Isolation and characterization marine bacteria capable of degrading lignin-derived compounds.

Lignin, a characteristic component of terrestrial plants. Rivers transport large amounts of vascular plant organic matter into the oceans where lignin can degrade over time; however, microorganisms involved in this degradation have not been identified. In this study, several bacterial strains were isolated from marine samples using the lignin-derived compound vanillic acid (4-hydroxy-3-methoxybenzoic acid) as the sole carbon and energy source. The optimum growth temperature for all isolates ranged from 30 to 35°C. All isolates grew well in a wide NaCl concentration range of 0 to over 50 g/L, with an optimum concentration of 22.8 g/L, which is the same as natural seawater. Phylogenetic analysis indicates that these strains are the members of Halomonas, Arthrobacter, Pseudoalteromonas, Marinomonas, and Thalassospira. These isolates are also able to use other lignin-derived compounds, such as 4-hydroxybenzoic acid, ferulic acid, syringic acid, and benzoic acid. Vanillic acid was detected in all culture media when isolates were grown on ferulic acid as the sole carbon source; however, no 4-hydroxy-3-methoxystyrene was detected, indicating that ferulic acid metabolism by these strains occurs via the elimination of two side chain carbons. Furthermore, the isolates exhibit 3,4-dioxygenase or 4,5-dioxygenase activity for protocatechuic acid ring-cleavage, which is consistent with the genetic sequences of related genera. This study was conducted to isolate and characterize marine bacteria of degrading lignin-derived compounds, thereby revealing the degradation of aromatic compounds in the marine environment and opening up new avenues for the development and utilization of marine biological resources.

Introduction

Lignin, a major component of vascular plant tissue, is the second most abundant natural substance in nature after cellulose [1]. It plays a protective role against pathogenic and saprophytic organisms by guarding cellulose polymers from hydrolytic attack. Furthermore, it also allows for terrestrial plant growth by providing rigidity in the stems and supports the circulation of sap by waterproofing the vascular tissue [2].

The complex aromatic lignin heteropolymer is formed when crosslinked lignin monomers, such as guaiacyl, syringyl, and p-hydroxyphenyl, are further polymerized via β-aryl ether linkages, biphenyl bonds and heterocyclic linkages, by oxidase radicals. Although this molecule is highly recalcitrant, several enzymes with the ability to depolymerize lignin have been identified, such as lignin peroxidases (LiPs), manganese peroxidases (MnPs) and laccases [1]. These extracellular fungal enzymes attack lignin substructures in decaying plant cell walls, thereby releasing different lignin-derived breakdown products [2].

Vanillic acid, ferulic acid, p-hydroxybenzoic acid, syringic acid, benzoic acid and biphenyl are known lignin-derived compounds [3]. Detailed studies have been performed on the degradation of these low molecular compounds by soil bacteria [4, 5]. Research suggests that protocatechuic acid (PCA) is a key intermediate in the metabolism of lignin-derived compounds, resulting in aromatic ring cleavage. Three PCA metabolic pathways have been reported: 2,3-cleavage [6], 3,4-cleavage and 4,5-cleavage [7, 8]. The ring cleavage products from each pathway eventually enter the tricarboxylic acid cycle.

Rivers transport large amounts of vascular plant organic matter into the oceans, resulting in a carbon flux that is estimated to make up approximately 0.7% of terrestrial primary production [9]. The fact that lignin is only produced by terrestrial plants, and because it is a highly stable compound, makes it an excellent biomarker for the presence of terrestrial organic matter in lacustrine and marine environments [10]. Furthermore, when buried in sediments, this biomarker can aid in assessing the impact of land composition on marine environments [11, 12]. Marine microbes from the sea that are capable of degrading lignin potentially contribute to organic matter cycling [13]. Additional information can be inferred by determining the ratios of lignin-derived compounds. For instance, the synringyl/ vanillyl ratio, and the cinnamyl/ vanillyl phenols ratio can indicate the vegetative source; whereas the vanillic acid/ vanillin ratio and the syringic acid/ syringaldehyde ratio indicates the degradation state of lignin materials [14]. Research suggests that the average cycling time of dissolved lignin in marine environments is approximately 90 years [15], thereby highlighting the importance of understanding the degree and mechanisms of lignin degradation in the oceans, which, in turn allows, for a better understanding of carbon cycling within the marine environment.Some studies have examined lignin degradation by mixed microorganisms in marine environments [16, 17]; however, the microorganisms responsible for the degradation were not identified.

In this study, we isolated several bacterial strains from marine samples using the lignin-derived compound vanillic acid as the sole carbon and energy source. We then investigated the physiological characteristics of the isolates to determine their role in lignin mineralization in the marine environment.

Materials and methods

All experiments in the materials and methods were conducted according to protocols approved by Jiangsu Normal University and Anhui Normal University and completed by researchers of the manuscript. Field sampling is funded by the National Natural Science Foundation of China, and belongs to the research content of the project. The study area is not a special marine protected area and does not involve endangered or protected species.

Study site and sample collection

Seawater samples were collected from Lianyungang and sediments samples were collected from the East China Sea in Zhoushan, Zhejiang (30°58′20.71″N, 122° 95 41′22.10″E), People's Republic of China. The depth of the sediment sample was 10 m below sea level, the water temperature was 20°C. Further, the sediments samples were placed in sealed plastic boxes surrounded with ice and brought to the laboratory within 24h.

