Distillers’ grains organic fertilizer alters soil bacterial composition and co-occurrence patterns in a tobacco-growing field

Abstract

In recent years, numerous studies have indicated that the combination of organic and inorganic fertilizers can effectively improve soil fertility and soil productivity. Distillers’ grain (DG), the primary by-product of Chinese spirits production, has a high utilization value for producing organic fertilizer. We investigated the effects of distillers’ grain organic fertilizer (DGOF) on soil chemical properties and microbial community composition, as well as the effects of chemical properties on the abundance of keystone species. The results indicated that the application of DGOF significantly increased tobacco yield by 14.8% and mainly affected the composition rather than the alpha diversity of the bacterial community. Ten amplicon sequence variants (ASVs) were identified as keystone species in the bacterial communities, and most of their relative abundance was influenced by the DGOF addition through affecting soil chemical properties. Our results elucidated the alterations in soil chemical properties and microbial community composition resulting from DGOF application, which is of great importance to better understand the relationship between DGOF and soil microorganisms in the flue-cured tobacco cultivation field.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01229-2.

Introduction

Tobacco (Nicotiana tabacum L.) is an important economic crop and is widely cultivated in more than 100 countries [1]. In China, most of the tobacco-growing soil has a high multiple cropping index. Long-term input of chemical fertilizers alone has led to tobacco-growing soil compaction, reduced biological activity, and low utilization efficiency of phosphorus, potassium, and other mineral elements [2]. Over the past decades, extensive research has been conducted to explore sustainable agronomic management practices aimed at coping with these challenges. Among these practices, the substitution of chemical fertilizers with organic fertilizers not only minimizes the losses of nutrients but also improves nutrient use efficiency and crop productivity [3, 4]. Meanwhile, the recycling and composting of organic waste materials can also alleviate the detrimental impacts on air and water environments [5].

China is the biggest producer and consumer of distilled spirits in the world, with an average annual production of Chinese spirits reaching 1.0 × 10⁷ tons from 2015 to 2020 according to the National Bureau of Statistics of China (http://www.stats.gov.cn/english/). As the main by-product of Chinese spirits production, the yield of distillers’ grain (DG) was estimated at about 2.4 × 10⁷ tons every year because the ratio of Chinese spirits to DG is approximately 1:3 [6]. DG generally contains a substantial number of nutrient components, including starch, protein, lipid and water-soluble vitamins and so on [7]. Direct landfilling or discarding without prior treatment is still a very prevalent style of DG disposal, thus leading to environmental pollution and waste of resources [8]. Therefore, numerous studies have been carried out to explore its potential utilization value, such as protein feed, bio-organic fertilizer, chemical products, bacterial cellulose, and fuel [9–12].

Soil microorganism is the most active part of soil and serves as a crucial indicator for evaluating soil quality, which plays a vital role in nutrient transformation and material cycling [13]. The addition of organic fertilizer has been shown to significantly enhance the soil bacterial diversity and the relative abundance of phyla Proteobacteria, Chloroflexi, Firmicutes, Acidobacteria, and Planctomycetes [14]. Organic fertilization increased the rhizosphere microbial quantity and carbon source utilization and significantly changed the function of the microbial community in the rhizosphere of flue-cured tobacco [15]. Recently, there have been many studies on the effects of organic fertilizer on soil physicochemical properties and tobacco quality and yield. However, the application of DGOF and its effects on flue-cured tobacco are rarely reported. Therefore, in this study, the objectives were to investigate: (1) What are the effects of the DGOF application on tobacco yields and soil chemical properties? (2) how does the application of DGOF affect soil bacterial community composition, diversity, and the relationships between soil chemical properties and microbial communities?

