Potential microbial strains for composting cattle and horse manure

Potenciais cepas microbianas para compostagem de esterco de gado e cavalo

Abstract

The potential of effective microorganisms in composting animal manure might be significant. This work aimed at isolating promising microbial strains for composting cattle and horse manure. A total of forty-five isolates have been isolated from soils of Northern Kazakhstan as well as cattle and horse manure. These microorganisms were extensively studied for their cellulose-degrading capacity, non-pathogenicity, protease, nitrogenase and catalase activities, as well as their growth stimulation, antagonistic ability, and growth rate. As a result, 21 potent strains were selected and genotyped for the creation of microbial consortia. These consortia were then used in small-scale composting experiments with cattle and horse manure. Further studies evaluated the effectiveness of the composting process and the quality of the compost produced. The study yielded promising results, identifying effective microbial strains that could enhance the composting of horse and cattle manure.

Keywords:
effective microorganisms; soil and manure isolates; composting; horse and cattle manure

Resumo

O potencial de microrganismos eficazes na compostagem de esterco animal pode ser significativo. Este trabalho teve como objetivo isolar cepas microbianas promissoras para compostagem de esterco de gado e cavalo. Um total de 45 isolados foi obtido a partir de solos do norte do Cazaquistão, bem como de esterco de gado e cavalos. Esses microrganismos foram amplamente estudados por sua capacidade de degradação de celulose, não patogenicidade, atividades de protease, nitrogenase e catalase, bem como sua estimulação de crescimento, capacidade antagônica e taxa de crescimento. Como resultado, 21 cepas potentes foram selecionadas e genotipadas para a criação de consórcios microbianos. Esses consórcios foram então usados ​​em experimentos de compostagem em pequena escala com esterco de gado e cavalo. Estudos posteriores avaliaram a eficácia do processo de compostagem e a qualidade do composto produzido. O estudo produziu resultados promissores, identificando cepas microbianas eficazes que podem melhorar a compostagem de esterco de cavalo e gado.

Palavras-chave:
microrganismos eficazes; isolados de solo e esterco; compostagem; esterco de cavalo e gado

1. Introduction

Organic farming has become a priority for ensuring food safety and long-term sustainability, serving as a response to environmental pollution caused by excessive pesticide use (Milestad and Darnhofer, 2003; Aktar et al., 2009). Fertilization with animal manure is a cost-effective and widely adopted practice in organic farming. However, animal manure contains a wide range of pathogens that can pose potential risks to human and animal health (Bicudo and Goyal, 2003). Composting is an effective method for controlling pathogens in manure, as aerobic respiration during the process generates high temperatures that result in manure sanitization (Heinonen-Tanski et al., 2006). While composting occurs naturally, it is not always efficient. The application of effective microorganisms could play a crucial role in accelerating manure maturation into organic fertilizer (Hidalgo et al., 2022).

Microorganisms native to a specific area are typically better adapted to the local environment. As a result, they can rapidly colonize soil and manure, contributing beneficial qualities (Avila et al., 2021). Both soil and animal manure are rich in effective microorganisms, which can be isolated and enriched to create biopreparations for manure composting and soil quality improvement (Higa and Wididana, 1991). In agriculture, effective microorganisms are defined as decomposers of organic waste, fixers of atmospheric nitrogen, enhancers of nutrient cycling, controllers and suppressors of plant pathogens, and stimulators of plant growth (Hidalgo et al., 2022). When introduced into the soil over a certain period, these endogenous microorganisms can enhance crop yields and protect plants (Higa and Wididana, 1991).

Previous research has identified microbial decomposers such as Lactobacillus sp., Actinomycetes sp., and Aspergillus in cattle manure (Idham et al., 2016; Ropiatningsuari et al., 2018). Additionally, cattle manure has been shown to be rich in fungi capable of degrading cellulose (El Shishtawy and Ageez, 2021). However, little data is available on the isolation of effective microorganisms from horse manure.

Soil, on the other hand, is well known for harboring prominent microbial strains that can be isolated and utilized in agriculture. Examples include soil-endogenous fungi and bacteria with decomposition and cellulose-degrading capabilities (Deacon et al., 2006; Berg and Laskowski, 2005). Some soil microorganisms also possess the ability to control plant pathogens (Ikotun and Adekunle, 1990), while others can stimulate plant growth by producing phytohormones (Jeon et al., 2003).

