First reported quantitative microbiota in different livestock manures used as organic fertilizers in the Northeast of Thailand

Nutrient, FOM, and physiochemical contents of the fecal manures

For the three main fertility nutrients (N, P and K) in the fecal manures (Table 1a) the E manure contained the highest N level at 80 µg/g, 16-fold more than the other samples. In addition, it had a high P level of 100 µg/g (along with PC1 and PC3) compared with an average of 34.66 µg/g for the others, and a moderate K level of 150 µg/g (others ranged from 125.00 to 350.00 µg/g. The FOM content ranged from 1.00 to 1.50% for all the animal manures (Table 1a). For the general physiochemical properties, chicken manures showed a relatively high salinity (Table 1b: 3.52 ± 0.28 to 4.01 ± 0.64 ppt), and these ionic salts caused a high conductivity (11.27 ± 0.69 to 12.79 ± 0.08 mS/cm) and high water saturation (73.29 ± 0.40 to 80.56 ± 3.17 mS/cm), giving less available water for plant roots. A moderate level of salinity, conductivity and water content, such as those in E, are generally preferred for farming soil. The water content of the manure was lowest in G2, goats fed with P. purpureum (23.75 ± 3.07%), but increased two-fold in G1 goats fed with D. eriantha (49.35 ± 6.08%), suggesting a different type of grass feed might confer different fecal liquid and dry contents, or the chemical composition influenced the water activity in the goat’s gut. The R manure also had a low water content (41.53 ± 5.57%).

The visual observation of each manure was in accord with the reported water content, with a dry and hard texture (insufficient moisture) for G1, G2, and R manures, and a wet texture for the chicken, cattle, pig, and buffalo manures. Too much water in the soil can adversely affect aeration, where the plant’s roots receive insufficient oxygen and rot. Moreover, most plants prefer a slightly acidic soil pH of 6.2–6.8, which was found in the R, MB and E composts (Table 1b). The manure pH can change through reactions such as organic matter decomposition that affects the availabilities of NPK³⁹.

From the overall comparison of the NPK nutrients and physiochemical properties, the fecal manures from the Phuparn chicken species might be less suitable as a fertilizer because of the poor N level, and high level of salts and conductivity. Indeed, the manures from most species contained a low N level, and those from goat and rabbit also had a relatively low water content (insufficient water for a plant to absorb nutrients from the soil through the roots, to the trunk, leaves and fruits). A moderate water content in the manure was appropriate. But of course, a somewhat low water level could be adjusted by adding water. The biophysiochemical quality analyses of the manures suggested that the E manure would be appropriate as a fertilizer because of the enhanced NPK levels, low salinity, low conductivity, slightly acidic-neutral pH, and a moderate water content (Table 1b).

Quantification of total bacteria in the fecal manures

The total number of bacteria in copies/g DW fecal manure was derived from the 16S rRNA gene qPCR. Figure 1a showed the data were consistent between independent triplicate samples, and that the E and R manures had the lowest bacterial counts (at 2.1 × 10⁹ and 2.5 × 10⁹ copies/g DW, respectively). The other animal fecal manures contained more than 5 × 10⁹ copies/g DW, and the greatest bacterial load was found in PC2 and MB at 2.6 × 10¹⁰ and 1.9 × 10¹⁰ copies/g DW, respectively, while PC1 and PC3 had a significantly lower level (p < 0.001 [PC1:PC2] and p = 0.001 [PC3:PC2]) (Fig. 1b).

Bacterial taxonomic profiles by 16S rRNA gene sequences

The 16S rRNA gene V3-V4 library preparation and next generation sequencing were successful in that the number of raw and quality reads allowed data normalization (N = 7945 quality reads per sample) that covered > 99% and > 98% of the sequencing coverage of taxonomic compositions at the genus and species levels, respectively (Table 2). The average Good’s coverage indices were 99.54% and 99.43% for the genus and species levels, respectively (Supplemental Table 1). This was consistent with the plateau rarefaction curves, showing the frequencies of OTUs become constant despite increasing sequencing reads, meaning a sufficient sequencing coverage was obtained (Supplemental Fig. 1).

Combining the count of total 16S rRNA gene copies with the OTU percent compositions gave the quantitative number of copies of each OTU in a community. These quantitative microbiota data were then used to compute the alpha and beta diversity measurements. The alpha diversity revealed that the microbiota of E had the relatively most diverse OTUs at both the genus and species levels (Chao richness, Fig. 2a,c), regardless of having the lowest total bacterial count (Fig. 1). The low total bacterial abundance but high diversity in E underlined that the various OTUs of bacteria in E might be present in small numbers when compared to the copy numbers of OTUs in the other animal manures. Note that the alpha diversity obtained by considering the distribution of general OTUs among the different animal manures were similar (Shannon diversity, Fig. 2b,d).

Figure 3a described the quantitative number of bacterial phylum OTUs. Proteobacteria, Firmicutes and Bacteroidetes were common in all manure samples. PC2 and CC were predominantly Proteobacteria (at 9.41 × 10⁹ and 7.24 × 10⁹ copies/g DW, respectively), and MB and also PC2 were predominantly Bacteroidetes (at 8.22 × 10⁹ and 9.03 × 10⁹ copies/g DW, respectively). Firmicutes were also moderately common in all the manures except for E. The percentage abundance of genera above 1% were demonstrated in Fig. 3b, and was comprised of a total of 23 genera. The genus Ignatzschineria were responsible for the high Proteobacteria levels in PC2, MB and PC1, while the genus Acinetobacter were responsible for the high Proteobacteria levels in the other animal manures except for the E. Manures PC1, PC2 and MB contained all Bacteroides genera. Streptococcus was only found in PCO and was relatively abundant (20.04%). Escherichia were relatively low in R, G2, E, DC and MB, while Treponema were responsible for the high Spirochaetes levels in PC3, PCO and R at 3.63, 3.20 and 6.75%, respectively. Noted that the diversified OTUs in E belonged to Bacteroidetes at 6.28%. Moreover, a proportionate percentage of unclassified OTUs were presented in G1, G2, DC and E at 19.62, 41.86, 21.71 and 27.22%, respectively.