Materials and methods

Experimental site information

The field experiment was conducted at the Bozhou County, Zunyi, Guizhou Province, China (106°81´E, 27°51´N). Zunyi belongs to the subtropical humid monsoon climate region. The soil type of the experimental site is yellow loam, and classified as Alfisols according to the soil taxonomy of the USA. Zunyi has an average annual precipitation of 968 mm and an average temperature of 15.0˚C with a tobacco cultivation history of over 70 years. The current tobacco cultivation areas are about 150,000 ha, accounting for 5.05% of the crop-sown areas and contributing 8.42% to the agricultural output value [16].

Field experiments designs and fertilizer application

The DGOF was provided by Zixing Organic Fertilizer Factory of Bozhou, Zunyi, China. The DGOF had the following characteristics: pH 6.9, total nitrogen 29 g/kg, total phosphorus 15 g/kg, total potassium 18 g/kg, and organic matter 632 g/kg. In the field experiment, the application rate of DGOF was 0 and 1.5 t/ha, and each treatment was repeated 3 times. Each plot had an area of 200 m² and followed a completely randomized design in the experimental field. Agronomic practices, including fertilizer application and field management, followed the local high-quality tobacco production technical specifications. A compound fertilizer (N-P₂O₅-K₂O:10–10-24) of 525 kg/ha was applied before ridging in a strip as basal fertilizer. The compound fertilizers were purchased from Migao (Zunyi) Technology Fertilizer Co., LTD. After 10 days, the round pits with a diameter of 8 cm, a depth of 20 cm, and a hole distance of 50 cm were drilled into the field ridges using a well-cellar type transplanter. On May 5, 2022, tobacco plants from seedling trays were transplanted into these well-cellar holes. The tobacco variety of Yunyan 87 was used in this study. Subsequently, a compound fertilizer (N-P₂O₅-K₂O:22–14-10) of 45 kg/ha as seedlings fertilizer was dissolved by water and sprayed on the soils close to the seedlings. A compound fertilizer (N-P₂O₅-K₂O:15–0-30) of 105 kg/ha dissolved in water was top-dressed into soils approximately 15 cm away from the plants at 7 days after transplanting seedlings, and the fertilizers (N-P₂O₅-K₂O:15–0-30) of 120 kg/ha was applied at 15 days after transplanting. After another 5 days, DGOF was applied to the well cellars and covered with soil on the organic fertilizers. The final harvest of tobacco was conducted on August 29, 2022.

Soil sample collection

Soil samples from 0 to 20 cm soil layer were collected using a hollow auger with scale at the rosette stage (Time1, June 5, 2022), at the vigorously growing stage (Time2, July 12, 2022), and mature stage (Time3, August 15, 2022). Soil samples of six points in each plot were collected and mixed into a single sample. The sampling points had a distance of 5 cm away from tobacco plants. The soil samples were placed into sterile zip-lock plastic bags, then quickly transferred to an insulated container with ice packs and transported to the laboratory as soon as possible. All fresh soil samples were sieved through a 2.0-mm mesh screen to remove fine roots and litter. About 25 g of each sample was conserved at − 80℃ until DNA extraction. The remaining soil samples were air-dried in the shade and milled to pass through a 0.85- and 0.149-mm sieve for the following analysis.

Analysis of soil chemical properties

The soil pH, Olsen P (available phosphorus, AP), organic matter (OM), total carbon (TC), total nitrogen (TN), ammonium (NH₄⁺), and nitrate (NO₃⁻) were determined. The soil pH was measured in a soil/water ratio of 1:2.5 (w/v) suspension using a pH meter (F2-Standard, Mettler Toledo, Shanghai, China). The AP was extracted with sodium bicarbonate (0.5 M) in a reciprocating shaker for 30 min, and determined by molybdenum blue method [17]. The OM was determined according to the K₂Cr₂O₇ volumetric method released by China in 1988 (GB9834–88). Soil TC and TN were measured via the dry combustion method on an elemental analyzer (Elementar Vario EL CUBE, Langenselbold, Germany). NH₄⁺-N and NO₃⁻-N were extracted by shaking 5 g of fresh soil with 20 mL of 1 M KCl for 1 h and then analyzed by a continuous flow autoanalyzer (Skalar San⁺⁺, Breda, Holland).