Chemicals and material

4-hydroxybenzoic acid (99.9%purity), benzoic acid (99.9% purity) and syringic acid (99.8% purity) were purchased from Sangon Biotech (Shanghai, China) at the highest purity available. Methanol (99.9%purity), trifluoroacetic acid (99.5%purity), and acetonitrile (99.9%purity) were purchased from Sigma-Aldrich (St. Louis, MO). Vanillic acid (99.9%purity), ferulic acid (99.9%purity) and protocatechuic acid (99.9%purity) were purchased from TCI Fine Chemicals (Tokyo, Japan). Marine Broth 2216 (DSMZ medium 604) was used as the nutrient medium. ONR7a medium (DSMZ medium #950, DSMZ, Germany), which contains only inorganic ions, was used as the defined seawater medium [18].

Isolation and characterization of vanillic acid degraders

Both the direct plating method and the enrichment technique were used to isolate vanillic acid-utilizing bacteria. The autoclaved medium was allowed to cool at room temperature and the filter sterilized vanillic acid was added into the medium. Briefly, direct plating was carried out on ONR7a agar plates amended with 0.2% vanillic acid. Cultures were incubated at 25°C until colonies appeared. The individual colonies that utilized vanillic acid as the sole carbon source for growth were selected and purified by re-streaking 3 times. The enrichment technique was performed in liquid ONR7a media amended with 0.2% vanillic acid. Cultures were incubated with shaking at 25°C until turbid. They were then plated on ONR7a agar after repeating this enrichment technique 5 times to obtain pure colonies [19].

To isolate the total DNA, each isolate was grown to exponential phase in 10 ml Marine Broth at 25°C. The resulting cells were washed twice with TES buffer (pH 8.0) containing 0.1 M NaCl, 10 mM Tris-HCl, and 1 mM EDTA and then suspended in 2.5 ml of the same buffer amended with 5 mg of lysozyme. After being kept at 37°C for 1h, 256 μl of 10% (wt/vol) sodium dodecyl sulfate (SDS) and 0.5 ml of TES containing 0.5 mg of protease K were added, and the mixture was incubated for 1 h at 65°C. Then, 1.8 ml of 10% SDS was added, and the mixture was incubated for another 1 h at 65°C. Total DNA was precipitated by adding 2.5 volumes of ethanol at room temperature. The DNA obtained by centrifugation at 6000 g for 10 min at room temperature was washed with 70% ethanol and dissolved in 1 ml TES for use.

The 16S rRNA gene-containing fragment of approximately 1.5 kb was amplified with the 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT) primers using 2 μl DNA as the template, and the PCR products were sequenced [20]. PCR was done with a total volume of 50 μl, which contained 27.8 μl Sterilized H2O, 5 μl 10×PCR Buffer, 5 μl dNTP Mix, 4μl MgCl2, 3 μl Primer1, 3 μl Primer2, 0.2 μl Taq DNA Polymerase and 2 μl Template DNA. Amplification of the DNA was done for 35 cycles under the following conditions: denaturation, 95°C for 30 s; primer annealing, 56°C for 30 s; and primer ex tension, 72°C for 90 s. Miseq platform was used and the concentration of each sample was about 300ng μl-1. The almost-complete 16S rDNA gene sequences of these isolates were obtained and used for initial BLAST searches in GenBank and for phylogenetic analyses. The sequences were aligned to their nearest phylogenetic neighbors using Genetyx software (GENETYX Corporation, Tokyo, Japan), and calculation of pairwise 16S rDNA gene sequence similarities was achieved using the EMBL sever (http://www.ebi.ac.uk/Tools/psa/). Phylogenetic trees were generated using same software. The robustness of the inferred topologies was tested by using 1,000 bootstrap re-samplings.

Optimum growth conditions for temperature and NaCl concentration

Each bacterium was grown to the stationary phase in Marine Broth 2216 at 25°C. The resulting cells were collected by centrifugation (5000×g, 5 min) and resuspended in 100 ml of the ONR7a medium to an optical density at 600 nm (OD600) of 0.1. The cultures were incubated at different temperatures (20, 30, 35, 40, and 45°C) and NaCl concentration (0 g L-1, 15 g L-1, 22.8 g L-1, 40 g L-1, and 50 g L-1). A portion of the culture was collected and measured at OD600 after being incubated with shaking. Each experiment was done in parallel three times.

Degradation of different lignin-derived compounds by isolates

Each bacterium was grown to the stationary phase in Marine Broth 2216 at 25°C. The resulting cells were washed twice with ONR7a medium and then suspended in 5 ml of the same medium to give a turbidity of 0.05–0.1 at 600 nm. Each of the lignin-derived compounds (4-hydroxybenzoic acid, benzoic acid, ferulic acid and syringic acid) was added to a final concentration of 0.1%, and the mixture was shaken (150 rpm) at 30°C. Each experiment was done in one time.

Degradation products of ferulic acid by isolates

Isolates were inoculated into ONR7a medium supplemented with ferulic acid and incubated for 24 h under conditions optimized. The concentrations of degradation products were determined with HPLC.

