Open Access

Complete genome sequence of the lignin-degrading bacterium Klebsiella sp. strain BRL6-2

  • Hannah L Woo1, 2, 3,
  • Nicholas R Ballor1,
  • Terry C Hazen1, 3, 4, 5,
  • Julian L Fortney1, 3,
  • Blake Simmons1, 6,
  • Karen Walston Davenport7,
  • Lynne Goodwin7,
  • Natalia Ivanova8,
  • Nikos C Kyrpides8,
  • Konstantinos Mavromatis8,
  • Tanja Woyke8,
  • Janet Jansson9,
  • Jeff Kimbrel1, 10 and
  • Kristen M DeAngelis11Email author
Standards in Genomic Sciences20149:19

DOI: 10.1186/1944-3277-9-19

Received: 21 May 2014

Accepted: 3 November 2014

Published: 8 December 2014

Abstract

In an effort to discover anaerobic bacteria capable of lignin degradation, we isolated Klebsiella sp. strain BRL6-2 on minimal media with alkali lignin as the sole carbon source. This organism was isolated anaerobically from tropical forest soils collected from the Bisley watershed at the Ridge site in the El Yunque National Forest in Puerto Rico, USA, part of the Luquillo Long-Term Ecological Research Station. At this site, the soils experience strong fluctuations in redox potential and are characterized by cycles of iron oxidation and reduction. Genome sequencing was targeted because of its ability to grow on lignin anaerobically and lignocellulolytic activity via in vitro enzyme assays. The genome of Klebsiella sp. strain BRL6-2 is 5.80 Mbp with no detected plasmids, and includes a relatively small arsenal of genes encoding lignocellulolytic carbohydrate active enzymes. The genome revealed four putative peroxidases including glutathione and DyP-type peroxidases, and a complete protocatechuate pathway encoded in a single gene cluster. Physiological studies revealed Klebsiella sp. strain BRL6-2 to be relatively stress tolerant to high ionic strength conditions. It grows in increasing concentrations of ionic liquid (1-ethyl-3-methyl-imidazolium acetate) up to 73.44 mM and NaCl up to 1.5 M.

Keywords

Anaerobic lignin degradation Tropical forest soil isolate Facultative anaerobe

Introduction

Lignin is one of the biggest barriers to efficient lignocellulose deconstruction because it occludes the action of cellulases. It is also a major waste stream after lignocellulose deconstruction. Tropical forest soils are the sites of very high rates of decomposition, accompanied by very low and fluctuating redox potential conditions [1, 2]. Because early stage decomposition is typically dominated by fungi and the free-radical generating oxidative enzymes phenol oxidase and peroxidase [3, 4], we targeted anaerobic tropical forest soils with the idea that they would be dominated by bacterial rather than fungal decomposers. Bacteria grow faster than fungi, allowing higher recombinant enzyme production for commercial use [5]. To discover organisms that were capable of breaking down lignin without the use of oxygen free radicals, we isolated Klebsiella sp. strain BRL6-2 under anaerobic conditions using lignin as the sole carbon source. In addition, this strain was observed to withstand moderately high concentrations of ionic liquids, and thus was targeted for whole genome sequencing.

Organism information

Klebsiella sp. strain BRL6-2 was isolated from soil collected from the Bisley watershed at the Ridge site in the El Yunque experimental forest, part of the Luquillo Long-Term Ecological Research Station in Luquillo, Puerto Rico, USA. A soil slurry was made with 1 gram of soil sample diluted in 100 ml of MOD CCMA media without carbon source, serially diluted and inoculated to roll tubes containing MOD CCMA media with alkali lignin as the C source. MOD CCMA media consists of 2.8 g L-1 NaCl, 0.1 g L-1 KCl, 27 mM MgCl2, 1 mM CaCl2, 1.25 mM NH4Cl, 9.76 g L-1 MES, 1.1 ml L-1 filter sterilized 1 M K2HPO4, 12.5 ml L-1 trace minerals [6, 7], and 1 ml L-1 Thauer’s vitamins [8]. Tubes were incubated at room temperature for up to 12 weeks, at which point the colony was picked from a roll tube that had been inoculated with a 10-4 dilution of soil slurry, grown in 10% tryptic soy broth (TSB), and characterized.