DNA extraction and PCR amplification

The soil DNA was extracted from 0.5 g of fresh soil samples according to the manufacturer’s protocol using the FastDNA SPIN Kit (MP Biomedicals, Santa Ana, CA, USA). The V4-V5 hypervariable regions of 16S rRNA genes were amplified using the primers 515F (5’-GTGCCAGCMGCCGCGG-3’) and 907R (5’-CCGTCAATTCMTTTRAGTTT-3’) for sequencing [18]. Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq PE300 platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database under the accession number from SRR24179538 to SRR24179555.

Data analysis

The demultiplexed paired raw sequences were quality-filtered and aligned using the DADA2 algorithm in R software to get the end product amplicon sequence variant (ASV) table [19]. Bacterial community alpha and beta diversity, principal coordinates analysis (PCoA), redundancy analysis (RDA), and PerMANOVA were analyzed using the vegan package [20] and visualized using microeco package [21] in R 4.2.3 [22]. Bacterial co-occurrence network analysis was conducted using the WGCNA package [23]. A global network based on 18 samples was constructed to identify keystone species. The Spearman’s correlation at p < 0.01 and r > 0.6 was used for network construction. Network visualization was performed using the Gephi platform [24]. Pearson correlation relationship between soil chemical properties and keystone ASVs was analyzed using the Hmisc package and visualized using the linkET package in R 4.2.3 [22].

Results

Effect of DGOF on soil properties at different sampling times

The application of DGOF significantly increased the maximum leaf length, width, and yield of tobacco (14.8%, Table 1). Adding DGOF led to a significant increase in SOM and TC across all three sampling periods (Table 2). Soil TN exhibited a significant increase in DGOF compared to CK only at Time3 (Table 2). However, soil inorganic nitrogen and pH were not significantly different between CK and DGOF across all three sampling times (Table 2).

Table 1.

Effect of DGOF application on tobacco leaf characteristics and yields

Treatments	Yield (kg/ha)	Maximum leaf length (cm)	Maximum leaf width (cm)
CK1	1800.0	72.0	28.2
CK2	1884.0	67.3	26.8
CK3	1711.5	71.1	27.6
Average (CK)	1798.5 ± 86.3 b	70.1 ± 2.5 b	27.5 ± 0.7 b
DGOF1	2080.5	74.5	30.5
DGOF2	2143.5	75.5	31.3
DGOF3	1969.5	73.9	31.3
Average (DGOF)	2064.5 ± 88.1 a	74.6 ± 0.8 a	31.0 ± 0.5 a

CK, no distillers’ grain organic fertilizer application; DGOF, applying distillers’ grain organic fertilizer. Different lowercase letters within a column indicate significant differences based on paired t-test at p < 0.05 between CK and DGOF

Table 2.

Effect of DGOF addition on soil properties at different sampling times

Sample time (t)	Treatment	pH	SOM (g kg−1)	TC (g kg−1)	TN (g kg−1)	NO3− (mg kg−1)	NH4+ (mg kg−1)	AP (mg kg−1)
Time1	CK	7.06 ± 0.11 a	26.17 ± 0.75 b	15.81 ± 0.55 b	1.35 ± 0.05 a	7.53 ± 0.51 a	10.13 ± 0.87 a	88.74 ± 6.43 b
	DGOF	7.09 ± 0.17 a	28.69 ± 0.97 a	17.29 ± 0.51 a	1.48 ± 0.11 a	9.08 ± 0.45 a	8.37 ± 0.78 a	104.48 ± 6.18 a
Time2	CK	6.65 ± 0.12 a	27.43 ± 0.62 b	16.80 ± 0.15 b	1.27 ± 0.15 a	7.40 ± 0.83 a	10.19 ± 0.62 a	73.57 ± 3.78 b
	DGOF	6.76 ± 0.10 a	29.73 ± 0.92 a	18.38 ± 0.61 a	1.44 ± 0.11 a	8.64 ± 1.48 a	11.82 ± 1.28 a	91.42 ± 3.94 a
Time3	CK	6.72 ± 0.01 a	26.66 ± 0.27 b	15.48 ± 0.35 b	1.36 ± 0.04 b	9.93 ± 0.58 a	6.55 ± 0.67 a	73.23 ± 5.95 a
	DGOF	6.86 ± 0.53 a	29.61 ± 0.51 a	18.00 ± 0.3 a	1.59 ± 0.04 a	9.41 ± 0.67 a	6.73 ± 0.16 a	77.69 ± 2.09 a

