Abstract
Symbiotic bacteria in the termite gut system may play an important role in lignin degradation that can assist the subsequent saccharification process. Pseudocitrobacter anthropi MP-4, which is capable of degrading lignin components and rapidly growing on various lignin analogue dyes, was successfully screened from the gut of a wood-feeding termite Microtermes pakistanicus. Further decolorization tests with this strain showed that the strain MP-4 potentially produced some relevant extracellular enzymes to participate in lignin degradation. The removal rate of chemical oxygen demand by this strain was recorded as high as 52.1% when it was incubated in a mineral-salt medium with lignin as the sole carbon source. For the degrading process of MP-4 on lignin, it was proposed through a series of evaluations by field emission scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and pyrolysis gas chromatography/mass spectroscopy, that the lignin degradation mechanism of the strain MP-4 would primarily include the cleavage of various chemical linkages and the demethylation reactions. This resulted in a change in the S/G ratio and the disappearance of the biphenyl structure in the lignin components. Thus, these findings suggested that the strain MP-4 uniquely presented an attractive capability to deconstruct lignin components from biomass, which may be potentially valuable for a future industrial exploration.
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Biodegradation of Lignin via Pseudocitrobacter anthropi MP-4 Isolated From the Gut of Wood-feeding Termite Microtermes pakistanicus (Isoptera: termitidae)
Feng Li, Rongrong Xie, Nian Liang, Jianzhong Sun,* and Daochen Zhu *
Symbiotic bacteria in the termite gut system may play an important role in lignin degradation that can assist the subsequent saccharification process. Pseudocitrobacter anthropi MP-4, which is capable of degrading lignin components and rapidly growing on various lignin analogue dyes, was successfully screened from the gut of a wood-feeding termite Microtermes pakistanicus. Further decolorization tests with this strain showed that the strain MP-4 potentially produced some relevant extracellular enzymes to participate in lignin degradation. The removal rate of chemical oxygen demand by this strain was recorded as high as 52.1% when it was incubated in a mineral-salt medium with lignin as the sole carbon source. For the degrading process of MP-4 on lignin, it was proposed through a series of evaluations by field emission scanning electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and pyrolysis gas chromatography/mass spectroscopy, that the lignin degradation mechanism of the strain MP-4 would primarily include the cleavage of various chemical linkages and the demethylation reactions. This resulted in a change in the S/G ratio and the disappearance of the biphenyl structure in the lignin components. Thus, these findings suggested that the strain MP-4 uniquely presented an attractive capability to deconstruct lignin components from biomass, which may be potentially valuable for a future industrial exploration.
Keywords: Lignin degradation; Termite gut symbionts; Pseudocitrobacter anthropi; Microtermes pakistanicus
Contact information: Biofuels Institute of Jiangsu University, School of Environmental and Safety Engineering, Jiangsu University, No. 301 Xuefu Road, Jingkou District, Zhenjiang 212013, Jiangsu Province, China; *Corresponding authors: jzsun1002@ujs.edu.cn; dczhucn@hotmail.com
INTRODUCTION
Lignin is considered one of the most abundant renewable and natural aromatic compounds that can be potentially applied as an alternative petroleum-derived feedstock for various bio-based and value-added chemicals. Despite its abundance, the utilization of lignin has been particularly challenging due to its recalcitrant structures (Zhao et al. 2016). Moreover, lignin is a complex amorphous phenylpropanoid polymer that is mainly synthesized from three monomeric precursors (p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units), and those monomers are incorporated in the form of various types of chemical bonds or interunit linkages such as β–O–4, α–O–4/β–5, β–β, 4–O–5, and 5–5 (Munk et al. 2015).