In recent years, the application of beneficial microorganisms to cereal crops has demonstrated their positive effects on yield and quality under unfavorable environmental conditions (Mumtaz et al., 2019; Hussain et al., 2019; Ashrafuzzaman et al., 2009). Certain bacteria are also capable of assimilating soluble forms of mineral phosphorus, synthesizing siderophores, auxins, cytokinins, and vitamins, which significantly enhance plant growth by improving phosphorus absorption efficiency (Bulut, 2013; Hayes et al., 2000; Elkoca et al., 2010).

We propose that isolating and combining endogenous microorganisms from soil and manure into consortia for manure composting offers a promising and cost-effective approach to advancing organic farming. A consortium, a mixed culture of effective microorganisms, can encompass a broad range of agronomically valuable microbial traits and deliver more substantial benefits when applied.

The objectives of this study were: (1) to isolate and characterize the functional potential of cultivable microorganisms from soil and animal manure based on substrate utilization, and (2) to evaluate the efficiency of isolate-based consortia in composting cattle and horse manure.

2. Material and Methods

2.1. Microbial isolation

The microorganisms used in this study were isolated from various soils in northern Kazakhstan, including chernozem, chestnut, dark chestnut, light chestnut, and alkali soils, as well as from horse and cattle manure. Soil samples were collected from three depths (0-10 cm, 10-20 cm, and 20-30 cm) under sterile conditions. Soil moisture was determined by oven drying at 105°C, while pH was measured using a salt extract method. General microbiological techniques, including serial dilution and inoculation on selective media, were employed for the isolation of microorganisms from soil and manure samples (Mokarenko and Vorobyova, 2004). The number of bacteria utilizing organic nitrogen was determined using meat-peptone agar (MPA), while bacteria and actinomycetes utilizing mineral nitrogen were counted on starch-ammonium agar (SAA). Mycelial fungi were isolated using acidified Chapex-Dox agar. Aerobic cellulose-degrading microorganisms were identified on Hutchinson's medium, followed by differentiation into bacteria, fungi, and actinomycetes (Netrusov et al., 2005). Colony-forming units (CFU) were counted, and the isolated microorganisms were morphologically studied (Bergey, 1994). Pure cultures were obtained using the streak plate method.

2.2. Microbial studies

Proteolytic activity of bacteria and actinomycetes was assessed based on their ability to liquefy gelatin. Cellulolytic activity was studied using Hutchinson's medium with filter paper. Egg yolk-salt agar was used to assess catalase activity and pathogenicity, excluding harmful strains. Nitrogenase activity was measured using established methods (Netrusov et al., 2005). The growth-promoting effect of broth filtrates from actinomycetes and bacteria on crop seedlings was evaluated according to Berestetsky (Berestetsky, 1982). For each treatment, 30 healthy seeds were soaked in broth filtrates for 24 hours, rinsed, and then transferred to Petri dishes lined with filter paper. Seeds were incubated for 7 days at 20-22°C, and germination rate, sprout, and root lengths were recorded. The cross-streak method (Rudakov, 1981) was used to assess the antagonistic properties of actinomycetes and bacteria against major crop pathogens. Pathogenic and antagonistic cultures were co-inoculated on Chapex-Dox agar and monitored on the 3rd, 5th, 7th, and 10th days. Inhibition of pathogen growth by antagonists was calculated on the 7th day using the formula (1):

$$Р=\left(К-А\right)/К100$$, (1) Equation 1

where P is the percentage of pathogen growth inhibition, K is growth in the control, and A is growth in the cross-streak culture.

Biparasitic properties (%) of antagonists were evaluated using the following scale (Velikanov et al., 1994): 0 – no growth; 1 – antagonist covers up to 25% of the pathogen's area; 2 – 25-50%; 3 – 51-75%; 4 – complete coverage; “+” – antagonist growth; “++” – pathogen colonies exhibit actinomycete sporulation.

2.3. Species identification

Species of potent microorganisms isolated from soils and animal manure were identified by sequencing 16S rRNA and comparing the sequence with those in the GeneBank database.