Values are means ± SD (n = 3). SOM soil organic matter, TC total carbon, TN total nitrogen, AP available phosphorus. At each sampling time, different lowercase letters indicate significant differences between CK and DGOF based on paired t-test (p < 0.05).

Diversity and composition of soils with and without the application of DGOF

A total of 20,813 ASVs were identified from the 18 soil samples, and 85.8% of shared sequences between CK and DGOF treatments were clustered into 4,309 ASVs (Figure S1). At the phylum rank, across all the soil samples, the bacterial communities were dominated by Proteobacteria (mean relative abundance, 33.57%, same below), Actinobacteriota (24.37%), Acidobacteriota (9.60%), Chloroflexi (8.01%), Fimicutes (7.26%), Planctomycetota (4.77%), and Bacteroidota (2.93%). The combined relative abundance of these seven phyla exceeded 90% (Fig. 1). The relative abundance of these dominant phyla remained similar between CK and DGOF at Time1 and Time2. However, at Time3, the relative abundance of Chloroflexi, Firmicutes, Planctomycetota, and Gemmatimonadota was higher in DGOF than in CK, while the relative abundance of Bacteroidota was lower in DGOF compared to CK (Fig. 1). At the genus rank, Pseudarthrobacter, Marmoricola, Nocardioides, Gaiella, Lysobacter, and Bacillus had a higher relative abundance across all three sampling times regardless of whether DGOF was added. DGOF addition increased the relative abundance of Rhodanobacter, especially at Time2 and Time3 (Figure S2).

Fig. 1.

The relative abundance of dominant bacterial phyla (top10) in the soils at different sampling times

One-way analysis of variance revealed no significant differences in Chao1, Shannon, and PD values between CK and DGOF (Fig. 2). However, the PCoA result showed that there were significant differences in the bacterial community structure between CK and DGOF (PerMANOVA, F = 4.05, p = 0.001). The CK and DGOF samples were separate along the PCoA1 axis, and the Time3 samples were separate from the Time1 and Time2 samples along the PCoA2 axis. However, Time1 and Time2 samples were overlapped (Fig. 3).

Fig. 2.

Soil bacterial Chao1 and Shannon values. Different lowercase letters indicated no significant difference between CK and DGOF at each sampling time based on a paired t-test (p < 0.05)

Fig. 3.

Principal coordinate analysis (PCoA) plots of bacterial communities of soils with or without DGOF addition at different sampling times

Relationship between soil properties and bacterial communities

The db-RDA analysis showed that the first two axes explained 35.9% variation in the bacterial communities (Fig. 4). Soil AP was the dominant factor driving the changes in soil bacterial communities, which can independently explain 6.68% of the variation (Table S1). This was followed by TN (6.01%), NH₄⁺ (5.94%), TC (5.60%), SOM (5.27%), NO₃⁻ (4.87%), and pH (4.46%) (Table S1).

Fig. 4.