Despite the fact that lignin is highly recalcitrant towards biological degradation, many nature biomass conversion systems, including white rot fungi, bacteria, and wood-feeding termites, are indeed capable of utilizing lignin through the secretion of various lignolytic enzymes (Xie et al. 2014). The microbial degradation of lignin has been well studied in the fungal systems, but it is a real challenge for fungal protein expression and its genetic manipulation. Compared with conventional fungal systems, bacteria have in recent years attracted increased attention due to their unique advantages, such as rapid growth, stable adaptability in a wide range of environmental conditions, and a relatively easygoing genetic manipulation (Bugg et al. 2011; Ma et al. 2016).
Within the past few years, a variety of investigations have been conducted on the role of bacteria in lignin degradation. For example, the bacteria Novosphingobium sp. B-7, Comamonas sp. B-9, Pandoraea sp. B-6, and Cupriavidus basilensis B-8 were successfully isolated from the eroded bamboo slip steeping fluid derived, and those bacteria were identified to have a remarkably higher laccase and peroxidase activity, as well as the ability to aerobically degrade kraft lignin (Chen et al. 2012; Shi et al. 2013a,b; Chai et al. 2014). Among bacteria lignin degraders, Sphingomonas paucimobilis SYK-6 is considered one of the best characterized lignin-degrading bacteria that can utilize a wide variety of lignin-related biaryls and monoaryls as a sole carbon source, and it contains the metabolic pathways for deconstruction of the β-aryl ether, biphenyl, and other components of lignin (Kamimura et al. 2017). Enterobacter lignolyticus SCF1 has the ability to use lignin in both assimilatory and dissimilatory pathways (DeAngelis et al. 2013). The extremophile Bacillus ligniniphilus L1 was able to degrade lignin under the alkali and salty conditions, which was beneficial for the treatment of wastewater containing lignin such as black liquor from the papermaking industry (Zhu et al. 2017). Those reports indicated that those novel lignin-degrading enzymes are mainly to be identified as laccases, lignin peroxidase, glutathione-S-transferase, ring cleaving dioxygenases, monooxygenases, quinone oxidoreductase, esterases, and so on. Compared to lignolytic fungal systems, bacterial systems have been found to be less oxidatively powerful but may provide a plentiful source for illustrating novel accessory enzymes that could act synergistically with the major oxidative enzymes to trigger and uncap various sites (Brown and Chang 2014). Therefore, the exploration of novel bacteria with higher delignification efficiency or a unique degrading strategy may open a new window to learn from a natural mechanism for a better lignin deconstruction process (Sun et al. 2014).
Termites can dissimilate 74% to 99% of the cellulose and 65% to 87% of the hemicelluloses, leaving the modified lignin residues as feces (Li et al. 2017). To date, previous investigations have demonstrated that the fungus-growing termites, as a unique type of wood-feeding termite, have developed a viable mechanism to degrade lignocellulose, particularly with important and complementary assistance from their gut symbionts (Geng et al. 2018). The different types of cellulolytic, hemicellulolytic, and lignolytic symbionts, especially for some symbiotic bacteria interacted with biomass degradation processing in its digestive system, have been successfully isolated and identified (Cibichakravarthy et al. 2017; Tikhe et al. 2017). As a growing research interest, this type of fungus-growing termite may have evolved a unique group of bacterial symbionts to cope with the lignin recalcitrance in their food, which is assumed to be largely dependent on their environment and various food resources. The unique symbiotic relationship between termite hosts and their gut bacteria, which are potentially associated with lignin component depolymerization, remains lacking and unclear in the existing literature. Hence, to better understand a possible function of those involved bacterial symbionts towards lignin component deconstruction, it is essential for the relevant gut bacterial composition profiles, functions, as well as their potential applications for the biorefinery industry to be revealed.
The aim of this investigation was to isolate some novel lignolytic bacterial strains from the gut of a fungus-growing termite species, Microtermes pakistanicus, and further to understand the involved lignin degrading mechanism for these bacterial symbionts assisted with a variety of analyzing tools to characterize their physiochemical properties.