Analysis of metabolism of lignin-derived compounds by HPLC and GC-MS

0.5 ml of the culture was collected and mixed with equal amounts of methanol. The mixture was filtered through a Millipore Filter Membrane (0.22 μm/50 mm) and then subjected to high-performance liquid chromatography (HPLC) analysis. An Alliance HPLC instrument (Waters, Milford, Mass.) with an octadecyl silica reverse-phase column (30 cm in length and 4.6 mm in diameter; Waters) was used at 40°C. A mobile phase consisting of solvent A (0.1% trifluoroacetic acid in water) and solvent B (acetonitrile) was used at a flow rate of 1 ml/min. After injection of 10 μl of a sample, 90% solvent A was run through the column for 4 min, after which the percentage of solvent B was gradually increased from 10% to 100% over 15 minutes. Finally, 100% of solvent B was run for 2 min. The absorbance spectrum of the eluate was monitored from 200 to 500 nm with a photodiode array detector (Waters 2998). The retention times of vanillic acid and ferulic acid were 6.237 and 7.332 min, respectively. All vanillic acid and ferulic acid we have used are standard. The test samples were extracted by ethyl acetate and dried over anhydrous Na2SO4 and the solvent was allowed to evaporate at 40°C. The residual was dissolved in an equal volume of methanol to original liquid sample and analyzed by gas chromatography-mass spectrometry (GC-MS) (Shimadzu QP5050A instrument) with a DB-5 column (length, 30 m; diameter, 0.25 mm) (J&W Scientific). The column temperature was initially kept at 50°C for 5 min before being increased to 300°C at a rate of 2.5°C per min. The injector and detector temperatures were both 300°C. Determination of vanillic acid, vanillin, vanillate, protocatechuate, and fumarate through SIM (selective reaction monitoring) with referenced ion fragments information as showed as following: 168.15m/z, 152.15m/z, 151.03m/z, 154.12m/z and 115.05m/z, respectively.

Enzyme assays for PCA dioxygenase

Isolates were grown in 600 ml of Marine Broth for 12 h at 25°C with shaking and then washed twice with ONR7a. We centrifuged the pre-cultured bacterial solution (5000× g, 5 min), removed the supernatant, then washed twice with seawater medium, removed the supernatant, and blotted dry with a pipette tip. Finally, the bacterial solution was dissolved in a seawater medium of 5 ml. The resulting cells were resuspended in 200 ml of ONR7a containing 0.2% vanillic acid, and the mixture was incubated with shaking at 25°C. The cells were harvested when approximately half of the vanillic acid was degraded, washed twice with 50 mM Tris-HCl (pH 8.0), and disrupted by ultrasonication. The amount of vanillic acid was determined by spectrophotometer. The cell-free extracts were obtained by centrifugation. The reaction mixture for assaying PCA dioxygenase activity consisted of 1900 μl of 50 mM Tris-HCl (pH 8.0), 40 μl of 1 mM PCA, and 60 μl of cell-free extract (100 μg ml-1). The reaction mixture was incubated at 25°C and monitored spectrometrically from 200 to 500 nm with an EVOLUTION 260 BIO UV-Visible spectrophotometer (ThermoFisher, USA).

Results

Isolation of vanillic acid degraders

In this study, vanillic acid was used as lignin-derived low molecular weight compound to isolate vanillic acid-utilizing bacteria from marine and sediment samples collected from the coast of Lianyungang and the China East Sea, respectively. The predominant strains, which have adapted to their marine environment, could be isolated using the direct plating method, which resulted in isolation of bacteria with the highest growth rate in laboratory conditions. Three strains with the ability to use vanillic acid as the sole carbon and energy source were successfully isolated by direct plating, namely, strains ZXY12VA01, ZXY12VA14, and ZXY12VA15 (Table 1). Three additional strains with the ability to use vanillic acid as the sole carbon and energy source were isolated using the enrichment technique; namely, ZXY12VA05, ZXY12VA16, and ZXY12VA17.

Table 1

Strains isolated in this study.

Strain name	Isolation method	Sampling site	Relative type strain	Identity (%)
ZXY12VA01	Enrichment	Lianyungang	Halomonas titanicae BH1T(FN433898)	99.87
ZXY12VA05	Direct plating	China East Sea	Arthrobacter nicotianae	99.80
ZXY12VA14	Enrichment	China East Sea	Pseudoalteromonas prydzensis	95.36
ZXY12VA15	Enrichment	China East Sea	Marinomonas brasilensis	97.22
ZXY12VA16	Direct plating	Lianyungang	Thalassospira profundimaris	99.85
ZXY12VA17	Direct plating	China East Sea	Pseudoalteromonas nigrifaciens	99.98

The colony morphology of the isolates when plated with vanillic acid are as follows: ZXY12VA01, white and small; ZXY12VA05, milk yellow, uplifted with a smooth edge, large; ZXY12VA14, milk yellow, smooth edge, uplifted; ZXY12VA15, white and small, uplifted, rapid growth; ZXY12VA16, small, dark brown, slow growth, secreted brown pigment; ZXY12VA17, primrose yellow and turned to yellow. All strains are Gram-negative, except strain ZXY12VA05, which is Gram-positive (Fig 1).