2.4. Consortia preparation and composting

Two microbial consortia were developed for small-scale composting trials using cattle and horse manure. The consortia were cultured in liquid media for 12 hours under shaking conditions. Concentrated suspensions were diluted 1:10 with warm water and applied to manure piles at a rate of 10 ml of the working solution per kilogram of manure. Manure with 30-35% moisture content was composted in piles of 1.2-1.4 kg (horse manure) and 1.9-2.0 kg (cattle manure), placed in pots with a diameter of 22 cm and height of 18-20 cm. Parameters such as compost temperature, air temperature, precipitation, odor, color, consistency, and the number of turns were recorded. Manure piles were turned every 3 days for aeration, and pots were covered during unfavorable weather. The experiment was conducted in five replicates with the following design:

• Variant 1: Control (cattle manure without bio-preparation)

• Variant 2: Cattle manure + Consortium A

• Variant 3: Cattle manure + Consortium B

• Variant 4: Cattle manure + biopreparation “Agromix”

• Variant 5: Control (horse manure without bio-preparation)

• Variant 6: Horse manure + Consortium A

• Variant 7: Horse manure + Consortium B

• Variant 8: Horse manure + biopreparation “Agromix”

Daily temperature and pH values were monitored for all compost piles.

2.5. Chemical analysis of compost

Chemical analysis of compost was performed using a CHNS/O analyzer, based on the classical Dumas-Preyglé method, which involves burning the sample in the presence of an oxidizer in an inert gas stream.

3. Result

3.1. Microbial studies

A total of forty-five isolates were selectively obtained from soils of Northern Kazakhstan, as well as cattle and horse manure, and were extensively studied for various agronomically valuable properties. The isolates exhibiting the highest abundance and earliest visible colony formation were prioritized for analysis. Both bacterial and fungal isolates were purified through repeated sub-culturing. A summary of the microbial studies is presented in Figure 1. Several key properties were used for the selection of microorganisms: including cellulose-degrading capacity, non-pathogenicity, protease, nitrogenase and catalase activities, growth stimulation, antagonistic ability, and growth rate.

Figure 1
Selection criteria of potent microorganisms based on bio-physio-chemical properties. This table presents the functional traits of bacterial trains, including cellulase, protease, catalase, nitrogenase activity, growth stimulation, antagonistic activity, and growth rate. Each strain is identified by a unique number.

Out of the forty-five strains studied, 84% demonstrated cellulose-degrading ability and non-pathogenic characteristics. Protease and catalase activities were observed in 93% and 91% of isolates, respectively. In addition, 87% of the strains exhibited plant growth-promoting potential, while only 31% showed antagonistic activity against the causative agent of helminthosporiosis in grain seeds, Bipolaris sorokiniana. Nitrogenase activity was detected in 47% of isolates, and active microbial biomass was formed in 42% of strains.

Interestingly, the studied species displayed multiple properties rather than a single function. Further species identification allowed for a more detailed analysis of the microbial population present in manure. All species identified had been previously reported to possess various agronomically valuable properties.

3.2. Sequencing data

The 16S rRNA gene sequences of all strains were submitted to the NCBI GeneBank for Accession Numbers. Sequencing allowed for the identification of microorganisms from different taxa present in soil and manure (Table 1). Most of the isolates were identified as bacteria and actinomycetes: Streptomyces, Achromobacter, Priestia, Bacillus, Sphingomonas, and Pseudarthrobacter. Only two isolates were identified as fungi of the same species Trichoderma.

Table 1
Species identification based on the analysis of 16S rRNA sequence of the isolates. This table lists bacterial and fungal strains identified in the study, along with their species names and previously reported functional activities. References are provided for each reported activity.