For initial genotyping and for validating the isolation, the small subunit ribosomal RNA gene was sequenced by Sanger sequencing using the universal primers 8 F and 1492R [9]. The 16S rRNA gene sequence places Klebsiella sp. strain BRL6-2 in the domain Bacteria, phylum Proteobacteria, class Gammaproteobacteria, and order Enterobacterales (Figure  1A). However, small subunit ribosomal RNA (16S rRNA) sequence is not sufficient to clearly define the evolutionary history of this region of the Gammaproteobacteria, so we have also constructed a hierarchical clustering of whole genomes based on pfams [10] (Figure  1B). This clustering supports the placement of Klebsiella sp. strain BRL6-2 within the order Enterobacterales.
Figure 1

Phylogenetic trees highlighting the position of Klebsiella sp. strain strain BRL6-2 relative to other type and non-type strains within the Gammaproteobacteria, based on (A) 16S ribosomal RNA phylogeny, and (B) whole genome classification based on pfams. Strains are shown with corresponding NCBI genome project ids listed within [11]. The 16S tree uses sequences aligned by the RDP aligner, the Jukes-Cantor corrected distance model to construct a distance matrix based on alignment model positions without the use of alignment inserts, and a minimum comparable position of 200. The tree is built with RDP Tree Builder, which uses Weighbor [12] with an alphabet size of 4 and length size of 1000. The building of the tree also involves a bootstrapping process repeated 100 times to generate a majority consensus tree [13]. The whole genome classification is a hierarchical clustering of pfams groups that was generated using the Integrated Microbial Genomes (IMG) system [14]. Succinimonas amylolytica DSM2873 , Succinatimonas hippei YIT12066, and Tolumonas auensis TA 4 DSM9187 are type strains with genomes available in IMG. All others are non-type strains.

Table 1

Classification and general features of Klebsiella sp. strain BRL6-2

MIGS ID

Property

Term

Evidence code a

 

Current classification

Domain Bacteria

TAS [15]

Phylum Proteobacteria

TAS [16]

Class Gammaproteobacteria

TAS [17]

Order Enterobacteriales

TAS [18]

Family Enterobacteriaceae

TAS [19]

Genus Klebsiella

TAS [20, 21]

Species Klebsiella sp. s train BRL6-2

TAS [18, 19, 22]

 

Gram stain

negative

NAS

 

Cell shape

rod

IDA

 

Motility

motile via flagella

IDA

 

Sporulation

non-sporulating

IDA

 

Temperature range

Mesophile

IDA

 

Optimum temperature

30°C

IDA

 

pH range; Optimum

8-10; 8

IDA

 

Carbon source

glucose, xylose, others (Table  7)

IDA

MIGS-6

Habitat

Tropical forest soils

TAS [23]

MIGS-6.3

Salinity

Can tolerate up to 9% NaCl, 6% KCl. Growth in 10% trypticase soy broth is improved with 0.125 M NaCl

IDA

MIGS-22

Oxygen

facultative aerobe; grows well under completely oxic and anoxic conditions

IDA

MIGS-15

Biotic relationship

free-living

IDA

MIGS-14

Pathogenicity

no

 

MIGS-4

Geographic location

Soil collected from a subtropical lower montane wet forest in the Luquillo Experimental Forest, part of the NSF- sponsored Long-Term Ecological Research program in Puerto Rico

IDA

MIGS-5

Sample collection time

July 2009

IDA

MIGS-4.1 MIGS-4.2

Latitude – Longitude

(18.268 N, 65.760 W)

IDA

MIGS-4.3

Depth

10 cm

IDA

MIGS-4.4

Altitude

375 m

IDA

(a) Evidence codes - IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [24].

Genome sequencing information

Genome project history

The genome was selected based on the ability of Klebsiella sp. strain BRL6-2 to grown on and degrade lignin anaerobically (Table  1). The genome sequence was completed on 1 February 2013, and presented for public access on April 17, 2014 by Genbank. Finishing was completed at Los Alamos National Laboratory. A summary of the project information is shown in Table  2, which also presents the project information and its association with MIGS version 2.0 compliance [25].
Table 2

Project information

MIGS ID

Property

Term

MIGS-31

Finishing quality

Permanent draft

MIGS-28

Libraries used

Illumina Std PE, Illumina CLIP, PacBio

MIGS-29

Sequencing platforms

Illumina HiSeq 2000, PacBio

MIGS-31.2

Fold coverage

Illumina Std PE 765x

Illumina CLIP PE 626x

PacBio 57x

MIGS-30

Assemblers

AllpathsLG

MIGS-32

Gene calling method

Prodigal 1.4, GenePRIMP

 

