Screening and Isolation of Microbes for Promoting Humification of Agricultural Organic Wastes ^†

Abstract

Agroecosystems play a crucial role in carbon sequestration and reducing atmospheric CO₂ emissions by storing organic carbon in soil. Soil fertility and productivity are essential for global crop demands and depend on soil organic matter, particularly humic substances (HS). HS is crucial for soil health and carbon sequestration as it involves the carbon cycle, supplies nutrients for plants, and reduces the emission of pollutants through microbial processes for the enhancement of CO₂ sequestration. Humification is a natural process of organic matter stabilization, playing a crucial role in maintaining soil organic content and carbon storage. Laccase is used to polymerize monomeric compounds such as phenols and their derivatives into highly polymerized HS. We screened five cellulose-degrading isolates, among which three strains demonstrate lignin-degrading capabilities. WSC-7 exhibited the highest laccase activity and showed a high similarity [99.51%] to Arthrobacter defluvii based on 16S rDNA analysis. Strain LiPK-078-5 in nutrient broth medium and WSC-7 in Difco Sporulation Medium exhibited optimal catalytic activity for catechol, indicating their efficiency for aromatic polymerization of soluble organic carbon. The addition of rice husk biochar with strains LiPK-078-5, WSC-6, and WSC-7 increased organic carbon content effectively. The synthesis of humic substances in soil through microbial processes increases soil carbon sequestration and reduces greenhouse gas emissions in the environment.

1. Introduction

Human activities have increased carbon dioxide in the atmosphere rapidly from 280 ppm before the Industrial Revolution to 415.48 ppm in June 2021 [1]. Soil stores the most carbon emitted. The carbon storage in the “soil carbon pool” on the earth’s surface is as high as 250 billion tons, including about 1550 Gts (gigatons) of soil organic carbon and 950 gigatons of soil inorganic carbon. The soil carbon pool is about 3.3 times larger than that of the atmospheric carbon pool (760 Gts) and 4.5 times larger than that of the biological carbon pool [2]. The increasing rate of humus formation in the soil affects the long-term storage of carbon in the soil. Soil is a huge source of carbon emissions. The rate of soil carbon dioxide (CO₂) emissions is approximately 60 petagram (Pg)/yr, which is 10 times higher than that of fossil fuels [3]. Soil carbon emission fluxes are 6 to 10 times higher than emissions from human activities [4]. Changes in land use and inappropriate agricultural practices, such as improper farming or excessive use of chemical fertilizers, cause large soil carbon emission fluxes. Soil carbon flux management is important to mitigate global warming [2,5,6,7] and has a significant impact on biodiversity, agricultural productivity, and food security. It is also important for many countries to practice sustainable development.

Soil carbon sequestration is determined based on the total organic carbon (TOC) content in the soil. Carbon with high stability is estimated for soil carbon sequestration [8]. Through the biochemical reactions of microorganisms in the soil, organic matter is converted into a stable form that is difficult for organisms to decompose, as converted organic matter is bonded to soil minerals or is protected by micro aggregates formed by biological debris. The process usually includes the following: (1) microorganisms and their extracellular enzymes, such as laccase, manganese peroxidase, and lignin peroxidase, which decompose cellulose and lignin to form aromatic small molecules; (2) condensation with amino acids, quinones, or reducing sugars to form melanin-type compounds. The substances formed through biochemical reactions are more resistant to biological decomposition and form a stable organic carbon pool in the soil [9]. The contribution of microorganisms to the soil carbon sink depends on the interaction between the number of microorganisms, community structure and their metabolites, and soil properties such as texture, minerals, and pore distribution. The formation of humus matter in the soil is conducive to soil carbon sinks. Humus is formed by the oxidative polymerization of phenolic substances in the soil as a stable organic matter structure in the soil [10]. Microorganisms assist in the humification of biochar, which provides soil carbon sinks and makes the effect of soil carbon sequestration more stable.

In this study, we screened and isolated microorganisms to effectively promote the humification of organic matter and enhance soil carbon sequestration. The results provide a reference for future sustainable agricultural practices.

2. Materials and Methods

2.1. Collection of Soil Samples

Surface soil samples were collected from forest, organic farms, rhizosphere soil, and mature compost. The soil sampling locations included Jiadong Township, Pingtung County, Rende District, Tainan City, and Rende District, Tainan City. These soil samples were used for the isolation and screening of microorganisms which promoted the humification of organic matter.