Strain number	Species name	Reported activities
Nº6, Nº8, Nº9, Nº10, Nº19, Nº33, Nº45, Nº22, Nº13	Priestia megaterium	Growth stimulation (Biedendieck et al., 2021)
Nº24	Streptomyces macrosporeus	Cellulolytic activity (Soeka et al., 2019)
Nº37	Streptomyces griseorubens	Cellulolytic activity (Xu and Yang, 2010; Prasad et al., 2013)
Nº41	Streptomyces flavofuscus	Cellulolytic activity (Ventorino et al., 2016)
Nº34, Nº38	Streptomyces pratensis	Antagonistic activity (Lian et al., 2017; Hu et al., 2023)
Nº32	Streptomyces albogriseolus	Antagonistic activity (Zeng et al., 2013)
Nº40	Achromobacter xylosoxidans	Growth stimulation (Ma et al., 2009)
Nº21	Pseudarthrobacter oxydans	Growth stimulation (Oubohssaine et al., 2022)
Nº18	Bacillus paramycoides	Bioremediation (Rashid et al., 2022)
Nº44	Sphingomonas paucimobilis	Biodegradation (Abrusci et al., 2011)
Trichoderma –KZ, Trichoderma Nº3	Trichoderma asperellum	Antagonistic activity (de los Santos-Villalobos et al., 2013; Díaz-Gutiérrez et al., 2021)

Based on the obtained nucleotide sequences of bacterial and fungal strains, a phylogenetic analysis was conducted, resulting the construction of phylogenetic trees (Figures 2 and 3).

Figure 2
The phylogenetic tree depicts the evolutionary relationships among Streptomyces and Achromobacter strains based on sequence analysis. Reference sequences retrieved from GenBank are labeled with accession numbers. Newly identified strains (marked with red circles) are clustered with their closest reference sequences. Bootstrap support values (%) indicate the reliability of branching. The scale bar represents genetic distance.

Figure 3
The phylogenetic tree illustrates the evolutionary relationships among Trichoderma strains based on sequence analysis. Reference sequences from GenBank are indicated with accession numbers. Bootstrap support values (%) are shown at key nodes. The newly identified strains (OR528067 and OR528068) cluster with Trichoderma asperellum, indicating their close genetic relationship with known reference sequences. The scale bar represents genetic distance.

The evolutionary history was inferred using the maximum likelihood method and the Tamura-Nei model. The tree with the highest log probability (-3486.12) is shown. The initial tree(s) for the heuristic search were obtained by applying the neighbor-joining method to a matrix of pairwise distances estimated using the Tamura-Nei model.

The tree was drawn to scale and the length of branches was measured by the number of substitutions per site. The proportion of sites where at least one unambiguous base was present in at least one sequence for each descendant clade was shown next to each internal tree node. This analysis involved 14 nucleotide sequences. There were a total of 1522 items in the final data set. Evolutionary analysis was carried out in MEGA11.

Consortium A (strains # 40, 41, 37, 38, 32, 24, 34, 18, 8, and 21) was made of actinomycetes and bacteria growing on starch-ammonium agar. Bacterial consortium B (strains # 10, 44, 13, 9, 6, 19, 22, 33, and 45) was prepared based on bacteria that consume organic forms of nitrogen. Additionally, previously developed “Agromix” biopreparation consisting of effective microorganisms was also used for composting cattle and horse manure.

The pH values in all variants of composting cattle and horse manure and were within the physiological range for microbial growth and development – pH 6-8 (Figure 4). A two-way ANOVA was performed to assess the effects of manure treatment (eight treatments) and composting duration (five time points) on pH. The analysis revealed significant main effects of both time points (F(4, 80) = [1434], p < 0.0001) and manure treatment (F(7, 80) = [34.99], p < 0.0001), indicating that pH varied significantly over time and among treatments. Additionally, a significant interaction effect was observed (F(28, 80) = [11.22], p < 0.0001), suggesting that the impact of manure treatment on pH depended on the composting stage.

Figure 4
pH dynamics during the composting of horse and cattle manure with and without biopreparations. The bars indicate pH measurements at different time intervals (1–5, 6–10, 11–15, 16–20, and 21–25 days). The experimental treatments include manure without biopreparations, as well as manure treated with Consort. A, Consort. B, and Agromix. Statistical analysis using one-way ANOVA followed by Tukey’s multiple comparison test revealed no significant differences among treatments (p < 0.05). The pH values are presented as the mean ± standard deviation (SD) of three replicates. Superscript letters denote significant differences.

