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Depolymerization and decomplexation of soil humic acids by an Antarctic Pseudomonas strain
Korean J. Microbiol. 2023;59(4):252-258
Published online December 31, 2023
© 2023 The Microbiological Society of Korea.

Dockyu Kim1* , Ui Joung Youn1, Eungbin Kim2, and Hyoungseok Lee1

1Division of Life Sciences, Korea Polar Research Institute, Incheon 21990, Republic of Korea
2Department of Systems Biology, Yonsei University, Seoul 03722, Republic of Korea
Correspondence to: *E-mail: envimic@kopri.re.kr; Tel.: +82-32-760-5525; Fax: +82-32-760-5509
Received September 19, 2023; Revised October 4, 2023; Accepted October 5, 2023.
Abstract
With recent rapid warming in the maritime Antarctic, the susceptibility of soil organic matter to microbial decomposition is an emerging topic related to CO2 emission and vegetation changes. Soil humic acids (HA) enter the Antarctic ecosystems as small-soluble organic compounds resulting from microbial decomposition. Antarctic and commercial HAs were incubated with Antarctic Pseudomonas sp. PAMC 29040 with an esterase activity and structural changes in the resulting HA samples were then investigated. Gel permeation chromatography revealed the depolymerization of the large molecular fractions of both Antarctic and commercial HAs. Solid-state 13C-nuclear magnetic resonance spectroscopy showed an increase in the aromaticity degree which can be attributed to the increased intensity of aromatic-C signals upon the cleavage of interior C–C and C–O–C linkages (e.g., decomplexation by esterase activity), while the Fourier-transform infrared spectroscopy did not provide any information on the changes in the functional groups, such as Ar–OH and Ar–COOH, or bonding patterns in the HAs. To the best of our knowledge, this is the first report showing direct evidence of HA decomposition capacity by soil bacteria in an Antarctic terrestrial ecosystem.
Keywords : Pseudomonas, Antarctic tundra, bacterial degradation, humic substances, structural changes
Body

Humic substances (HS) are supramolecular polyaromatic compounds constituting the largest proportion (60%–80%) of soil organic matter (SOM). HS are formed by the spontaneous condensation reactions between the decomposition products of mainly lignocellulosic plant material and are thus considered as modified lignin. In particular, humic acids (HA), which include aromatic and aliphatic compounds such as amino acids and polysaccharides, are the major extractable component of HS. They possess two major functional groups, hydroxyl (Ar–OH) and carboxyl (Ar–COOH), on the aromatic rings, which are known to stimulate plant growth (García et al., 2019) and can possibly affect vegetation development and succession.

Some portion of the ice-free coastal region of the Barton Peninsula on King George Island, maritime Antarctic, is covered with various mosses and lichens but only two Antarctic vascular plants. With the recent rapid Antarctic warming, the coverage and species diversity of lichens and mosses have significantly increased and the vascular plants are expanding their habitats (Kim et al., 2016). The two vascular plants are small in population size, and the dominant mosses and lichens lack lignin, which constitutes a major structural portion of typical HA. Therefore, the Antarctic tundra vegetation provides soil with organic material with higher amounts of aliphatic compounds mostly derived from these mosses, lichens, and vascular plants. Lower amounts of aromatic tannins and flavonoids from lichens and mosses are incorporated into the humification process (Abakumov et al., 2022). Rencoret et al. (2021) recently reported that lignin-like fractions isolated from several temperate mosses are derived from flavonoids. Therefore, the resulting Antarctic soil HAs are mainly composed of low lignin content materials, which is common among those from the Arctic and Antarctic tundra regions (Abakumov and Alekseev, 2018). Our group has previously reported that natural Antarctic HA (KS1-3-HA) can enhance the growth and photosynthesis of Ceratodon purpureus, one of the dominant mosses in the Antarctic flora (Byun et al., 2021). Spectroscopic analysis results revealed that KS1-3-HA and commercial HA (SiA-HA; Sigma-Aldrich, Cat. No. 53680) share similar compositional and structural properties.

The susceptibility of HA to microbial decomposition owing to their intrinsic conformational flexibility is an emerging topic related to the rapid warming and subsequent vegetation changes in the maritime Antarctic region. HA are decomposed by soil microbes (fungi and bacteria) that secrete diverse extracellular enzymes. Remarkably, Proteobacteria, one of the major bacterial phyla in Antarctic tundra soils, plays an important function in the decomposition of Antarctic HA and other refractory aromatic compounds (Kim et al., 2022; Pradel et al., 2023). Bacterial decomposition results in smaller humic fractions with more exposed functional groups which are more soluble than the larger fractions, diffusible in water and easily absorbed by plants.