2.2. Isolation and Purification of Strains

Collected samples were placed in sterile water and shaken at room temperature for 1 h. The supernatant was diluted to a concentration of 10⁻⁴ to 10⁻⁵ CFU/mL and spread on sterile mineral salt medium (MSM) plates with 100 mg/L of cellulose, hemicellulose, or lignin as the carbon source. The samples were incubated at 30 °C and shaken at 120 rpm. The culture solution was continuously diluted and spread on modified MSM agar plates for incubation at a constant temperature. Colonies with different morphologies and phenotypes on the agar plates were selected, streaked repeatedly on plates for isolation, and preserved on a slant agar medium.

2.3. Catechol Catalytic Reaction and Dark Reaction

To understand the humification ability of each strain on organic matter, the crude protein from each strain was used to catalyze the reaction of catechol. Then, the humification degree of organic matter was measured over time. The strains were cultured separately in nutrient broth (NB) medium and WSC-7 in Difco Sporulation Medium (DSM) medium or in a catechol catalytic reaction for 20 days. The products of the reaction were obtained on days 3, 7, 10, 14, 17, and 20 for humic substance extraction tests and for the recording of changes in the humification index. A 1 mL sample of the culture was obtained and centrifuged, and 100 µL of the supernatant was diluted to 10 mL with ultrapure water. The changes in chromophores and the formation of brown substances during the aromatization and humification processes of organic matter were measured at the absorbance at 254, 465, and 665 nm.

2.4. Determination of Laccase Activity

Laccase activity was measured by monitoring the oxidation of 1 mM of guaiacol (Hi-Media, Mumbai, India) buffered with 0.2 M sodium phosphate at pH 4.5 and 420 nm for 1 min. The reaction mixture of 900 µL contained 300 µL of 1 mM guaiacol, culture filtrate, and 0.2 M sodium acetate buffer of pH 4.5. One unit of enzyme activity was defined as the amount of enzyme that oxidized 1 µmol of guaiacol per minute. The enzyme activity was expressed in U/mL.

2.5. Bacterial Identification

Identification of the selected strains was carried out using 16S rDNA sequence analysis. The selected strains were grown in nutrient broth, and harvested cells were centrifuged to obtain cell pellets for genomic DNA extraction using the Power Soil DNA kit (MOBIO, Jefferson City, MO, USA) following the manufacturer’s instructions. PCR amplification was performed in 25 mL consisting of 10 mM of Universal 16S rRNA bacterial primers 27F (5′-AGAGTSTTGATCCTGGCTCAG-3′) and 1392R (5′-GGTSTACCTTGTSTACGACTT-3′) to amplify the gene using 10 ng of genomic DNA isolated from each strain. Polymerase chain reaction (PCR) products were visualized on a 1% agarose gel stained with ethidium bromide under ultraviolet (UV) light to confirm the presence of up to a 1350 bp band. The PCR products were purified using a kit from Ambion, (Emeryville, CA, USA) before bidirectional sequencing with primers 27F and 1392R. Sanger sequences were generated at the UCSF Genomics Core Facility using an ABI PRISM 3730xl DNA Analyzer (Life Technologies Corporation, (Emeryville, CA, USA). The PCR products were separated in 0.8% agarose gel and observed on a UV transilluminator. The collected DNA bands from the gel were purified by a QIA quick gel extraction kit (Qiagen, Germantown, MD, USA) following the manufacturer’s instructions. The purified PCR products were sequenced using an automated DNA sequencer. The partial 16S rDNA sequences were compared with the GenBank database on the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 27 August 2024) by using the Basic Local Alignment Search Tool (BLAST).

3. Results and Discussion

3.1. Isolation and Selection of Test Strains

We isolated strains from soils and composts from different regions. Seventeen strains were selected based on their activity in lignin peroxidase, laccase, and manganese peroxidase. Strains with higher enzyme activities were identified through 16S rDNA sequencing. The test strains were classified as Pseudomonas sp., Mesorhizobium sp., Cupriavidus sp., and others. The selection of test strains was based on whether the strains decomposed lignin and cellulose and exhibited laccase and/or humification activities. Five selected strains demonstrated a higher increase in the aromaticity of soluble organic carbon in soil. The functions of each test strain are described in

Table 1. 16S rRNA percent identity and functions of the selected strains.