Locus tag

G360

 

Genbank ID

ARVT00000000

 

Genbank Date of Release

April 17, 2014

 

GOLD ID

Gi0021863

 

BIOPROJECT

PRJNA185290

 

Project relevance

Anaerobic lignin, switchgrass decomposition

MIGS-13

Source Material Identifier

DSM 25465

Growth conditions and DNA preparation

Klebsiella sp. strain BRL6-2 grows well aerobically and anaerobically, and was routinely cultivated aerobically in 10% tryptic soy broth (TSB) with shaking at 200 rpm at 30°C. DNA for sequencing was obtained using the Qiagen Genomic-tip kit and following the manufacturer’s instructions for the 500/g size extraction. Three column preparations were necessary to obtain 50 μg of high molecular weight DNA. The quantity and quality of the extraction were checked by gel electrophoresis using JGI standards.

Genome sequencing and assembly

The draft genome of Klebsiella sp. strain BRL6–2 was generated at the DOE Joint genome Institute (JGI) using a hybrid of the Illumina and Pacific Biosciences (PacBio) technologies. An Illumina standard shotgun library and long insert mate pair library was constructed and sequenced using the Illumina HiSeq 2000 platform [26]. All general aspects of library construction and sequencing performed at the JGI can be found at http://www.jgi.doe.gov. All raw Illumina sequence data was passed through DUK, a filtering program developed at JGI, which removes known Illumina sequencing and library preparation artifacts [27]. Filtered Illumina and PacBio reads were assembled using AllpathsLG (PrepareAllpathsInputs: PHRED 64 = 1 PLOIDY = 1 FRAG COVERAGE = 125 JUMP COVERAGE = 25; RunAllpath- sLG: THREADS = 8 RUN = standard pairs TARGETS = standard VAPI WARN ONLY = True OVERWRITE = True) [28]. For the Std PE, 25,559,315 reads were generated as raw data, and 25,511,030 (99.811%) reads were output after quality control. For the CLIP PE, 35,554,143 reads were generated as raw data, and 35,548,398 (100% but really 99.984%) reads were output after quality control. A Pacbio SMRTbellTM library was constructed and sequenced on the PacBio RS platform. 81,950 raw PacBio reads yielded 105,417 adapter trimmed and quality filtered subreads totaling 294.3 Mb. The final draft assembly contains one contig in one scaffold. The total size of the genome is 5.8 Mb, and the final assembly provides an average 1199.1X Illumina coverage and 50.7X PacBio coverage of the genome, respectively.

Genome annotation

Genes were identified using Prodigal [29] as part of the DOE-JGI annotation pipeline [30] followed by a round of manual curation using the JGI GenePRIMP pipeline [31]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform (http://img.jgi.doe.gov/er) [32].

Genome properties

The genome consists of one 5,801,355 bp circular chromosome with no discernable plasmids, and a GC content of 55.24% (Table  3). Of the 5,495 genes predicted, 5,296 were protein-coding genes, and 199 RNAs; 64 pseudogenes were also identified. The majority of the protein-coding genes (86.3%) were assigned with a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table  4.
Table 3

Genome statistics

Attribute

Value

% of Total a,b

Genome size (bp)

5,801,355

100.00%

DNA coding region (bp)

5,144,694

88.68%

DNA G + C content (bp)

3,204,653

55.24%

DNA scaffolds

1

 

Total genes

5,495

100.00%

Protein-coding genes

5,296

96.38%

RNA genes

199

3.62%

Pseudo genes

64

1.16%

Genes in internal clusters

NA

 

Genes with function prediction

4,740

86.26%

Genes assigned to COGs

4,599

83.69%

Genes assigned Pfam domains

4,904

89.24%

Genes with signal peptides

582

10.59%

Genes coding for transmembrane helices

1,330

24.20%

CRISPR repeats

NA

 

a) The total is based on either the size of the genome in base pairs or the total number of protein coding genes in the annotated genome.

b) Also includes 54 pseudogenes and 5 other genes.