Redundancy analysis (RDA) of soil bacterial communities and chemical properties. SOM, soil organic matter; TC, total carbon; TN, total nitrogen; AP, available phosphorus

Bacterial co-occurrence network

A global bacterial co-occurrence network was constructed using all soil samples, and sub-network for CK and DGOF treatments were compared across all three sampling times (Fig. 5a, b, and c). The addition of DGOF resulted in a more intricate microbial community co-occurrence network. This was characterized by an increased number of vertices interconnected under the same construction parameters, a higher edge count, elevated average degree, reduced average path length, heightened clustering coefficient, greater density, increased heterogeneity, and a higher value of centralization (Table S2).

Fig. 5.

Soil bacterial co-occurrence networks and their related characteristics. (a) global network; (b) sub-network for CK; (c) sub-network for DG; (d) within-module and among-module connectivity; (e) the relative abundance of keystone ASVs, different lowercase letters indicated no significant difference between CK and DGOF at each sampling time based on paired t-test (p < 0.05); (f) Pearson relationship between the relative abundance of keystone ASVs and soil chemical properties

The topology roles of nodes and their taxonomy information are depicted in Fig. 5d and Table S3. The majority of these keystone ASVs’ relative abundance was affected by the application of DGOF (Fig. 5e). Most soil chemical properties were significantly positively or negatively correlated with the relative abundance of these keystone ASVs. For instance, soil pH exhibited a significant negative correlation with ASV23272 but a significant positive correlation with ASV13787 and ASV28004. NO₃⁻ displayed a significant negative correlation with the relative abundance of ASV25556 and ASV12037. AP demonstrated a significant positive correlation with the relative abundance of ASV13787 and ASV28004. However, NH₄⁺ was not significantly correlated with the relative abundance of any keystone ASVs.

Discussion

Effect of DGOF application on soil chemical properties

As a cereal byproduct of the distillation process, distiller’s grains usually have a high content of organic matter, nitrogen and phosphorus nutrients, etc. [25]. The addition of DGOF significantly enhanced tobacco yield as well as the maximum leaf length and width, which might benefit from increased soil nutrients (Table 1). This study revealed a significant increase in SOM and TC content following the application of DGOF (Table 2). However, only at Time3, we observed a significant alteration in soil TN. A previous study has shown that the nitrogen uptake by tobacco plants mainly occurs at the early stage of tobacco growth [26]. There were no significant changes in soil inorganic nitrogen (NH₄⁺ and NO₃⁻), which may be attributed to the released inorganic nitrogen by microbial mineralization being easily absorbed by the tobacco plants. Compared with CK, soil AP was higher at the early stage of tobacco growth after DGOF addition. Organic materials such as DGOF can improve soil P availability but may have a time effect because the soil available P may be bound by soil particles at later period of tobacco growth after organic materials addition [27].

Effect of DGOF application on soil bacterial communities

Decaying organic materials provide bioavailable organic carbon and essential nutrients for soil microorganisms [28]. Microbial composition and characteristics may be altered by changes in soil environments [29]. In our study, there are approximately 15% differences in the bacterial ASVs between DGOF and CK treatments. However, at the phylum rank, soil microorganism composition is much more similar at Time1 than at Time3, indicating that it may take time for soil microorganisms to establish a new community balance. Furthermore, the addition of DGOF does not change soil bacterial Chao1 and Shannon diversity indexes (Fig. 2), suggesting that soil bacterial species around the tobacco plant roots might be at a relatively stable status, and the appearance or disappearance of species have little effect on bacterial diversities after the addition of DGOF.

The dominant bacterial phylum abundance pattern is similar between CK and DGOF treatments but slightly changed with the tobacco growth period. Proteobacteria became the most dominant phyla at Time3, probably due to the specific selection of tobacco plants to microbial communities through root exudates [30]. The relative abundance of Acidobacteriota decreased in DGOF treatment, which could be linked to the fact that this phylum is recognized as oligotrophic bacteria with lower abundance in environments rich in organic carbon [31]. At the genus level, the most significant differences in bacterial composition were observed in the relative abundance of Azotobacter and Rhodanobacter. In this study, the TN and NO₃⁻ contents were higher at Time3, and studies have shown that Azotobacter activity was negatively correlated with nitrogen levels [32]. Furthermore, Rhodanobacter has been documented to participate in nitrogen cycling and exhibit antagonistic properties against pathogens such as Fusarium solani [33]. These findings suggest that the addition of DGOF may not only influence soil nutrient dynamics but also contribute to enhancing soil health.