Tukey’s multiple comparison test revealed significant pH differences among most manure treatments (p < 0.0001). However, certain treatments exhibited similar pH trends at specific time points, indicating that some formulations influenced pH more consistently than others. During the first five days of composting, pH differences among treatments were mostly insignificant, except for significantly higher pH in cattle manure + Consort. A compared to horse manure + no biopreparation and horse manure + Consort. B. By days 6–10, a major pH increase was observed in cattle manure + Agromix, which differed significantly from cattle manure + no biopreparation, cattle manure + Consort. A, horse manure + no biopreparation, horse manure + Consort. B, and horse manure + Agromix.

Over the first 10 days, the average pH across all treatments remained around 6. The most pronounced pH differences emerged during days 11–20, where pH values sharply increased to 7–8. On days 11–15, control manures without biopreparations exhibited significantly lower pH than other treatments, with an average difference of 0.8. Similarly, in horse manure, pH in horse manure + no biopreparation was significantly lower (by 0.3) compared to the other three treatments. By days 16–20, pH declined to 6–7 in most treatments, with statistically significant differences observed across groups, except between cattle manure + no biopreparation and cattle manure + Consort. B, which showed no significant variation. During the final composting phase (days 21–25), no significant pH differences were observed among cattle manure treatments. However, in horse manure, horse manure + Agromix exhibited a significantly higher pH than the control.

The temperature reached the highest values between the 6^th and 15^th day of composting for both cattle and horse manures (Figure 5). A two-way repeated measures ANOVA was performed to assess the effects of manure treatment and composting duration on temperature. The analysis revealed a significant main effect of time (F(4, 80) = [129952], p < 0.0001), a significant main effect of manure treatment (F(7, 80) = [3344], p < 0.0001), and a significant interaction effect (F(28, 80) = [286.9], p < 0.0001), indicating that temperature changes over time depended on manure treatment. Post hoc Tukey’s multiple comparison test showed significant differences among manure treatments (p < 0.0001) with a few exceptions. No significant temperature differences were observed between cattle manure + Consort. A and cattle manure + Consort. B at all time points except days 21–25. Additionally, temperature differences were not significant between:

Figure 5
Temperature dynamics during the composting of horse and cattle manure with and without biopreparations. Bars represent temperature variations across different time intervals (1–5, 6–10, 11–15, 16–20, and 21–25 days). Treatments include manure without biopreparations and manure treated with Consort. A, Consort. B, and Agromix. Temperature values are expressed as the mean ± standard deviation (SD) of three replicates. Statistical analysis was performed using two-way ANOVA. Statistical significance is indicated by superscript letters (e.g., a, b, c) above the bars, based on Tukey’s multiple comparison test (p < 0.05). Significant temperature difference were observed within the same compost type.

• Cattle manure + Agromix and horse manure without biopreparation (days 1–5),

• Cattle manure + Agromix and horse manure + Consort. A (days 6–10),

• Cattle manure without biopreparation and cattle manure + Agromix (days 11–15),

• Horse manure + Consort. B and horse manure + Agromix (days 16–20),

• Cattle manure without biopreparation and cattle manure + Consort. B,

• Cattle manure + Agromix and horse manure + Consort. B,

• Cattle manure + Agromix and horse manure + Agromix (days 21–25).

NPK content is a key indicator of compost quality (Figures 6 and 7). The graphs summarize the effects of cattle and horse manure composting on NPK levels. One-way ANOVA revealed significant differences in nitrogen (F(9, 20) = [33.18], p < 0.0001), phosphorous (F(9, 20) = [318.7], p < 0.0001), and potassium (F(9, 20) = [120], p < 0.0001) among treatments. For both manure types, nitrogen content significantly decreased after composting compared to raw manure (p < 0.0001).

Figure 6
Effect of composting on NPK content in cattle manure. The bar graph represents phosphorus (P) and potassium (K) concentrations (mg/kg), while the line graph shows nitrogen (N) content (%). Treatments include raw cattle manure, composted manure without biopreparation, and manure treated with Consort A, Consort B, and Agromix. Values are expressed as mean ± standard deviation (SD) of three replicates. Statistical significance is indicated by superscript letters (e.g., a, b, c) above the bars, based on Tukey’s multiple comparison test (p < 0.05). Treatments sharing the same letter are not significantly different, while those with different letters exhibit significant variation.