Table 4

Number of genes associated with general COG functional categories

Code

Value

% of total a

Description

J

204

3.92

Translation

A

2

0.04

RNA processing and modification

K

489

9.39

Transcription

L

167

3.21

Replication, recombination and repair

B

0

0

Chromatin structure and dynamics

D

38

0.73

Cell cycle control, mitosis and meiosis

V

61

1.17

Defense mechanisms

T

213

4.09

Signal transduction mechanisms

M

261

5.01

Cell wall/membrane biogenesis

N

128

2.46

Cell motility

U

178

3.42

Intracellular trafficking and secretion

O

149

2.86

Post-translational modification, protein turnover, chaperones

C

306

5.88

Energy production and conversion

G

644

12.37

Carbohydrate transport and metabolism

E

508

9.76

Amino acid transport and metabolism

F

113

2.17

Nucleotide transport and metabolism

H

207

3.98

Coenzyme transport and metabolism

I

133

2.55

Lipid transport and metabolism

P

329

6.32

Inorganic ion transport and metabolism

Q

138

2.65

Secondary metabolites biosynthesis, transport and catabolism

R

533

10.24

General function prediction only

S

405

7.78

Function unknown

-

896

16.31

Not in COGs

a) The total is based on the total number of protein coding genes in the annotated genome.

Metabolic characterization using biolog phenotypic microarray

The Biolog phenotypic microarray was used to test Klebsiella sp. strain BRL6-2’s utilization of a variety of carbon, nitrogen, phosphorus, and sulfur sources. Different modifications of the isolation medium, MOD CCMA [33], were used to resuspend cells when inoculating different PM plates (Table  5). The scheme is similar to that used with D. vulgaris in S. Borglin et al. [34]. Plates were done iteratively to optimize each component before proceeding to the next. For all runs, a cell suspension at 0.1 OD600 and Biolog redox Dye Mix G were used to inoculate the plates. All plates were prepared in duplicate, incubated at 30°C, and read every 15 minutes for 4.5 days. PM1 and PM2 (carbon sources) were prepared anaerobically and aerobically to compare respiration. The anaerobic plates were prepared anaerobically in the anaerobic chamber in degassed medium and sealed in gas tight Whirlpak bags before loading into the Omnilog reader.
Table 5

Inoculation Fluid used for each PM plate type

PM#

Substrates on Plate

Inoculating Fluid

1

Carbon sources

MOD CCMA

2

Carbon sources

MOD CCMA

3

Nitrogen sources

20 mM Mannose MOD CCMA without NH4Cl

6

Nitrogen sources

20 mM Mannose MOD CCMA without NH4Cl

7

Nitrogen sources

20 mM Mannose MOD CCMA without NH4Cl

8

Nitrogen sources

20 mM Mannose MOD CCMA without NH4Cl

4

Phosphorus and Sulfur sources

20 mM Mannose MOD CCMA without KH2PO4 or vitamins

9

Osmolytes

20 mM Mannose MOD CCMA

10

pH

20 mM Mannose MOD CCMA

Carbon sources

190 different carbon substrates were tested using phenotypic microarray plates. The list of chemical additives that produced the highest increase in respiration relative to background is presented in Table  6. This was measured by the change in redox dye color. D-mannose was used in subsequent plates because of its convenient powder form compared to the viscous Tween solutions, which are mixtures of polyoxyethylene sorbitan esters of saturated fatty acids (predominantly 12:0, 14:0, and 16:0). They are typically used as a surfactant. Although the strain was isolated on lignin, D-cellobiose was utilized at almost the same rate as simpler carbohydrates glucose and xylose, which could suggest possible high cellulolytic activity as well.
Table 6

Carbon sources most utilized by Klebsiella sp. strain BRL6-2

Chemical Name

KEGG

Ratio to background

Tween 20

C11624

3.764

Tween 40

N/A

3.573

D-Mannose

C00159

3.678

D-Ribose

C00121

3.425

D-Fructose

C00095

3.602

D-Trehalose

C01083

3.700

N-Acetyl-D-Glucosamine

C03000

3.501

D-Xylose

C00181

3.512

Dulcitol

C01697

3.138

a-D-Glucose

C00031

3.473

D-Cellobiose

C00185

3.434

Background

 

1

Anaerobic vs. aerobic carbon source utilization

There were no significant differences between the aerobic and anaerobic utilization of the PM carbon sources. There is a vertical shift in the respiration curves, which is due to a difference in the starting OL at t = 0, as seen in negative control well A01.