The soil samples showed a clear separation between DG and CK treatments in PCoA plots, indicating a difference in the bacterial community composition between these two treatments (Fig. 3). We further tested the soil chemical properties' effects on the bacterial communities using RDA (Fig. 4). The result showed that only a small portion of the variation in the bacterial communities could be explained by each of the chemical properties, but they together can explain 35.9% of the variation. Among all the soil chemical properties, SOM highly correlated with the RDA1 axis, indicating its importance in explaining soil bacterial community variation, which has been proven by many other studies [34, 35].

Effect of DGOF application on soil bacterial co-occurrence network patterns

In this study, compared with CK treatment, the bacterial community network of DGOF treatment showed a more topology characterized by increased edges, nodes, and complexities (Table S2). The increased SOM due to the addition of DGOF may strengthen the connections between bacteria [36]. Furthermore, the genus UTCFX1 was identified as both module hub and connector in the bacterial network (Fig. 5d; Table S3), and its relative abundance was higher in the CK than in the DGOF treatment. This is probably due to the addition of DGOF increasing the organic acids content, which might suppress the abundance of this particular genus related to anammox [37, 38]. The relative abundance of the connector Bryobacter was higher in DGOF than in CK treatment. Despite belonging to the Acidobacteria phylum, known for favoring oligotrophic environments, Bryobacter is strictly aerobic, and the addition of DGOF may improve soil porosity conditions [39]. Another connector in the network, the genus Skermanella, is a bacterium highly resistant to antimony, suggesting that the studied tobacco field might be exposed to antimony-contaminated conditions [40]. These keystone genera were found to be correlated with most of the tested soil chemical properties (Fig. 5f), indicating that the addition of DGOF may have significant effects on the soil microbial communities through altering soil conditions.

Conclusions

Our study demonstrated that the bacterial community in the tobacco field was affected by the addition of DGOF. However, the DGOF addition mainly affects the composition rather than the alpha diversity of the bacterial community. Proteobacteria, Actinobacteriota, Acidobacteriota, Chloroflexi, and Firmicutes are the dominant phyla in both CK and DGOF treatments. Furthermore, the difference in soil bacterial communities also increased with the tobacco growing period. Each of the soil chemical properties can explain the differences in bacterial communities between CK and DGOF treatments to a small extent, while the collective influences of them can elucidate over 30% of the observed variation. Ten ASVs were identified as keystone species in the bacterial communities, and their relative abundance was affected by the addition of DGOF through altering soil chemical properties. Our results provide fundamental insights into the bacterial communities affected by the DGOF addition in tobacco fields. Nonetheless, it's essential to recognize that our study only carried out a specific DGOF application rate. Future studies need to be conducted to explore different DGOF application rates and methods combined with the reduction of chemical fertilizers that are of great importance to the improvement of tobacco quality and production.

Author contribution

ZT and AH conceived and designed the experiments. FH and CZ were responsible for the field management and soil sampling. HH, YL and JS conducted the determinations of soil chemical properties. ZT, AH, and JL analyzed the data. ZT, AH, and JL wrote the manuscript. All authors contributed to the manuscript revision and approved the submitted version.

Funding

This research was supported by the Scientific and Technological Project “Effects of Distillers' Grains Organic Fertilizer with Humic Acid on Tobacco Yield and Quality and Its Optimization Application” (NO.202207) from the Technology Center, China Tobacco Jiangsu Industrial Co. Ltd.

Data availability

The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database with the accession number from SRR24179538 to SRR24179555.

Declarations

Conflict of interest

The authors declare no competing interests.