Fig 1

Gram stain of isolates ZXY12VA01 (A), ZXY12VA05 (B), ZXY12VA14 (C), ZXY12VA15 (D), ZXY12VA16 (E), ZXY12VA17 (F).

Electrophoresis results of PCR products (1500 bp) of isolated bacteria are shown in S1 Fig. Phylogenetic analysis based on the neighbor-joining algorithm revealed that strain ZXY12VA01 clusters with the Halomonas genus (Fig 2). The most closely related type strain is Halomonas titanicae BH1T (99.87% 16S rDNA sequence similarity). Other related type strains are Halomonas variabilis DSM 3051T (99.0%), Halomonas olivaria C17T (99.0%), and Halomonas neptunia Eplume1T (99.0%). Based on these data, strain ZXY12VA01 likely belongs to Halomonas, which belongs to the family Halomonadaceae. This heterogeneous family, originally proposed by Frenzmann et al. (1988), to date includes more than 60 species [21]. They have been isolated from many water and soil environments within saline, hypersaline or alkaline habitats. Halomonas titanicae BH1T was isolated from rusticle samples collected at the site of the wreck using Bacto marine agar 2216 medium (Difco). It grows optimally at 30–37°C in the presence of 2–8% (w/v) NaCl.

Fig 2

Phylogenetic tree of thermophilic isolates based on 16S rDNA sequences.

The most closely related type strain to ZXY12VA05 is Arthrobacter nicotinanae DSM 20123T (99.86%). Other related type strains include Arthrobacter mysorens LMG 16219T, Arthrobacter arilaitensis Re117T, and Arthrobacter uratoxydans DSM 20647T. Based on these data, strain ZXY12VA05 belongs to the genus Arthrobacter. Members of the Arthrobacter genus have a high G+C content, are actinomycete-coryneform bacteria, and are all Gram-positive. This genus contains more than 40 species of bacteria isolated from diverse environments, including soil and clinical specimens. Most Arthrobacter strains are mesophiles; however, some members have been isolated from Antarctic lakes and deep-sea sediment [22].

Both ZXY12VA14 and ZXY12VA17 strains cluster with the genus Pseudoalteromonas (Fig 2). The most closely related type strain for ZXY12VA14 and ZXY12VA17 are Pseudoalteromonas mariniglutinosa KMM 3635T (95%) and Pseudoalteromonas shioyasakiensis SE3T (98%), respectively. Other related type strains are Pseudoalteromonas prydzensis MB8-11T for ZXY12VA14; and Pseudoalteromonas arabiensis k53T, Pseudoalteromonas lipolytica LMEB 39T, and Pseudoalteromonas byunsanensis FR1199T for ZXY12VA17. Members of Pseudoalteromonas are characteristically Gram-negative, rod-shaped, motile bacteria with one or two polar flagella. Most Pseudoalteromonas strains are mesophiles; however, some halotolerant psychrophilic members have been isolated from Antarctic sea ice [23]. Strain KMM 3635T was isolated from a Chaetoceros lauderi diatom, which was collected from seawater in the Marseille Gulf [24]. Strain SE3T was isolated from Pacific Ocean sediment [25].

Phylogenetic analysis revealed that the most closely related type strain to ZXY12VA15 (Fig 2) is Marinomonas communis LMG 2864T (99.15% 16S rDNA sequence similarity). Other related type strains include Marinomonas vaga ATCC 27119T, Marinomonas aquimarina CECT 5080T, and Marinomonas fungiae AN44T. Based on these data, strain ZXY12VA15 belongs to the genus Marinomonas, which was created in 1983 and comprises 15 species. The members are Gram-negative, aerobic bacterium and have mainly been isolated from geographically diverse marine environments [26].

Strain ZXY12VA16 clusters with the Thalassospira genus (Fig 2). The most closely related type strain is Thalassospira povalilytica Zumi 95T (99.62% 16S rDNA sequence similarity). Other related type strains include Thalassospira profundimaris WP0211T, Thalassospira tepidiphila 1-1BT, and Thalassospira permensis NBRC 106175T. Members of the Thalassospira genus are vibrioid to spiral cells; they are strict aerobes that are unable to grow under anaerobic conditions by fermentation, respiration or photoheterotrophy [27]. Thalassospira povalilytica Zumi 95T was isolated from plastic rope litter found in the Tokyo Bay, Japan, and exhibits the ability to utilize polyvinyl-alcohol as a carbon source [28].

The sequence data reported in this study appear in the EMBL, GenBank and DDBI sequence databases under the accession numbers. The GenBank accession number of strains ZXY12VA01, ZXY12VA05, ZXY12VA14, ZXY12VA15, ZXY12VA16 and ZXY12VA17 are KT274805, KT274806, KT717632, KT274807, KT274808 and KT274809, respectively.

Marine and terrestrial environments are highly variable; however, microorganisms have the ability to rapidly evolve and adapt to changing environments. The optimum growth conditions of terrestrial bacteria in terms of temperature and NaCl concentration are generally 30°C and 0.5%, respectively. In contrast, marine microorganisms prefer to grow at temperatures under 30°C and at a high NaCl concentration. Thus, by investigating the optimal temperature and salt concentrations of the isolates, it may be possible to infer if these organisms originated from the terrestrial or the marine environment.