The microbial HA decomposition has been conducted mostly with various bacterial communities using microcosm incubation or field observation methods, and the HA decomposition capacity at a single cell level has been rarely evaluated. This study reports HA transformation using a single bacterial culture of the Pseudomonas strain (Proteobacteria). The changes in the structure and composition of the transformation products were investigated. The in situ capacity of individual bacteria to decompose HA was also evaluated.

Materials and Methods

1-Naphthyl acetate (NAC) transformation by PAMC 29040

The Antarctic soil bacterium, Pseudomonas sp. PAMC 29040 (= KCTC 72094), was isolated from organic soil (KS1-3) on Kaya Hill, located on King George Island, maritime Antarctic (Fig. 1A and B) and its HA-degradative pathway has been previously proposed (Kim and Lee, 2019). PAMC 29040 was grown in a mineral salts basal (MSB, 400 ml) medium with glucose (5 mM) for 3 days at 25°C under shaking, and the cells were harvested and washed with water by centrifugation (8,000 × g for 15 min). The pellet was transferred to an Erlenmeyer flask (250 ml) containing MSB (40 ml) with NAC dissolved in dimethyl sulfoxide to a final concentration of 0.05% (w/v). The control was prepared without cell pellet inoculation. The flasks were incubated with shaking at 25°C for 5 min, and the transformation product (1-naphthol, NOH) owing to the esterase activity of PAMC 29040 was monitored using high-pressure liquid chromatography (HPLC).

Fig. 1. (A) Location of the Kaya Hill on the Barton Peninsula, King George Island.
(B) Photos of the sampling site of humic substances (HS)-rich soil (KS1-3) and the lichens and mosses which cover the surface area.

HA transformation by PAMC 29040

PAMC 29040 was grown in an MSB medium (40 ml) with glucose (5 mM) for 2 days at 25°C with shaking, and the cells were harvested by centrifugation. The pellet was transferred to an Erlenmeyer flask (250 ml) containing MSB with 4.5 ml of either KS1-3-HA or SiA-HA to a final concentration of 0.9% (w/v). The control was prepared without cell pellet inoculation. Each HA stock solution was prepared by dissolving the HA powder in distilled water (10% w/v) and the solution was left under stirring for 24 h. The undissolved fraction was removed by centrifugation (10,000 × g for 10 min). The flasks were incubated with shaking at 25°C, and the structural changes in the HA owing to the degradative activity of PAMC 29040 were analyzed using different chromatographic and spectroscopic techniques: gel permeation chromatography (GPC), Fourier-transform infrared (FTIR) spectroscopy, and solid-state 13C-nuclear magnetic resonance (13C-NMR) spectroscopy.

Structural characterization of the HA transformation products

After an 11 day-incubation for the HA transformation by PAMC 29040, a small portion (0.7 ml) of the culture was centrifuged (10,000 × g for 10 min). The supernatant was filtered through a hydrophilic membrane (0.2 µm), and the filtrate (10.0 μl, ~9.0 mg/ml) was separated using Shodex OHpak SB-G 6B guard (6.0 mm ID × 50 mm length), SB-804 HQ (8.0 mm × 300 mm), and SB-805 HQ columns (8.0 mm × 300 mm) tandemly connected to an Agilent Technology 1200 HPLC. The flow rate of the mobile phase (degassed water) was 0.4 ml/min, and the GPC eluates were examined with a diode array detector at a wavelength of 254 nm, which is representative of the absorption of aromatic moieties present in HAs (Deflandre and Gagné, 2001; Leenheer and Croue, 2003).

After an incubation for 11 days with shaking, the culture (50.0 ml) was centrifuged (10,000 × g for 10 min) to eliminate the cells. The solution was acidified to a pH of 2.0 with HCl (5 N) to extract HA from the supernatant. The insoluble HA fraction was separated by centrifugation (10,000 × g for 10 min) and freeze-dried. To investigate the changes in functional groups and chemical bonding, the FTIR spectra of the KS1-3-HA and SiA-HA powders were recorded over the range of 400–4,000 cm-1 (spectral resolution of 4 cm-1) using a Bruker Vertex 80v FTIR spectrometer with the KBr disc method.