Nitrogen, phosphorus, and sulfur sources

380 nitrogen sources were tested using phenotypic microarray plates. The most utilized nitrogen sources are reported in Table  7. Dipeptide amino acids were some of the most utilized sources, but ammonia from the original MOD CCMA was used in subsequent plates to avoid adding any other potential carbon source. Based on similar reasoning, phosphate was used for subsequent plates (Table  8). Within the sulfur wells, there was robust respiration in the negative control background well indicating that the buffer MES in the MOD CCMA media can serve as a possible sulfur source (Table  9). Since none of the other sulfur sources produced respiration significantly higher than background, MES will serve as the sulfur source in following plates.
Table 7

Nitrogen sources most utilized by Klebsiella sp. strain BRL6-2

N source

Ratio to Background

Gly-Asn

2.867

L-Cysteine

2.835

Gly-Gln

2.766

Allantoin

2.758

Urea

2.749

Ala-Arg

2.677

Ala-Gln

2.650

Thr-Arg

2.634

Trp-Ala

2.631

Table 8

Phosphorus sources most utilized by Klebsiella sp. strain BRL6-2

P source

Ratio to Background

Adenosine 2',3'-Cyclic Monophosphate

1.877

O-Phospho-D-Tyrosine

1.732

Thiophosphate

1.736

Tripolyphosphate

1.810

Phosphoenol Pyruvate

1.733

Cytidine 5'-Monophosphate

1.671

Pyrophosphate

1.767

Phosphate

1.757

Thymidine 5'-Monophosphate

1.677

Guanosine 2',3'-Cyclic Monophosphate

1.686

Guanosine 3'-Monophosphate

1.668

Phospho-Glycolic Acid

1.634

Background

1

Table 9

Sulfur sources most utilized by Klebsiella sp. strain BRL62

S source

Ratio to Background

Tetramethylene Sulfone

1.202

Methane Sulfonic Acid

1.122

L-Methionine Sulfoxide

1.139

N-Acetyl-D,L-Methionine

1.091

L-Djenkolic Acid

1.048

L-Methionine Sulfone

1.114

2-Hydroxyethane Sulfonic Acid

1.039

L-Cysteine Sulfinic Acid

1.090

Gly-Met

1.097

L-Methionine

1.072

Taurocholic Acid

1.020

Thiourea

1.019

Taurine

0.995

Glutathione

1.050

D,L-Lipoamide

1.002

Hypotaurine

1.007

Butane Sulfonic Acid

1.012

N-Acetyl-L-Cysteine

1.005

1-Thio-b-D-Glucose

0.966

Background

1

p-Aminobenzene Sulfonic Acid

0.993

L-Cysteine

1.018

Sulfate

0.990

Osmolyte stress response

Klebsiella sp. strain BRL6-2 was tested for respiration in a variety of osmolyte stressors and a range of pH (Table  10), with and without osmoprotectants (Table  11). For these assays, 20 mM D-Mannose MOD CCMA was used to inoculate the osmolyte response assays in Omnilog PM plates 9 and 10. Klebsiella sp. strain BRL6-2 is relatively halotolerant as it grew in increasing concentrations of NaCl up to 9%, which 1.5 M. The addition of trehalose, glycerol, octopine, and trimethylamine-N-oxide aided respiration in presence of 6% NaCl. The strain was found to be particular sensitive to sodium benzoate out of all the osmolytes tested. Klebsiella sp. strain BRL6-2 was found to respire at faster rates in pH 8–10, with the optimum at pH 8.
Table 10

Osmolyte Stress Response of Klebsiella sp. strain BRL6-2

Assay Name

Klebsiella sp. strain BRL6-2 response

NaCl Tolerance

Respiration up to 9% (1.5 M)

NaCl Tolerance with various osmoprotectants

See next table for normalized area under the curve

Potassium chloride

Respiration up to 6%

Sodium sulfate

Respiration up to 5%

Ethylene glycol

Respiration up to 20%

Sodium formate

Respiration up to 2%

Urea

Respiration up to 7%

Sodium lactate

Longer lag phase with addition of sodium lactate up to 12% but roughly same final yield.