The optimum growth temperature of the isolates rang from 30 to 40°C (Fig 3). The optimum growth temperature for strains ZXY12VA01, ZXY12VA15, ZXY12VA16, and ZXY12VA17 is 35°C; whereas strains ZXY12VA05 and ZXY12VA14 grow optimally at 30°C. These isolates do not grow above 45°C or below 20°C.

Fig 3

Optimum growth temperatures of isolates ZXY12VA01 (A), ZXY12VA05 (B), ZXY12VA14 (C), ZXY12VA15 (D), ZXY12VA16 (E), ZXY12VA17 (F).

All isolates grow well in a wide NaCl concentration range from 0 to 50 g L-1, with the optimum concentration being 22.8 g L-1, which is the same as natural seawater (Fig 4). These 6 strains appear to have adapted to the NaCl concentration of seawater; therefore, they are not terrestrial microorganisms that were recently deposited into the marine environment.

Fig 4

Optimum growth in different NaCl concentrations of isolates ZXY12VA01 (A), ZXY12VA05 (B), ZXY12VA14 (C), ZXY12VA15 (D), ZXY12VA16 (E), and ZXY12VA17 (F).

Ability to utilize lignin-derived compounds

In addition to vanillic acid, it is possible for some organisms to utilize other lignin-derived low molecular weight compounds, such as benzoic acid, 4-hydroxybenzoic acid, ferulic acid, and syringic acid. Thus, the isolates were cultured for 10 days at 30°C in the selected lignin-derived compounds to test their ability to use these compounds as their sole carbon and energy source. 4-Hydroxybenzoic acid was the best substrate for all isolates because it resulted in cell densities of approximately 1.0 of OD600 (Table 2). The cell densities grown on syringic acid and ferulic acid were much lower than those on 4-hydroxybenzoic acid; however, strains ZXY12VA05, ZXY12VA14, ZXY12VA16 grew as well on benzoic acid as on 4-hydroxybenzoic acid, while ZXY12VA01 and ZXY12VA15 did not. These results demonstrate that the strains isolated in this study have the ability to grow on several lignin-derived compounds and likely play an important role in lignin mineralization in marine environments.

Table 2

Vanillate-degrading isolates grown on different lignin-derived carbon sources.

Strain name	Carbon source	OD600
		1 d	2 d	3 d	6 d	10 d
ZXY12VA01	Benzoate	0.068	0.551	--	--	--
	4-Hydroxybenzoate	1.061	--	--	--	--
	Ferulate	0.281	0.314	--	--	--
	Syringate	0.241	0.283	--	--	--
ZXY12VA05	Benzoate	0.138	--	1.222	--	--
	4-Hydroxybenzoate	1.325	--	--	--	--
	Ferulate	0.355	--	0.392	--	--
	Syringate	0.298	--	0.307	--	--
ZXY12VA14	Benzoate	--	--	--	--	1.232
	4-Hydroxybenzoate	1.992	--	--	--	--
	Ferulate	--	--	--	--	0.45
	Syringate	--	--	--	--	0.316
ZXY12VA15	Benzoate	--	--	--	0.741	--
	4-Hydroxybenzoate	0.941	--	--	--	--
	Ferulate	--	--	--	0.392	0.443
	Syringate	--	--	--	0.307	0.334
ZXY12VA16	Benzoate	--	--	--	1.172	--
	4-Hydroxybenzoate	1.022	--	--	--	--
	Ferulate	0.336	--	--	--	0.524
	Syringate	0.366	--	--	--	0.523
ZXY12VA17	Benzoate	--	--	1.102	--	--
	4-Hydroxybenzoate	--	--	0.952	--	--
	Ferulate	--	--	0.420	--	0.447
	Syringate	--	--	0.290	--	0.439

Detection of ferulic acid intermediates

Ferulic acid, an easily available component of lignin, plays an important role in the cross-linking of cell walls in various plants. The first step in ferulic acid degradation is the removal of its side chain. Two types of side chain removal have been reported for a number of microorganisms [29]. One type, which results in a 4-hydroxy-3-methoxystyrene intermediate, is catalyzed by a non-oxidative decarboxylase that eliminates one carbon from the ferulic acid side chain. The 4-hydroxy-3-methoxystyrene is subsequently transformed to vanillin and then to vanillic acid. The other type, which results in the direct formation of vanillin, involves the elimination of two carbons from the ferulic acid side chain. Vanillin is subsequently oxidized to vanillic acid by vanillin dehydrogenase.

To determine which type of metabolism is carried out by these strains, metabolites from cultures grown with ferulic acid as the sole carbon source were investigated using HPLC. Vanillic acid was detected in all culture media when isolates were grown on ferulic acid as the sole carbon source; however, no 4-hydroxy-3-methoxystyrene was detected (Figs 5 and 6), indicating that ferulic acid metabolism by these strains occurs via the elimination of two side chain carbons. Through this study, we found that ZXY12VA16 produced the most amount of vanillic acid. The ZXY12VA15 strain is a dominant strain of aromatic degrading bacteria. Normally, feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase are found in most FA-degrading strains. These strains convert ferulic acid to vanillin by a non-β-oxidative pathway [30]. It demonstrates that vanillic acid is an important intermediate in the metabolism of lignin and plays a leading role in the metabolism of aromatic compounds.