The one-dimensional solid-state cross-polarization magic-angle-spinning 13C nuclear magnetic resonance (13C-CP/MAS NMR) spectra of the powdered HAs, which were prepared using the same procedures as for FTIR, were measured using a Bruker Avance 500 MHz system. The aromaticity degree (AD) was used to describe the extent of changes in the aromatic and aliphatic carbon connectivity in the HAs. The areas of the 0–48 (alkyl-C), 48–105 (protein and anomeric-C), and 105–165 ppm (aromatic-C and phenolic-C) regions were integrated, and the AD was calculated using the equation (105–165)/(0–105) (Pizzeghello et al., 2020).

Results and Discussion

NAC degradation by PAMC 29040 esterase activity

Using HPLC, the HA degradative ability of PAMC 29040 was examined against a bi-aromatic NAC with one ester linkage between the aryl-OH and -COOH (Fig. 2A), which is one of the most prevalent linkages in biodegradable biopolymers such as lignin and HA. The results (Fig. 2B) revealed that PAMC 29040 was able to degrade NAC to NOH and acetate possibly by esterase activity. The HPLC peak of the product NOH appeared shortly after the addition of the substrate NAC to the PAMC 29040 cell suspension, with a small amount of NAC still remaining, while the concentration of NAC in the no-cell control, rarely decreased during the same time indicating that PAMC 29040 secreted esterase(s) to cleave the ester linkage to produce both aryl-OH and -COOH compounds.

Fig. 2. (A) Degradative route for NAC catalyzed by esterase.
(B) HPLC elution profile of metabolites formed after NAC incubation with the Antarctic bacterial strain Pseudomonas sp. PAMC 29040. The transformation reaction was performed for less than 5 min.

HA transformation by PAMC 29040

After incubation for HA transformation, a small portion of the culture was centrifuged and the subsequent supernatant was analyzed by GPC to investigate the changes in the molecular weight distribution. The GPC elution profile was divided into three nominal fractions based on the elution order: high (0–40 min), medium (40–50 min), and low (50–60 min) molecular weight. For KS1-3-HA from the PAMC 29040-inoculated test sample, the peak area at retention times of 15–30 min shifted slightly later compared to that of the control (without cells), with a slight increase in the peak area at 40–50 min (Fig. 3A), which is an indicative of the depolymerization of the high-molecular-weight HA fraction to form a lower molecular weight fraction owing to bacterial decomposition. For SiA-HA, the peak area of the high-molecular-weight fraction at 15–30 min significantly decreased compared to that of the control, indicating that soil bacteria can decompose HAs from different environmental sources if they have similar structural and compositional properties (Fig. 3B).

Fig. 3. Gel permeation chromatograms for changes in the molecular mass distribution of (A) Antarctic KS1-3-HA and (B) commercial SiA-HA after incubation with the Antarctic bacterial strain Pseudomonas sp. PAMC 29040.

After incubation of HAs with PAMC 29040, the FTIR spectral pattern of KS1-3-HA was barely different from that of the control regarding the following major functional groups (Fig. 4A):1,475–1,600 cm-1 for Ar–C=C, 1,600–1,725 cm-1 for C=O of R–COO–R′ and R–CO–R′, and 3,000–3,500 cm-1 for R–OH and R–COOH (Byun et al., 2021). Furthermore, the SiA-HA spectra of PAMC 29040-treated and control samples were almost identical after incubation (Fig. 4B). Consequently, FTIR spectroscopy could not distinguish small structural changes in the HA functional groups after the transformation reaction using PAMC 29040.

Fig. 4. FTIR spectra of (A) Antarctic KS1-3-HA and (B) commercial SiA-HA after incubation with the Antarctic bacterial strain Pseudomonas sp. PAMC 29040.

Unlike the FTIR spectra, the NMR spectra can provide detailed information about the molecular structure of high-molecular-weight amorphous materials such as HA. Therefore, 13C-NMR spectroscopy was used to detect the structural changes in HA owing to the degradative activity of PAMC 29040. The NMR spectra of powdered HAs were measured and the AD was calculated. The AD value (0.41) of KS1-3-HA from the test sample was approximately twice that of the control (0.22) (Fig. 5A), while that of SiA-HA (4.18) increased by four times (1.02) (Fig. 5B). The aromaticity of the HA molecules increased, as indicated by the increase in the NMR signals in the 105–165 ppm region, which can be attributed to the increase in aromatic-C signals originating from the cleavage of C–C and C–O–C linkages. This result is consistent with the results of a previous study on the structural characterization of two different molecular size-HS fractions, high (> 3,500) and low (< 3,500); a negative correlation was found between the HS size and aromatic-C content, whereas positive correlations were found between the HS size and carbohydratic-C and aliphatic-C contents (Pizzeghello et al., 2020).