Treatment	Similar Strain	Lignin	Cellulose	Laccase (MAX) (μg/L)
LiPK-078-5	Pseudomonas sp.	+	+	0.0504
LiPK-078-8	Pseudomonas sp.	−	+	0.0352
RHCS-1	Cupriavidus sp.	−	+	0.0573
WSC-7	Arthrobacter sp.	+	+	0.0576
WSCCS-6	Mesorhizobium sp	+	+	0.0544

. All five strains decomposed cellulose, with three strains possessing both lignin and cellulose decomposition enzymes. Strain WSC-7 exhibited the highest laccase activity, producing 0.0576 U/L of laccase in the culture medium. Based on 16S rDNA sequence analysis, strain LiPK078-5 was identical to Pseudomonas sp. LiPK078-8 was 99.93% similar to Pseudomonas sp., RHCS-1 was 99.03% similar to Cupriavidus sp., WSC-7 was 93.51% similar to Arthrobacter sp., and WSC-6 was 99.86% similar to Mesorhizobium sp.

3.2. Spectral Characterization of Test Strains

3.2.1. E2/E4

The E2/E4 coefficient is used to characterize the degree of condensation of organic molecules during the humification process, which increases as the E2/E4 value decreases [11]. The test strains were cultured in NB and DSM media at a constant temperature, and the catalytic activity of the crude protein of the strain against catechol was tested periodically. On the 20th day of culture, the E2/E4 coefficient reached a peak value, ranging from 0.49 to 1.60. The E2/E4 coefficient index, acquired by measuring the UV spectrum, is an index describing the degree of humification of organic matter. A higher E2/E4 value means that there are relatively smaller aromatic molecules—such as monomers and low-density polymers—in the organic matter and that the degree of humification is low. A lower E2/E4 value means that there are relatively larger aromatic molecules—such as those of a high polymer value—in the organic matter and that the degree of humification is higher [12]. The catalytic activity of pyrogallol in different strains varied depending on the culture medium. The strain WSC-6 cultured in NB medium and the strain WSC-7 cultured in DSM medium exhibited lower E2/E4 values. This indicates that the WSC-6 and WSC-7 strains have a higher potential for the humification of organic matter (Figure 1).

3.2.2. E2/E6

Humic substances contain conjugated double-bond systems randomly distributed in macromolecules, which are responsible for their brown color. The E2/E6 coefficient is the ratio of initially humified structures to strongly humified ones. Higher E2/E6 values for humic acids (Has) derived from soil suggest more structures of low transformation, typical for lignins [13]. Among the test strains isolated in this study, the catalytic activity of pyrogallol varies in different strains and culture media. The test strains were cultured in NB and DSM media at a constant temperature until the 20th day, and the E2/E6 ratios of each strain ranged from 0.31 to 1.26. The strain WSC-6 cultured in NB medium and the strain LiPK-078-5 cultured in DSM medium exhibited lower E2/E6 values. This indicates that WSC-6, LiPK-078-5, and WSC-7 strains have better potential for the aromatization of organic matter. LiPK-078-5 and WSC-7 strain in NB medium and the WSC-6 strain in DSM medium exhibited the best catalytic activity for catechol, indicating that these strains have the highest capability for aromatization of soluble organic carbon (Figure 2).

3.2.3. E4/E6

The E4/E6 ratio measured at the absorbances at 465 and 665 nm is used to study the HA fraction. The E4/E6 ratio is inversely related to the degree of condensation and aromaticity of the humic substances and their degree of humification and characterizes the degree of polymerization of the benzene ring C framework [14]. Lower E4/E6 values indicate higher organic matter polymerization levels. The catalytic activity of pyrogallol varied for different strains and culture media in this study. The test strains were cultured in NB and DSM media at a constant temperature for 20 days, and the E4/E6 ratios of each strain ranged from 0.32 to 0.56. The strain WSC-6 cultured in NB and DSM medium showed lower E2/E6 values. This indicates that the WSC-6 strains have better potential for the humification of organic matter (Figure 3).

4. Conclusions

The results of this study showed the significant potential of the strain WSC-6 cultured in NB medium and the strain WSC-7 cultured in DSM medium for the humification and aromatization of organic matter. These strains showed lower E2/E4, E2/E6, and E4/E6 values, indicating higher degrees of condensation, polymerization, and aromaticity. Particularly, strain WSC-6 demonstrated the most consistent performance and superior performance across different indices and culture conditions, making it a promising candidate for enhancing the humification process in organic matter treatment.