Fig 5

The formation of the intermediates in the biodegradation of ferulic acid in ZXY12VA01 shown in the HPLC analysis.

The major metabolites were detected on the 0h (A), 12h (B) and 24h (C), respectively.

Fig 6

Metabolism of ferulic acid to vanillic acid by isolates ZXY12VA01 (A), ZXY12VA05 (B), ZXY12VA14 (C), ZXY12VA15 (D), ZXY12VA16 (E), ZXY12VA17 (F).

When strain ZXY12VA01, ZXY12VA05 and ZXY12VA14 was grown on 0.1% vanillic acid as the sole carbon, the formation of vanillin and protocatechuic acid was detected in the culture medium (Fig 7). Vanillic acid can be hydroformylated to form vanillin or demethylated to form protocatechuic acid. Protocatechuic acid is considered to be the target compound for ring fission. Protocatechuate has a vic-diol structure, which can be subjected to ring cleavage by dioxygenase. The formation of fumaric acid proves that the metabolite is finally decomposed into the tricarboxylic acid cycle (Fig 8).

Fig 7

Growth of ZXY12VA01 (A), ZXY12VA05 (B) and ZXY12VA14 (C) on vanillic acid. The four major metabolites were detected on the 12h and 24h, respectively.

Fig 8

Speculated biodegradation pathways of ferulic acid and vanillic acid by marine bacteria.

Enzyme assay for aromatic ring-fission reaction

Vanillic acid metabolism involves removal of the methoxy group by a demethylase, resulting in the formation of a protocatechuic acid (PCA) intermediate. Ring cleavage of PCA occurs by three known dioxygenases: PCA 2,3-dioxygenase, PCA 3,4-dioxygenase, and PCA 4,5-dioxygenase and results in the ring-cleavage products 5-carboxy-2-hydroxymuconate-6-semialdehyde (350 nm), 5-carboxymuconate (290 nm), and 4-carboxy-2-hydroxymuconate-6-semialdehyde (410 nm), respectively. Each ring-fission product has a specific absorption peak, making it easy to determine which ring-cleavage occurred.

To induce vanillic acid-metabolizing enzymes, ZXY12VA15 was grown on vanillic acid and harvested when approximately half of the carbon source was degraded. The cell-free extracts were applied to the enzyme assay using PCA as the substrate. Spectrophotometric assays provided evidence for the formation of 4-carboxy-2-hydroxymuconate-6-semialdehyde (λmax, 410 nm) at pH 8.0 (Fig 9). It was determined that strain ZXY12VA15 were capable of 4,5-cleavage.

Fig 9

Spectrophotometric analysis of ZXY12VA15 enzyme activity.

Absorbance increase of 4-carboxy-2-hydroxymuconate-6-semialdehyde (λmax, 410 nm).

Discussion

Genome-wide surveys were performed on the isolate representative species of related genera to identify genes associated with the PCA ring-cleavage pathway and the genomic information of strains were searched on KEGG.

Halomonas elongate DSM 2581 is a halotolerant bacteria isolated from a solar salt facility. It synthesizes the compatible solute Ectoine, thus allowing it to withstand NaCl concentrations well above 10% [31]. Contained in the H. elongata genome is a gene cluster (HELO_3955–3963) encoding PCA 3,4-dioxygenase and additional enzymes involved in the metabolism of ring-cleavage products.

Marinomonas mediterranea MMB-1 [32], Marinomonas sp. MWYL1 [33, 34], and Marinomonas posidonica IVIA-Po-181 [35] were isolated from marine samples. Their genomes have the same gene cluster, which encodes for PCA 4,5-dioxygenase and enzymes involved in the ring-cleavage product metabolism.

The members of Arthrobacter are among the most frequently isolated, indigenous, aerobic genera found in soils. Arthrobacter phenanthrenivorans Sph3 was isolated from a creosote-polluted site, where a wood-preserving factory had operated for over 30 years. It is able to metabolize phenanthrene as the sole carbon and energy source [36]. Arthrobacter aurescens TC1 was originally isolated from soil at an atrazine spill site [37]. Arthrobacter chlorophenolicus A6, a Gram-positive Actinobacterium, was enriched from a soil slurry using increasing concentrations of 4-chlorophenol and can degrade exceptionally high concentrations of 4-chlorophenol (up to 2.7 mM), along with other p-substituted phenols, including 4-nitrophenol and 4-bromophenol [38]. Arthrobacter arilaitensis Re117 was isolated from the surface of cheeses and possesses a salt-tolerance system [39]. Arthrobacter sp. FB24 was isolated from a soil sample that was obtained at a site contained by mixed waste that included both petroleum hydrocarbons and extremely high metal levels. The genomes of these Arthrobacter members have the same gene cluster encoding PCA 3,4-dioxygenase and enzymes involved in the ring-cleavage product metabolism.

Thalassospira xiamenensis M-5 (DSM17429) was isolated from an oil-waste pool at an oil storage dock. Strain M-5 has a gene cluster encoding PCA 3,4-dioxygenase and enzymes involved in the ring-cleavage product metabolism [40].

