn, K.; Patarapreecha, P.; Thip-uten, S.; Sawnongbue, P.; Subepang, S.; et al. Ensiling Cyanide Residue and In Vitro Rumen Fermentation of Cassava Root Silage Treated with Cyanide-Utilizing Bacteria and Cellulase. Fermentation 2023, 9, 151. https://doi.org/10.3390/fermentation9020151

AMA Style

Khota W, Kaewpila C, Suwannasing R, Srikacha N, Maensathit J, Ampaporn K, Patarapreecha P, Thip-uten S, Sawnongbue P, Subepang S, et al. Ensiling Cyanide Residue and In Vitro Rumen Fermentation of Cassava Root Silage Treated with Cyanide-Utilizing Bacteria and Cellulase. Fermentation. 2023; 9(2):151. https://doi.org/10.3390/fermentation9020151

Chicago/Turabian Style

Khota, Waroon, Chatchai Kaewpila, Rattikan Suwannasing, Nikom Srikacha, Julasinee Maensathit, Kessara Ampaporn, Pairote Patarapreecha, Suwit Thip-uten, Pakpoom Sawnongbue, Sayan Subepang, and et al. 2023. "Ensiling Cyanide Residue and In Vitro Rumen Fermentation of Cassava Root Silage Treated with Cyanide-Utilizing Bacteria and Cellulase" Fermentation 9, no. 2: 151. https://doi.org/10.3390/fermentation9020151

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