Figure 7
Effect of composting on NPK content in cattle manure. The graph shows phosphorus (P, mg/kg), potassium (K, mg/kg), and nitrogen (N, %) levels across different treatments: raw cattle manure, composted manure without biopreparation, and composted manure with Consort. A, Consort. B, and Agromix. P and K contents are shown as bars, while N content is represented by the line. The values are expressed as the mean ± standard deviation (SD) of three replicates. Statistical significance is denoted by letters (e.g., a, b, c) above the bars based on Tukey’s multiple comparison test (p < 0.05), where treatments sharing the same letter are not significantly different, while those with different letters exhibit significant variation.

Similarly, phosphorus content differed significantly between raw and composted manure, except for cattle manure + Consort A and horse manure + no biopreparation. Unlike nitrogen, phosphorus levels tended to increase after composting. Potassium content also nearly doubled in composted manure. However, no significant differences were observed in potassium content among manure treated with Consort A, Consort B, and Agromix.

A significant difference in carbon content was observed among cattle and horse manure treatments (F(9, 20) = [56.02], p < 0.0001). Cattle manure + Consort. A retained carbon levels comparable to raw cattle manure (~30%), while other cattle manure treatments exhibited a significant carbon decrease (p < 0.0001). Similarly, horse manure + Consort. B maintained the highest carbon content, comparable to cattle manure, whereas other treatments resulted in significant carbon loss. Carbon loss ranged from approximately from 2% to 10% for both manure types (Figure 8).

Figure 8
Percentage of carbon in cattle and horse manure under different treatments. The bars represent carbon content (%) in raw manure, composted manure without biopreparation, and composted manure with Consort. A, Consort. B, and Agromix. Blue bars indicate cattle manure, while orange bars indicate horse manure. The values are expressed as the mean ± standard deviation (SD) of three replicates. Statistical significance is denoted by letters (e.g., a, b, c) above the bars based on Tukey’s multiple comparison test (p < 0.05), where treatments sharing the same letter are not significantly different, while those with different letters exhibit significant variation.

The analysis of common heavy metals did not reveal the breakthrough concentration of heavy metals in composted manure (Table 2). Both Fe (F(3, 4) = [0.223], p = 0.88) and Mn (F(3, 4) = [0.163], p = 0.92) concentrations were shown not to change significantly between manures treated with different consortia. For the other two metals Zn and Cu, only cattle manure treated with Agromix demonstrated elevated levels of these heavy metals.

Table 2
The table presents the concentrations of iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn) in horse and cattle manure under different treatment conditions, including no biopreparation, microbial consortia (A and B), and Agromix. The limit of concern (LOC) for Zn, Cu, and Mn is provided for reference. Values are expressed in mg/kg. The values are expressed as the mean ± standard deviation (SD) of three replicates.

Manure treatment	Fe (mg/kg)	Zn (mg/kg)	Cu (mg/kg)	Mn (mg/kg)
LOC for the heavy metal	Not determined	110.0	66.0	1500.0
Horse manure + No biopreparation	10232.16±817.31	0	5.66±0.69	348.89±7.17
Horse manure + Consort. A	11617.45±580.40	0	2.59±0.24	350.55±1.98
Horse manure + Consort. B	7705.01±242.82	0	0	258.57±3.26
Horse manure + Agromix	8604.68±106.92	0	0	296.18±1.60
Cattle manure + No biopreparation	5802.34±145.37	0	0	457.72±48.58
Cattle manure + Consort. A	5418.01±114.13	0	0	452.49±24.71
Cattle manure + Consort. B	4941.69±107.04	0	0	422.18±12.54
Cattle manure + Agromix	7540.44±37.31	98.96±2.44	20.61±1.10	496.72±15.92

4. Discussion

The use of microorganisms in animal manure composting is widely adopted practice (Neklyudov et al., 2008). The use of EM in the bioconversion of manure into organic fertilizer provided a significant improvement in the compost by increasing the organic matter content (Cremeneac and Boclaci, 2018; Peng et al., 2024). All the species genetically identified in soil and cattle manure were previously reported to possess activities, which have been covered in this paper.