Pseudoalteromonas atlantica T6c is a biofilm-forming, Gram-negative, motile marine bacterium. It is chemoorganotrophic and secrets extracellular enzymes that are able to hydrolyze agar, alginate, and carrageenan [41]. Strain T6c has a gene cluster encoding PCA 4,5-dioxygenase and enzymes involved in the ring-cleavage product metabolism.

This study demonstrates that members of the genera Halomonas, Arthrobacter, Pseudoalteromonas, Marinomonas, and Thalassospira are involved in the degradation of lignin-derived compounds in marine environments. In addition, many environmental pollutants are aromatic compounds, and marine bacteria capable of degrading lignin can also degrade most environmental pollutants. The isolated lignin-degrading bacteria can be applied to environmental pollution treatment in a marine environment or a high-salt environment. Although we have isolated marine bacteria that can degrade vanillic acid, our further research will focus on applying to actual production.

Conclusion

In this study, 6 bacterial strains were isolated from marine samples using the lignin-derived compound vanillic acid as the sole carbon and energy source. It is concluded that these 6 strains have adapted to the NaCl concentration of seawater and are marine dominant bacteria widely present in the marine environment, rather than the soil microorganisms of the recent land. They are Phylogenetic analysis indicates that ZXY12VA01, ZXY12VA05, ZXY12VA14, ZXY12VA15, ZXY12VA16, and ZXY12VA17 are members of Halomonas, Arthrobacter, Pseudoalteromonas, Marinomonas, and Thalassospira, respectively. They are also able to use other lignin-derived compounds, such as 4-hydroxybenzoic acid, ferulic acid, syringic acid, and benzoic acid. The isolates showed 3,4-dioxygenase or 4,5-dioxygenase activity for protocatechuic acid ring-cleavage, which is consistent with information on related genera genome sequences.

Electrophoresis image of 16S rDNA PCR products.

(DOCX)

Click here for additional data file.

11 Mar 2020

PONE-D-19-35662

Isolation and Characterization Marine Bacteria Capable of Degrading Lignin-derived Compounds

PLOS ONE

Dear Mr. Feng,

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Reviewer #1: Yes

**********

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Reviewer #1: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: The paper is well written and reports the new knowledge on the lignin degrading marine bacteria isolated from East China Sea. Following comments should be taken into account before its consideration for publication elsewhere:

1. Remove the word "Isolation" from the list of keywords.

2. Revised sentence as "organic matter into the oceans" at Line No. 21.

3. It should be "sediments" instead of "ediment" at Line No. 93.

4. It should be revised as "East China Sea in Zhoushan, Zhejiang (30°58′20.71″N, 122° 95 41′22.10″E), People's Republic of China" at Line No. 94-95.

5. The sentence should be started with "The depth of sediments" instead of "Depth" at Line No. 95.

6. Revised this sentence as "Further, the sediments samples were placed in sealed plastic boxes surrounded with ice and brought to the laboratory within 24h" instead of "Placed in sealed plastic boxes surrounded with ice, the samples

97 were taken to the laboratory within 24h" at Line No. 95.

7. Kindly mention the a tentative purity of the Chinese chemicals within bracket at Line No. 101.

8. Kindly check the presence of reference both in the text as well as in the reference section.

9. Kindly also incorporate the HP-LC and GC-MS chromatogram in the manuscript.

**********

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Reviewer #1: No

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4 Jun 2020

Dear editors and reviewers:

Thank you for your letter and the reviewers’ comments on our manuscript entitled " Isolation and Characterization Marine Bacteria Capable of Degrading Lignin-derived Compounds" (PONE-D-19-35662). Those comments are very helpful for revising and improving our paper, as well as the important guiding significance to other research. We have studied the comments carefully and made corrections which we hope meet with approval. The main corrections are in the manuscript and the responds to the reviewers’ comments are as follows.

Replies to the editors’ comments:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response：Thank you for your suggestions and we have reorganized the manuscript to meet PLOS ONE's style requirements.

2. In your Methods section, please provide additional information regarding the permits you obtained for the work. Please ensure you have included the full name of the authority that approved the field site access and, if no permits were required, a brief statement explaining why.

Response：Thank you for your valuable comments. All experiments in the Methods were conducted according to protocols approved by Jiangsu Normal University and Anhui Normal University and completed by researchers of the manuscript. No additional authorization information is required for this work.

3. Please ensure that you refer to Figure 4 in your text as, if accepted, production will need this reference to link the reader to the figure.

Response：Fig 4 has been cited and all figures have been reordered and checked.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly.

Response：Captions of Supporting Information has been added and rematched.

Replies to the reviewers’ comments:

1. Remove the word "Isolation" from the list of keywords.

Response：The word has been removed.

2. Revised sentence as "organic matter into the oceans" at Line No. 21.

Response：The sentence has been modified.

3. It should be "sediments" instead of "ediment" at Line No. 93.

Response：The word has been modified.

4. It should be revised as "East China Sea in Zhoushan, Zhejiang (30°58′20.71″N, 122° 95 41′22.10″E), People's Republic of China" at Line No. 94-95.

Response：The full name of China has been added, which is very important.

5. The sentence should be started with "The depth of sediments" instead of "Depth" at Line No. 95.

Response：We have modified it according to your suggestion.

