Here we describe the screening and characterization of a lignin-degrading bacterium from an environmental sample. The bacterium was isolated from leaf soil and identified as Pseudomonas sp. strain CCA1 based on 16S rRNA gene sequencing. Although identified as able to degrade lignin in our screen, the ligninolytic activityof this strain was weak. Nonetheless, assessment of its utilization of lignin-associated aromatic monomers revealed that Pseudomonas sp. strain CCA1 assimilated at least ten lignin-associated aromatic monomers.

Akita H, Kimura ZI, Mohd Yusoff MZ, Hoshino T (2016) Isolation of Pseudomonas sp. Strain CCA1 from Leaf Soil and Preliminary Characterization Its Ligninolytic Activity. JSM Biotechnol Bioeng 3(4): 1062.

•    Pseudomonas
•    Lignin-associated aromatic monomer
•    Lignin-degrading bacterium

TOC: Total Organic Carbon

Biomass feedstocks represent a more sustainable carbon source, since these feedstocks grow largely through carbon dioxide fixation, and their burning does not change the level of greenhouse gas in atmosphere. In the production of biofuel, first-generation and second-generation biofuels are categorized based on the sources of the biomass used for their production. First-generation biofuels are directly produced from edible feedstocks such as cassava, corn and sugarcane [1,2]. Using edible feedstocks for biofuel production has several advantages: the sugar extraction methods are well established, the biofuel fermentations are easy to perform, and the production yields are relatively high. In fact, bio-ethanol is produced on a scale of 24 million gallons/year around the world [2]. However, edible feedstocks are also used to feed our animals and ourselves, which means production of first-generation biofuels is greatly influenced by our dietary needs.

On the other hand, second-generation biofuels are made from inedible feedstocks such as food waste, organic waste and lignocellulosic biomass, which are widely distributed around the world as available raw materials [1,2]. When second-generation biofuels are produced from lignocellulosic biomass, consecutive steps that include pretreatment, enzymatic hydrolysis and microbial fermentation are required. In the pretreatment step, lignocellulosic biomass is decomposed through heating, which releases cellulose, hemicellulose and lignin. In the enzymatic hydrolysis step, cellulose and hemicellulose are converted first to glucose, xylose, mannose and other sugars and then to saccharified solution, which includes fermentable sugars, aldehyde inhibitors and lignin. Finally, the saccharified solution is used as the carbon source in the fermentation step [1,2]. While aldehyde inhibitors inhibit microbial growth and interfere with subsequent fermentation, these compounds can be chemically or enzymatically detoxified. Despite these improvements, industrial host microorganisms find it difficult to degrade lignin. As potential alternatives, several lignin-degrading bacteria such as Serratialiquefacien PT01, Stenotrophomonasmaltophilia PT03 and Pseudomonas chlororaphis PT02 were studied on a lab scale [3]. However, their growth rates were slower, since these bacteria are not used on industrial scale for biofuel production [3]. Consequently, when biofuel is produced from lignocellulosic biomass, lignin is not effectively utilized.

Isolation of the bacterium strain

A lignin-degrading bacterium was isolated using lignin M9 plates (pH 7.2): 17 g·L-1 Na2 HPO4 ·12H2 O, 3 g·L-1 KH2 PO4 , 0.5 g·L1 NaCl, 1g·L-1 NH4 Cl, 0.24 g·L-1 MgSO4 ·7H2 O, 0.011 g·L-1 CaCl2 ·2H2 O 15 g·L-1 of agar and 1 g·L-1alkali lignin (Tokyo Chemical Industry, Tokyo, Japan). Environmental samples such as compost, forest soil, leaf soil and river side sand were collected from HigashiHiroshima City in Hiroshima Prefecture, Japan. After filtration of a 10% wash solution of the environmental sample (w/v), the filtrates were plated on lignin M9 plates and incubated for 2 days at 37°C. A single colony was selected from a lignin M9 plate and re-streaked on a Nutrient Broth plate (Kyokuto, Tokyo, Japan) at least three times to obtain a pure colony.

16S rRNA gene amplification and sequencing

The genomic DNA from the isolated microorganism was extracted and purified using an illustrate bacteria genomic Prep Mini Spin Kit (GE Healthcare, Buckinghamshire, UK) according to manufacturer’s instructions. The 16S rRNA gene was amplified using KOD -plus- DNA polymerase (TOYOBO, Osaka, Japan) with bacterial universal primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1391r (5′-GACGGGCGGTGTGTRCA-3′), after which the amplified PCR products were purified using a Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA). The purified fragments were cloned into pTA2 vector (TOYOBO), yielding 16SCCA1/pTA2, which was sequenced using universal primers M13 forward (5′-GTAAAACGACGGCCAGT-3′) and M13 reverse (5′-CAGGAAACAGCTATGAC-3′) on an Applied Bio systems model 3730xl DNA Analyzer at Fasmac (Kanagawa, Japan). The calculation of distances, multiple alignments and construction of neighbor-joining phylogenetic trees [4] were performed using CLUSTAL W version 1.83 [5].