Relative frequencies of plant symbiotic and pathogenic bacterial genera

Plant symbiosis and pathogenic bacteria were analysed across the different animal manures. Manures PC1-3, G1, G2 and CC demonstrated generally abundant symbiotic bacteria comprised of the genera Pseudomonas, Bacillus, Arthrobacter, Flavobacterium, Alcaligenes and Streptomyces (Fig. 4a). For examples, PC2 contained abundant Alcaligenes (1.16 × 10¹⁰ cells/g DW), Pseudomonas (3.35 × 10⁹), Flavobacterium (2.82 × 10⁸) and Arthrobacter (3.8 × 10⁸). Streptomyces was only found in G1, G2 and E in moderate numbers (5.9–29.0 × 10⁶ cells/g DW).

For the plant pathogenic bacteria, high levels were found in the manures that also contained high levels of plant symbiosis bacteria (PC1-3, G1, G2 and CC), except for E, plus the other manures, such as S, D and MB. The E had none of VFDB-listed plant pathogens. Thus, all manures except for E contained plant pathogenic bacteria, and these were from Escherichia, Shigella, Enterococcus, Clostridium, Acinetobacter, Treponema, Staphylococcus and Bacteroides (Fig. 4b). This finding correlated with the percent abundance of genera (Fig. 3b) where, examples, Treponema were relatively high in PCO and PC3 at 2.03 × 10⁸ and 1.31 × 10⁸ cells/g DW, respectively. Escherichia were relatively low in R and DC at 1.15 × 10⁵ and 2.06 × 10⁵ cells/g DW, respectively. In contrast, only E did not contain any VFDB-listed plant pathogenic bacteria suggesting that E offers a plant pathogen-free organic fertilizer.

Relationship among bacterial communities, and statistical correlation with the biophysiochemical properties

The NMDS demonstrated both the reproducibility of the data between the independent triplicate samples, except for R and PCO. The larger variation in bacterial communities were found generally between the manures from different animal species, while the minor variation in bacterial communities were found between the breeds, or the feeding diets. For examples, the variation in quantitative microbiota profiles between G1, G2 and E, compared with PC1-3 (p = 0.085) and MB (p = 0.59) (Supplemental Fig. 2). Indeed, the quantitative microbiota structures belonging to the six animal manures that had all the VFDB’s categorized plant pathogens (PC, S, R, D and MB, except PCO) were rather distant from E (p = 0.101, 0.052, 0.089, 0.104 and 0.099, respectively).

Seven parameters (NPK and four physiochemical properties) were analyzed for possible Pearson’s correlation with any of the quantitative microbiota structures. The N level was strongly correlated (p = 0.01) to the structures and in the same direction of the E (Fig. 5). The percent water, salinity and conductivity characteristics were significant and associated in the direction opposite to E, and also to the G1, G2 and PCO microbiota structures. On the other hand, the PC1, PC2 and MB microbiota structures were strongly associated with the water content, salinity and conductivity. These parameters are suggested to be important in controlling the diversity of microbiota structures.

Predicted functional profiles from quantitative microbiota

Clustering by metabolic profiles separated most of the PC communities, then PCO and CC, from the E clusters (Fig. 6a). The quantitative microbiota in E demonstrated enhanced levels of COGs defined as metabolism, for instances, carbon fixation, oxidative phosphorylation and photosynthesis pathways compared to the rests (Fig. 6b: p = 3.26 × 10⁻³, 0.01 and 0.012, in order). The degradation functions of toxic compounds (xenobiotics) including styrene, caprolactam, aminobenzoate, nitrotoluene, polycyclic aromatic hydrocarbon and benzoate were also high in E compared to the rest (p = 2.81 × 10⁻⁵, 8.73 × 10⁻⁴, 1.38 × 10⁻³, 1.65 × 10⁻³, 0.027 and 0.03, respectively), indicating that the earthworm manure had a greater potential potency to degrade toxic compounds contaminated in the soil.

The KEGG categories of bacterial toxin, Staphylococcus aureus infection, and plant-pathogen interaction were dominated statistically higher in the other manures than E (p = 1.6 × 10⁻³, 2.14 × 10⁻³ and 0.011, respectively), in which the findings were correlated with Fig. 4b. Within the same metabolic profile clusters with E (Fig. 6a) but carried all VFDB listed plant pathogens (Fig. 4b), the metabolic profile of D was selected for comparison with that of E to perhaps capture some metabolic differences that might involve pathogens. Consistently, anti-bacterial compounds (i.e. biosynthesis of penicillin and cephalosporin, and antibiotic secondary metabolites) were higher in E (Fig. 6c), supporting the ability of the manure’s bacterial community to potentially protect against plant pathogens40. Related plant pathogen functions (bacterial toxin, Staphylococcus aureus infection, and plant-pathogen interactions) were statistically higher in D (Fig. 6c), resembling those in the other manures (Fig. 6b: all other samples). Moreover, E had enhanced levels for carbon fixation and photosynthesis, which are essential for plant growth (p = 2.93 × 10⁻³ and 7.77 × 10⁻³, respectively).

Source: Ecology - nature.com