6. Revised this sentence as "Further, the sediments samples were placed in sealed plastic boxes surrounded with ice and brought to the laboratory within 24h" instead of "Placed in sealed plastic boxes surrounded with ice, the samples were taken to the laboratory within 24h" at Line No. 95.

Response：We have modified it according to your suggestion.

7. Kindly mention the a tentative purity of the Chinese chemicals within bracket at Line No. 101.

Response：The purity of all chemicals has been added in the method. Thank you for your suggestion.

8. Kindly check the presence of reference both in the text as well as in the reference section.

Response：References have been checked and re-marked.

9. Kindly also incorporate the HP-LC and GC-MS chromatogram in the manuscript.

Response：Thanks for your kindly and profssional comment. In this manuscript, Fig 5 shows the analysis of FA degradation by HPLC. We used SIM (selective reaction monitoring) mode to determinated the content variation of vanillic acid, vanillin, vanillate, protocatechuate, and fumarate. In this method, five referenced ion fragments information were monitored and determined the content through calculation of ionic strength. We have reflected upon your comment and addiion of the description of method in the manuscript.

Once again, thank you very much for your constructive comments and suggestions which would help us both in English and in depth to improve the quality of the paper.

Kind regards,

Zhaozhong Feng

E-mail: fzz2012@jsnu.edu.cn

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

23 Jul 2020

PONE-D-19-35662R1

Isolation and Characterization Marine Bacteria Capable of Degrading Lignin-derived Compounds

PLOS ONE

Dear Dr. Feng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 06 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Pankaj Kumar Arora

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: There has been limited work done to characterize marine microorganisms that are able to aerobically degrade lignin-derived compounds. This study describes the process of isolating and characterizing several strains that can degrade these compounds. The isolates were evaluated for their substrate range and enzyme activity. The authors addressed each point in their response to reviewers letter. There are, however, some points in the manuscript that require further clarification, especially with respect to the methods and typographical errors. Please see my specific comments below.

Specific comments:

Line 88 – Contribute

Line 115 – The depth of the sediment….

136 – Vanillic acid as the sole carbon source

Lines 139-140- Please clarify your methods for enrichment. It is unclear what “this process” is referring to.

Line 156- Please refer to the specific primer (ie 27 Forward) in your PCR recipe.

Experiments described in Lines 179-188, 217 – What were your controls? How many replicates were used?

Line 207- Please include more details about your GC-MS preparation, such as extraction protocol and if the samples were derivatized.

Line 355 – Please clarify why the table shows OD values on a time scale of up to 10 days if the text is stating that the cultures were grown for 3 days with each substrate.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

16 Sep 2020

Dear editors and reviewers:

Thank you for your letter and the reviewers’ comments on our manuscript entitled " Isolation and Characterization Marine Bacteria Capable of Degrading Lignin-derived Compounds" (PONE-D-19-35662). Those comments are very helpful for revising and improving our paper, as well as the important guiding significance to other research. We have studied the comments carefully and made corrections which we hope meet with approval. The main corrections are in the manuscript and the responds to the reviewers’ comments are as follows.

Replies to the reviewers’ comments:

1. Line 88 – Contribute

Response：The word has been modified.

2. Line 115 – The depth of the sediment….

Response：The sentence has been modified.

3. 136 – Vanillic acid as the sole carbon source

Response：The sentence has been modified.

4. Lines 139-140- Please clarify your methods for enrichment. It is unclear what “this process” is referring to.

Response：“This process” is referring to the enrichment technique. The enrichment technique was performed in liquid ONR7a media amended with 0.2% vanillic acid. Cultures were incubated with shaking at 25 ºC until turbid.

5. Line 156- Please refer to the specific primer (ie 27 Forward) in your PCR recipe.

Response：The reference cited in the primer has been corrected.

6. Experiments described in Lines 179-188, 217 – What were your controls? How many replicates were used?

Response：Each experiment is an independent experiment, which reflects the content of metabolites according to the time course.

7. Line 207- Please include more details about your GC-MS preparation, such as extraction protocol and if the samples were derivatized.

Response：The test samples were extracted by ethyl acetate and dried over anhydrous Na2SO4 and the solvent was allowed to evaporate at 40℃. The residual was dissolved in an equal volume of methanol to original liquid sample and analyzed by GC-MS. Determination of vanillic acid, vanillin, vanillate, protocatechuate, and fumarate through SIM (selective reaction monitoring) with referenced ion fragments information. All samples were not derivatized.

8. Line 355 – Please clarify why the table shows OD values on a time scale of up to 10 days if the text is stating that the cultures were grown for 3 days with each substrate.

Response：We have modified it and the cultures were grown for 10 days with each substrate.

Once again, thank you very much for your constructive comments and suggestions which would help us both in English and in depth to improve the quality of the paper.

Kind regards,

Zhaozhong Feng

E-mail: fzz2012@jsnu.edu.cn

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

22 Sep 2020

Isolation and Characterization Marine Bacteria Capable of Degrading Lignin-derived Compounds

PONE-D-19-35662R2

Dear Dr. Feng,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Pankaj Kumar Arora

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

28 Sep 2020

PONE-D-19-35662R2

Isolation and Characterization Marine Bacteria Capable of Degrading Lignin-derived Compounds

Dear Dr. Feng:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Pankaj Kumar Arora

Academic Editor

PLOS ONE