Characterization of ligninolytic activity

Ligninolytic activity was assessed by measuring the increase in OD600, which reflects cell growth. The OD600 was measured by monitoring the difference between the cell and cell-free turbidity values using an Eppendorf Bio Spectrometer (Eppendorf, Hamburg, Germany). The isolated microorganism was pre grown overnight in Nutrient Broth, after which the culture was diluted 3:100 in fresh M9 medium (pH 7.2) containing 1 g·L-1 alkali lignin or 5 mM lignin-associated aromatic monomers.

Quantification of TOC

To measure dissolved total organic carbon (TOC), samples were centrifuged at 20,000 × g for 1 min. The resultant supernatant was then saved, filtered through a membrane filter (pore size, 0.2 μm), and immediately analyzed. The concentration of TOC was measured using a Shimadzu model TOC-V analyzer with an ASI-V auto sampler (Shimadzu, Kyoto, Japan).

Screening for lignin-degrading bacteria

Microbial degradation of lignin has primarily been studied in the white-rot fungi, which are known to express the ligninolytic enzymes such aslaccase, lignin peroxidase, manganese peroxidase and versatile peroxidase [6]. The lignin degradation is achieved through successive reactions catalyzed by those ligninolytic enzymes. Brown-and white-rot fungi are able to produce free hydroxyl radicals from hydrogen peroxide using the Fenton reaction, after which the free hydroxyl radicals are used in the lignin degradation [7]. Although these fungi show high capabilities for lignin degradation, their growth rates are much slower than those of industrial host microorganisms, making them unsuitable industrial for biofuel production. These fungi require for long incubation times, which elevates the production costs and draws lower productivities. A few bacterial species belonging to the genera Arthrobacter, Burkholderia, Pseudomonas, Sphingobium, Streptomyces and Rhodococcus show capabilities for lignin degradation, but their activities are lower than those of fungi [8]. We therefore screened for lignin-degrading bacteria with rapid growth rates and high capacities for lignin degradation.

To obtain the lignin-degrading bacteria, filtrates were prepared from several environmental samples and plated onto lignin M9 plates (pH 7.2) containing alkali lignin as the sole carbon source, and incubated for only 2 days. Moreover, we tried more than 200 culture conditions, which were evaluated the effect of culture pH and temperature as well as oxygen utilization. When the lignin M9 plate with leaf soil filtrate was incubated aerobically at 37° C, a single colony was obtained. After standard dilution plating on Nutrient Broth plates, a purified colony was obtained and named strain CCA1. Strain CCA1 was then cultured in lignin M9 media to confirm its ability to assimilate lignin. However, strain CCA1 showed low growth (data not shown).

Phylogenetic analysis

The phylogeny of strain CCA1 was determined through 16S rRNA gene sequencing (1349 bp; accession number: LC145037). 16S rRNA gene analysis revealed that the strain CCA1 is phylogenetically related to P.citronellolis DSM 50332T (99.1%), P. delhiensis RLD-1T (99.1%) and P. jinjuensis Pss 26T (98.2%), and has lower sequence homologies with Serpensflexibilis ATCC 29606T (94.9%) and P. oleovorans IAM 1508T (94.7%). Neighborjoining phylogenetic tree reconstruction based on the 16S rRNA gene sequences revealed that strain CCA1 falls inside the cluster comprising members of the genus Pseudomonas, tightly clustering with two current members of Pseudomonas (Figure 1). Thus, strain CCA1 was identified as Pseudomonas sp. (strain number: HUT-8136).

Utilization of lignin-associated aromatic monomers

Lignin is the second-most abundant biopolymer on Earth. It is a structural component of plant cell walls and is constructed from heterogeneous aromatic macromolecules. Although the architectural components vary with the types of lignocellulosic biomasses, lignin generically consists of p-hydroxybenzene, guaiacyl (4-alkyl-2-methoxyphenol) and syringyl (4-alkyl-2,5- dimethoxyphenol) units, which are cross linked by C-C bonds (e.g., 5-5, β-1, β-5, β-β) and C-O-C bonds (e.g., 4-O-5, α-O-4, β-O-4) [9].