Nine isolates in this study were identified as Priestia megaterium – a well-known bacteria found in different habits (Biedendieck et al., 2021). Soil Priestia species can change cellular processes and produce a variety of substances to adapt to unfavorable environments including herbicide contamination (Dobrzanski et al., 2018). Additionally, P. megaterium has a variety of reported valuable properties actively exploited in agriculture including plant growth promotion (Feng et al., 2017), nitrogen fixation (Ding et al., 2005; Singh et al., 2020), and pathogen control (Chakraborty et al., 2006).

The second most abundant species among isolates in our study was Streptomyces. There has been extensive data on studying not only the agronomic value of Streptomyces (Chater, 2016). Several works showed prominent cellulolytic activities in S. macrosporeus (Soeka et al., 2019), S. griseorubens (Xu and Yang, 2010; Prasad et al., 2013), and S. flavofuscus (Ventorino et al., 2016). Ventorino et al. were able to demonstrate the presence of several enzymes in isolated Streptomyces species capable of lignocellulose conversion (Ventorino et al., 2016). Similarly, Xu and Soeka also reported multiple enzymatic activities for other members of Streptomyces (Soeka et al., 2019; Xu and Yang, 2010). While other Streptomyces species possessed biocontrol properties by releasing active compounds not only against fungal pathogens (Lian et al., 2017; Hu et al., 2023) but also against nematodes (Zeng et al., 2013).

The two bacterial strains identified in this study were Achromobacter xylosoxidans and Pseudarthrobacter oxydans, both known for their plant growth promotion. Achromobacter xylosoxida was capable of indole acetic acid production, which resulted in better plant biometrics (Ma et al., 2009), while Pseudarthrobacter oxydans stimulated plant growth by auxin release (Oubohssaine et al., 2022).

The other two bacterial species, identified in the present study, were reported as potent biodegrading agents: Bacillus paramycoides effective at degrading chemicals in wastewater (Rashid et al., 2022), and Sphingomonas paucimobilis efficient at degrading ionic liquids (Abrusci et al., 2011).

The single fungal representative in this study was identified as Trichoderma asperellum, which is quite well known for its antagonistic properties implying a great variety of acting mechanisms (de los Santos-Villalobos et al., 2013; Díaz-Gutiérrez et al., 2021).

All the species identified in this study have been previously characterized and reported as plant-beneficial microorganisms. In this work, we have also explored the capability of these species for composting cattle and horse manure.

Our findings suggest that manure inoculation with biopreparations significantly influenced composting temperature dynamics, with certain treatments exhibiting similar thermal profiles at specific time points. Among all treatments, Agromix exhibited the highest temperature values throughout the composting process for both cattle and horse manure. The composting process followed the three characteristic temperature stages: mesophilic, thermophilic, and maturation, as previously reported (Huang et al., 2017). However, thermophilic temperatures did not reach the higher values (50-60°C) observed in previous studies on composted cattle or horse manure (Huang et al., 2017; Wang et al., 2024). This may be due to the absence of bulking agents, which are commonly used to optimize the C/N ratio, typically in the form of straw or other organic materials (Paredes et al., 2015). The availability of organic substances in manure during the initial composting phase stimulates microbial activities, leading to a temperature increase (Jusoh et al., 2013).

The pH values stabilized within the normal range of 6-8, which was also explained by microbial activity resulting in the gradual release of substances in manure (Van Fan et al., 2018). These findings suggest that composting duration had the strongest influence on pH changes, while manure treatments exerted a smaller but significant effect. The significant interaction effect indicates that the efficacy of biopreparations in modifying pH varied over time, likely due to microbial activity and organic matter decomposition.

A pH increase during the thermophilic stage was observed in both cattle and horse manure, consistent with previous studies (Huang et al., 2017). When pH rises above 7, nitrogen volatilization occurs in the form of NH₃, contributing to a total nitrogen loss during composting (Huang et al., 2017). This was evident from the sharp decline in total nitrogen content in composted manure when compared to raw manure.

Cattle manure treatments containing Consort. A and B exhibited similar pH and temperature trends across most time points, suggesting comparable microbial activity. In contrast, horse manure treatments showed less pH variation, particularly in the later composting stages.