EXPERIMENTAL
Materials
Chemicals and medium
Alkali lignin (AL; catalog #370959), Azure B (AB), Ramazol Brilliant Blue R (RBBR), Methylene blue (MB), and Tetramethylammonium hydroxide (TMAH) were purchased from Sigma-Aldrich (St. Louis, USA). Phenol red (PR) and other chemicals in this study were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). The modified mineral salt medium (MSM) used in this study contained (g L-1 deionized water): K2HPO4, 1; KH2PO4, 1; (NH4)2SO4, 2; MgSO4, 0.2; CaCl2, 0.1; FeSO4, 0.05; MnSO4, 0.02; CuSO4, 0.001; ZnSO4, 0.001; and tryptone, 0.05. The medium was sterilized by autoclaving for 15min at 115 °C.
Methods
Isolation and identification of lignin-degrading bacteria from the termite gut
The termites were collected from a decayed tree stump in the tropical rainforest of the Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Xishuangbanna, China. A part of the termite samples was then identified in terms of the sequence of mitochondrial COII gene combined with the soldier sample’s morphology, which has been confirmed as a type of higher termite species, Microtermes pakistanicus.
Simultaneously, 50 worker termites of the M. pakistanicus species were surface sterilized by dipping them in 75% ethanol twice, followed by sterile water, and then they were dried on a piece of clean filter paper for gut system separation. After a dissection process for the termite gut, the internal contents in their gut system were suspended and homogenized in sterile phosphate buffer solution (PBS, pH 7.2, 1 mL). The homogenate samples were made in a serial dilution up to 10-8, and then 100 µL of the final diluted sample was spread on the AL-MSM agar medium. The plates were kept at 30 °C for 3 days and then observed for bacterial growth. The distinct colonies were picked up and purified via repeated spreading (at least three times) on the AL-MSM agar medium, which then were preserved for further study.
The DNA extraction of the bacterial strain, MP-4, was performed using the Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) according to the manufacturer’s instructions. Amplification of the 16S rRNA gene sequence was performed as described before (Weisburg et al. 1991), and the universal primers 27F and 1492R were used for the polymerase chain reaction. The purified DNA was then packed and sent to Sangong Biotech (Shanghai, China) for sequencing. The sequenced results were compared with the sequences deposited in the GenBank public database from the National Center for Biotechnology Information (NCBI).
Dye decolorization and lignolytic enzyme activity assays
The decolorization of the dyes as an indicator for the production of lignin-degrading enzymes was assessed in solid phase assays. Lignolytic indicator dyes were added to MSM using the methods described by Hooda et al. (2015). The following dyes were tested: PR, AB, RBBR, and MB at concentrations of 100, 50, 50, and 100 mg L-1, respectively. Bacteria were streaked onto dye-containing agar plates and incubated at 30 °C. The plates were observed for growth and decolorization zones after 5 days. Isolates that decolorized any of the dye were screened as positive for the lignolytic enzyme and selected for further lignin degradation studies. Laccase (Lac), lignin peroxidase (LiP) and Mn peroxidase (MnP) assay were performed by the methods as described by Shi (2013 a).
Measurement of bacteria growth and chemical oxygen demand (COD) reduction
Bacterial growth was determined by measuring the culture optical density (OD) at 600 nm with a UV-1200 spectrophotometer (Mapada Instruments, Beijing, China) using an uninoculated medium as a blank. The control and cultured samples were centrifuged at 12,000 rpm for 5 min to collect the supernatant, and then the COD was measured using a 5B-3B type COD analyzer (Lianhua Tech Co., Ltd., Beijing, China). The COD concentration was chosen as the decomposition index to characterize the lignin degradation (Hu et al. 2017). The COD removal ratio was calculated using Eq. 1:
CODremoval (%) = (InitialCOD － FinalCOD) / InitialCOD (1)
Biodegradation of AL
A single colony of MP-4 strain was inoculated into 50 mL of Luria broth (LB) medium and cultivated at 30 °C by shaking at 150 rpm until the OD600 of the inoculums reached approximately 1.0. Then, 2 mL of cells were centrifuged and washed twice with sterile PBS and then were re-suspended with 1 mL AL-MSM medium. The re-suspended cells were inoculated into 200 mL of sterile lignin fermentation medium (AL-MSM, pH 7.0 to 7.2). The fermentation was carried out at 30 °C with a shaking speed of 180 rpm for 7 days.