Fig. 5. Solid-state 13C-NMR spectra of (A) Antarctic KS1-3-HA and (B) commercial SiA-HA after incubation with the Antarctic bacterial strain Pseudomonas sp. PAMC 29040.
The areas of the 0–105 and 105–165 ppm regions were integrated, and the ratio of (105–165) to (0–105) is shown below the x-axis.

Soil fungi have been well characterized to decompose lignin (a model compound for studying HA-degradative enzymes and pathways) using lignin peroxidases, laccases, and manganese peroxidases. These enzymes can break down the complex structure of lignin into simpler compounds, and the resulting mono- and bi-aromatic compounds can be further degraded by bacteria. Remarkably, bacterial peroxidases (Tian et al., 2016), laccases, laccase-like enzymes (Granja‐Travez et al., 2018; Park et al., 2021), and beta-etherases (Husarcíková et al., 2018) have been detected in various lignin- and/or HA-degrading bacterial genera and are assumed to cleave C–C and C–O–C linkages within lignin. Pseudomonas sp. PAMC 29040 was able to grow on HA as a sole carbon and energy source. The whole genome was sequenced and used to propose an HA-degradative pathway and search for the catalytic enzymes, such as dye-decolorizing peroxidase, vanillin dehydrogenase, vanillate O-demethylase, and protocatechuate 3,4-dioxygenase (Kim and Lee, 2019). Noteworthily, we could detect one gene (GenBank ID, RUT39955; 201 amino acids; Sec signal peptide with 21 aa) as a potential arylesterase with high similarity to GDSL hydrolase family of serine esterases and lipases that possess a broad substrate specificity. It could play a role as the extracellular esterase catalyzing NAC degradation and subsequent HA decomposition.

Overall, combined with the fact that PAMC 29040 can grow on Antarctic soil HA and degrade bi-aromatic NAC to mono-aromatic and simpler acetate, the spectroscopic data presented here rigorously corroborate the capability of PAMC 29040 to decompose HAs. Until recently, the evidence that bacterial enzymes play a more significant role in HA decomposition than fungal lignin-degrading enzymes has been insufficient and ambiguous. HA decomposition is complicated owing to the multiple microbes and enzymes involved; however, synergistic decomposition by a fungal and bacterial consortium seems to occur in natural environments, including the cold Antarctic tundra soils. Smaller and more water-soluble humic fractions produced through microbial decomposition should be examined for their ability to stimulate plant growth and nutrition. Studies at a single cell level are essential for unraveling the complexities of HA decomposition, although such experimental approaches inevitably have limits for the prediction of the metabolic potential of entire bacterial communities. Furthermore, considering that the maritime Antarctic regions are under rapid warming, this study contributes to the urgent need of understanding the role of bacteria in HA decomposition in the vulnerable Antarctic terrestrial ecosystems.

적 요

해양성 남극권에서 최근의 급격한 온도상승과 더불어, 토양 유기물의 미생물 분해는 CO2 대기방출과 식생변화와 직접 관련되어 있는 새로운 연구 주제이다. 남극 식생 토양에 존재하는 천연고분자 화합물인 부식산(humic acids, HA)은 미생물에 의해 수용성의 작은 유기물들로 분해되어 육상생태계로 공급되고 있다. Esterase 활성을 보유한 남극토양 유래의 Pseudomonas sp. PAMC 29040를 이용하여 남극토양 HA와 상업용 HA의 미생물적 분해를 유도한 후, 분해산물의 다양한 구조변화를 분석하였다. Gel permeation chromatography는 두 종류 HA 내 고분자 부분에서의 저분자화 현상을 확인하였다. 고체상 13C-nuclear magnetic resonance spectroscopy에 의해 분해산물의 aromaticity degree 증가가 분석되었는데, PAMC 29040이 분비하는 esterase를 포함하는 가수분해효소들에 의해 HA 내부 C–C 혹은 C–O–C 결합의 절단으로 인해 aromatic-C 신호가 증가한 결과로 판단된다. 그러나, fourier-transform infrared spectroscopy 분석에서는 HA 내 작용기들 (aromatic–OH 혹은 aromatic–COOH) 혹은 화학 결합 부위에서의 작은 변화를 확인하지 못했다. 현재까지 이 연구 결과는 남극 육상생태계에 서식하는 토양 세균에 의한 HA 미생물적 분해를 증명하는 첫 사례이다.

Acknowledgments

This work was supported by Korea Polar Research Institute (KOPRI) grant funded by the Ministry of Oceans and Fisheries (KOPRI PE23130).

Conflict of Interest

The authors declare no conflict of interest.

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