In this study, all strains (21 out of 45) possessing nitrogenase activity have been selected for genetic analysis and were chosen as prominent strains. We believed that the inoculation with microorganisms capable of nitrogen fixation might compensate for the nitrogen loss during composting. The results of our study demonstrated no change in nitrogen content between manure composted without any consortia and with the addition of consortia. Inoculation with nitrogen-fixing bacteria did not mitigate nitrogen loss or improve nitrogen retention during composting of cattle and horse manure. It is contrary to what we have observed with nitrogen content, where composting cattle manure with effective microorganisms and in an anaerobic condition results in an increase in total nitrogen content in compost (Cremeneac and Boclaci, 2018). Nitrogen loss from compost is mainly due to ammonia (NH₃) evaporation, where the gas loss can account for about 50% of the original total N in manure (Michel Junior et al., 2004). In our study, the addition of nitrogen-fixing microorganisms did not compensate for the nitrogen loss. However, Liu and colleagues reported that inoculation with diazotrophs could enhance nitrogen retention in cattle manure compost by reducing ammonia and nitrogen oxide emissions (Liu et al., 2023).

Loss of carbon during composting is an inevitable process; where from 30% to 85% of carbon is mainly lost in the form of CO₂ from manure (Chang et al., 2019)_. The same authors claimed the prevention of extensive carbon loss by the addition of only biochar (Chang et al., 2019)_. Although consortia treatment of manure did not result in a total carbon increase, the carbon loss was not as dramatic as it was reported previously.

Apart from temperature and pH values, the C/N ratio is a key parameter influencing the composting of animal manure (Vochozka et al., 2017). In our study, raw manure prior to composting had a C/N ratio of approximately 11:1 for both cattle and horse manure. Notably, no bedding material was added to achieve the optimal composting C/N ratio of 20:1-35:1 (Macias-Corral et al., 2019). The same authors reported the lowest C/N ratio in cattle manure composted without bedding materials. Among treatments, the highest C/N ration was observed in cattle manure + Consort. A (17:1), and in horse manure + Consort. B (23.8:1), suggesting that microbial inoculation influenced carbon and nitrogen dynamics during composting.

On the other hand, we observed an increase in phosphorous and potassium content in composted cattle and horse manures. Similar trends have been reported for both manure types (Zhen et al., 2021; Wang et al., 2024). Phosphorous accumulation in composted manure is primarily due to the reduction in dry matter content, and the greater stability of phosphorous, compared to nitrogen, which is lost in a gas state (Zhen et al., 2021). Similarly, the stable increase in potassium content is explained by the preference and active consumption by microorganisms of other elements such as carbon, nitrogen, and phosphorous, leaving the high percentage of potassium unused (Zhen et al., 2021).

Manures contain a large amount of heavy metals, the concentration of which should be considered before applying manure to soil to avoid contamination. Heavy metal content in animal manure can be effectively reduced by composting (Eneji et al., 2003). Our results demonstrated the within-the-range concentrations of heavy metals in both composted manures (Brinton, 2000).

5. Conclusion

The repertoire of potentially beneficial microorganisms is vast, and ongoing research worldwide highlights the importance of continuously isolating and studying microorganisms from diverse habitats, including soil and manure. The microbial properties examined in this study hold significant potential for agricultural applications.

Our findings underscore the importance of isolating microorganisms from soil and manure with for potential use in composting cattle and horse manures. Both manure types were effectively composted with the inoculation of effective microorganisms. No dramatic differences were observed between cattle and horse manure in terms of temperature, pH, NPK content, or carbon levels during composting. However, incorporating bulking materials alongside microbial inoculants may be beneficial for optimizing the C/N ratio and enhancing compositing process.

Acknowledgements

This research has been funded by the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakhstan (Grant No. BR21882327) program, “Development of New Technologies for Organic Production and Processing of Agricultural Products,” within the subprogram “Organic Production and Processing of Agricultural Products.”

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Publication Dates

• Publication in this collection
  09 May 2025
• Date of issue
  2025

History

• Received
  20 Nov 2024
• Accepted
  25 Feb 2025