Analytical Techniques
Field emission scanning electron microscopy (FE-SEM) observation
For the FE-SEM analysis, the control and treated samples were frozen at -80 °C and vacuum-dried into powder. Then, the dried samples were fixed on the double-sided carbon tape, coated with gold-palladium alloy using a JFC-1600 auto fine coater (JEOL Co., Tokyo, Japan), and examined with a JSM-7800F scanning electron microscope (JEOL Co., Tokyo, Japan) operated at an accelerated voltage of 15 kV and the current emission of 84. 5 μA.
Fourier transform infrared (FTIR) characterization
The functional group of characteristics of the control and treated lignin samples were examined by a Nicolet nexus 470 FTIR spectroscope (Thermo Nicolet Corp., Boston, USA). The freeze-dried lignin samples were mixed with KBr at a ratio of 1/100 (w/w) and then milled to a mesh size of 0.1 mm. The powdered samples were made into slices by using a pellet maker (Jingtuo Instrument Science and Technology Ltd., Tianjin, China). The prepared pellets were scanned in the spectra range 4000 to 400 cm−1, and the spectra were recorded using 64 scans per sample at 4 cm−1 resolutions. A pellet prepared with an equivalent quantity of pure KBr powder was used as a background.
Thermogravimetric (TG) analysis
The thermogravimetric/derivative thermogravimetric (TG/DTG) experiments were performed with a TGA 4000 thermogravimetric analyzer (Perkin-Elmer Co., Ltd., Waltham, USA) for the slow pyrolysis of bacteria-treated lignin, as well as a control sample. Approximately 2 mg of the lignin sample were placed into the pan of the TGA microbalance and then were heated from 50 to 800 °C at a heating rate of 20 °C min-1 under low nitrogen of 20 mL min-1. Repeated experiments showed that the TG curves had good reproducibility.
Pyrolysis gas chromatography/mass spectroscopy (GC/MS) analysis for lignin substructure degradation
Pyrolysis-GC/MS was performed as modified by Ke et al. (2013). The pyrolysis processes were performed with a pyrolysis autosampler (model PY-2020Id; Frontier Laboratories, Fukushima, Japan) connected to a QP2010 Ultra GC/MS system (Shimadzu, Kyoto, Japan) equipped with a UA-30M- 0.25F capillary column (30 m in length, 250 µm in inner diameter, and 0.25 µm in film thickness) (Frontier Laboratories, Fukushima, Japan). Approximately 1 mg of each sample (raw lignin and treated lignin) was placed into a quartz tube and mixed with 5 µL of TMAH (25% in methanol, w/w), and then heated up to 610 °C for 30 s. The column used helium (99.999% purity) as the carrier gas (1 mL/min). The temperature program of the GC oven was programmed from 40 °C (hold 1 min) to 280 °C at 8 °C per minute and then held at this temperature for 5 min. The mass spectrometer was operated in electron impact (EI) mode at 70 eV, and the ion-source temperature was 230 °C. The compounds were identified by comparing their GC retention times and mass spectra with the NIST library and reported in the literature. The integration of the peak areas was calculated for each lignin pyrolysis product. The summed areas of the relevant peaks were normalized to 100% and the data were expressed as percentages.