As described above, Pseudomonas sp. strain CCA1 shows only weak growth when cultured in lignin M9 media. This may indicate that while Pseudomonas sp. strain CCA1 has ligninolytic activity, it lacks one or more enzymes necessary to break the cross links between the main building blocks. To confirm its ligninolytic activity, Pseudomonas sp. strain CCA1 was cultured with several lignin-associated aromatic monomers as the sole carbon source. The results summarized in Figure 2 show that Pseudomonas sp. strain CCA1 is able to assimilate at least ten lignin-associated aromatic monomers. In particular, strong growth was observed with three p-hydroxybenzene monomers: 4-hydroxybenzalchol, 4-hydroxybenzaldehyde and 4-hydroxybenzoic acid. Benzoic acid and catechol were also favorable carbon sources. By contrast, consumption of vanilloids was inefficient, and anisole, o-cresol, guaiacol, syringaldehyde, syringic acid, syringol and veratryl alcohol were not assimilated.

To further evaluate the utilization of lignin-associated aromatic monomers by Pseudomonas sp. strain CCA1, TOC levels were quantitated after cultivation of samples (Table 1). If ligninassociated aromatic monomers were completely metabolized by Pseudomonas sp. strain CCA1, they would be converted to growth energy, water and inorganic carbon, which means the TOC levels, would decline. Consistent with the observed utilization of lignin-associated aromatic monomers, TOC values for p-hydroxybenzene monomers, benzoic acid and catechol were all lower than those for vanilloids (Figure 2, Table 1). These results indicate that vanilloids were partially degraded, and their downstream metabolites were slightly utilized as carbon sources by Pseudomonas sp. strain CCA1. Indeed, Pseudomonas sp. strain CCA1 was able to use vanilloids as carbon sources, though growth under those conditions was weak (Figure 2).

Incomplete lignin degradation activity

Several Pseudomonas species show lignin-degradation activity [8]. Moreover, the lignin degradation pathways of P. paucimobilis SYK-6 [10], P. putida KT2440 [11] and P. putida CSV86 [12] have been characterized. Along these pathways, lignin is decomposed into its main building blocks by laccase, lignin peroxidase, manganese peroxidase, versatile peroxidase or their isozymes. Thereafter, the main building blocks are degraded into lignin-associated aromatic monomers, which are metabolized in central carbon metabolisms. To confirm lignin degradation pathways of Pseudomonas sp. strain CCA1, draft genome sequence was determined (accession numbers: BDGS01000001 to BDGS01000024). As a result, few laccase genes were found in the draft genome sequence, but other genes were not included. These results appear Pseudomonas sp. strain CCA1 may lack the activity needed to break the crosslinks between the guaiacyl and syringyl units. The β-O-4 linkage is the most frequent inter-unit linkage in lignin, comprising more than 50% of all linkages [13]. Moreover, formation of the β-O-4 linkage corresponds to the levels of guaiacyl and syringyl units [13].

The comparatively low metabolic activities toward vanilloids are another disadvantage. In the lignin degradation pathways of Pseudomonas species [10-12], protocatechuic acid is produced from vanillin via two continuous reactions [10]. In the first reaction, vanillin dehydrogenase, which is encoded by the vdh gene, catalyzes the NAD+ -dependent oxidation of vanillin to convert vanillic acid. In the second reaction, vanillated emethylase, encoded by vanAB genes, catalyzes the NADH-dependent demethylation of veratric acid to produce protocatechuic acid, which is then gradually degraded along ortho-or meta-cleavage pathways to produce growth energy [10-12]. In draft genome sequence of Pseudomonas sp. strain CCA1, vdh gene was also not found. We think that Pseudomonas sp. strain CCA1 has also incomplete degradation pathways toward vanilloids, which also incur low ligninolytic activity.

In this study, strain CCA1 was isolated from leaf soil by screening for lignin assimilation capability. The resultant isolate was identified as Pseudomonas sp. strain CCA1based on 16S rRNA gene sequence homologies. Pseudomonas sp. strain CCA1 exhibited growth on lignin-associated aromatic monomers. In particular, Pseudomonas sp. strain CCA1effectively utilized p-hydroxybenzene monomers, benzoic acid and catechol. On the other hand, this strain lacks the ability to break the crosslinks between guaiacyl and syringyl units, or to metabolize vanilloids. These results indicate that Pseudomonas sp. strain CCA1 possesses incomplete ligninolytic activity.

We are grateful to all members of the Bio-conversion Research Group at our Institute [Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Sciences and Technology (AIST)] for their technical assistance and valuable discussion. This work was supported in part by the Science and Technology Research Partnership for Sustainable Development (SATREPS), under Japan Science and Technology Agency (JST) and Japan International Cooperation Agency (JICA).

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