RESULTS AND DISCUSSION
Isolation and Identification of Lignin Degradation Bacteria From the Termite Gut
The lignin-degrading bacteria were isolated in MSM plates with alkali lignin as the sole carbon source. A total of 40 individual bacterial colonies were successfully isolated from the gut of M. pakistanicus and one of the colonies, named as MP-4 (GenBank accession: MF455197.1), presented a noticeable and outstanding degradation ability on the lignin substrate, which was then selected for further investigation regarding its degradation mechanism and characteristics. The 16S RNA sequencing and BLAST analysis showed that the strain MP-4 possessed more than 99% identities with the Pseudocitrobacter anthropi strain C138. In the neighbor-joining phylogenetic tree (Supplementary, Fig. S1), the strain MP-4 fell inside the cluster comprising members of the genus Pseudocitrobacter. Interestingly, as a new genus, the genus Pseudocitrobacter included only two species that were uniquely isolated from the fecal samples of hospitalized patients (Kampfer et al. 2014). However, a similar bacterial strain, MP-4, was the first to be isolated from the gut of a wood-feeding termite and uniquely presented a competitive ability to degrade lignin, which may indicate some distinctive evolution pathway to deal with different environments. As a matter of fact, with this bacterial strain, MP-4, its lignolytic potential is still largely unexplored.
Decolorization Performance with Lignolytic Indicator Dyes and lignolytic enzymes
To confirm that the strain MP-4 was able to produce lignolytic enzymes, the evaluations for a potential decolorization reaction were carried out in an agar plate with one of the four lignin-mimicking dyes, PR, AB, RBBR, or MB as indicators (the structure of dyes given in Fig. S2). The results showed that all four dye substrates were effectively decolorized by the strain MP-4 (Fig. 1). Further, the decolorization reactions with AB, MB, and PR dyes are actually applied to evaluate a potential peroxidase activity (Demidova and Hamblin 2005; Bandounas et al.2011; Yadav and Chandra 2015), while RBBR dye is more reliably used for a laccase promoted activity (Kiiskinen et al. 2004). Therefore, it can be inferred that the strain MP-4 potentially secreted the enzymes of laccase and peroxidase to degrade lignin components. Dye-decolorizing peroxidases (DyPs) in bacteria play a fundamental role in lignin degradation, which are equivalent to lignin peroxidase (LiP) and Mn peroxidase (MnP) in fungi (Bugg et al. 2011). It was speculated that the strain MP-4 might produce laccase and DyPs to support the degradation of lignin. However, it still needs to be further confirmed with a gene sequencing evaluation and an enzyme activity assay.
To better assess the lignolytic potential, three major enzymes (manganese peroxidase, lignin peroxidases and laccases) needed to be quantitatively evaluated. The results of Lac and LiP activity are shown in Fig. S3. MnP activity was not detectable during the 7-day fermentation. Lac and Lip reached maximum activities of 37.66U/L and 86.56 U/L in the lignin cultures, respectively. Compared with previous reported (Ma et al. 2016; Shi et al. 2013a), the strain MP-4 had a lower Lac and LiP activity, and if the enzyme activity is enhanced, optimal fermentation conditions need to be further investigated.
Fig. 1. Agar plate-based decolorization zones around the bacterial colony indicate the degradation process by certain lignolytic enzymes from MP-4 on the tested lignin analogues: a) MB, b) AB, c) PR, and d) RBBR
Bacterial Growth and COD Reduction
To determine the COD reduction and growth of strain MP-4, the bacterial cells were inoculated in a modified MSM medium and cultured for 7 days. The growth curve showed that there was a rapid growth of strain MP-4 at an early stage and until the 4th day; the cells reached a stationary phase between 5 to 7 days, as shown in Fig. 2. Because lignin as the sole organic source contributed to the entire COD load in liquid AL-MSM, the COD reduction could be directly correlated with its lignin degradation. The COD reduction curve indicated that the reduction of COD was not distinct until the 3rd day and then gradually reached its maximum reduction rate at 52.1% after 7 days of growth, which suggested that the degradation of lignin mainly occurred in its growth stationary phase (between 3 to 7 days). Previous reports have showed that the removal rate of COD of strain Cupriavidus basilensis B-8 and Comamonas sp. B-9 were 55.6% and 32%, respectively, with lignin as a single carbon source, after 7 days of fermentation. Similarly, the authors’ results indicated that the MP-4 strain also exhibited a fast growth and efficient degradation rates without any other added carbon sources. These results suggested that the strain MP-4 may demonstrate a potential value in the biorefinery industry.