Sodium phosphate

Respiration in 20–200 mM

Sodium benzoate

No respiration

Ammonium sulfate

Respiration in 10–100 mM

Sodium nitrate

Respiration up to 20 mM

Sodium nitrite

Respiration up to 40 mM

Table 11

Osmoprotectants utilized by Klebsiella sp. strain BRL6-2 in response to NaCl stress

Osmoprotectant

Ratio to NaCl 6% without osmoprotectant

NaCl 6%

1

NaCl 6% + KCl

0.968

NaCl 6% + Creatine

1.011

NaCl 6% + N-acethyl L-glutamine

1.031

NaCl 6% + Sarcosine

1.044

NaCl 6% + L-Carnitine

1.052

NaCl 6% + MOPS

1.121

NaCl 6% + Creatinine

1.131

NaCl 6% + gamma-amino-n-butyric acid

1.145

NaCl 6% + B-glutamic acid

1.150

NaCl 6% + Glutathione

1.154

NaCl 6% + L-proline

1.166

NaCl 6% + Trigonelline

1.195

NaCl 6% + Phosphoryl choline

1.204

NaCl 6% + Betaine

1.240

NaCl 6% + Dimethyl sulphonyl proprionate

1.262

NaCl 6% + Choline

1.285

NaCl 6% + Trimethylamine

1.329

NaCl 6% + N-N Dimethyl glycine

1.329

NaCl 6% + Ectoine

1.333

NaCl 6% + Trimethylamine-N-oxide

1.355

NaCl 6% + Octopine

1.365

NaCl 6% + Glycerol

1.371

NaCl 6% + Trehalose

1.371

Lignocellulose degradation

Because Klebsiella sp. strain BRL6-2 was initially isolated based on colony formation on minimal media with lignin supplied as the sole carbon source [35], we examined the genome to search for genes encoding putative proteins that would be associated with lignin degradation. It has a full protocatechuate pathway for processing catechol degradation to β-ketoadipate, as in Cupriavidus basilensis OR16 and Sphingomonas paucimobilis SYK6 [36, 37]. It has six putative peroxidase genes, encoding for glutathione peroxidases, DyP-type peroxidases, and catalases/peroxidases; all are potentially important for lignin degradation [38, 39]. It has two putative lactate dehydrogenase genes (EC:1.1.1.28) and two putative catalase genes (EC:1.11.1.6), and no laccase genes. It also has multiple cytochrome oxidase genes suggesting the possible use of lignin as a terminal electron acceptor as was previously observed for a related isolate Enterobacter lignolyticus SCF1 [40]. For the degradation of other relevant lignocellulose components like xylan and cellulose, Klebsiella sp. strain BRL6-2 has 2 xylanase genes, 6 β xylosidase genes, 12 β-glucosidase genes, and 2 endoglucanase genes.

Upon isolation of the strain on lignin, Klebsiella sp. strain BRL6-2’s ability to degrade several lignocellulose analogs in vitro was measured. Using a 4-methylumbelliferone based enzyme assay that has been previously used on bacterial isolates [35], cells grown in MOD CCMA plus 20 mM Mannose had high levels of β-glucosidase and xylosidase activity with 80% and 28% of the given substrate being degraded within 45 hours. However, it had low activity of cellobiohydrolase. Klebsiella sp. strain BRL6-2 was also tested for CMCase, another important class of cellulase, using a reducing sugar detection assay with 3,5-dinitrosalicylic acid (DNS) reagent and CMC [41]. No activity was detected on CMC. These low activities of cellulases could not be improved by growing cells in MOD CCMA plus 20 mM Mannose supplemented with 0.1% CMC. Although cellulose was a well-utilized substrate from the phenotypic microarray measurements, it may be due to Klebsiella sp. strain BRL6-2’s effective β-glucosidase.

Ionic liquid tolerance

Currently, ionic liquids are being investigated for their application to the bioenergy feedstock pretreatment; one of which is 1-ethyl-3-methyl-imidazolium acetate (Emim-Acetate). Klebsiella sp. strain BRL6-2 was tested for growth in 20 mM Mannose MOD CCMA in the presence of 0 mM, 36.72 mM, 73.44 mM, 146.88 mM, 293.75 mM, 587.51 mM Emim-Acetate. A 6% inoculum concentration from a 0.4 OD600 cell suspension was used to inoculate each treatment. Biolog Dye Mix G was used to monitor cell respiration during the incubation at 30°C within a Biolog reader. Klebsiella sp. strain BRL6-2 could tolerate up to 73.44 mM Emim-Acetate with increased lag phase and decreased final yields with increasing concentrations of Emim-Acetate. This is not as ionic liquid tolerant as Enterobacter lignolyticus SCF1, which was isolated in the same screen and showed tolerance of up to 500 mM 1-ethyl-3-methyl-imidazolium chloride [42]. However, Klebsiella sp. strain BRL6-2 tolerates ionic liquid concentrations higher than most bacterial strains, including E. coli, which were highly sensitive to concentrations as low as 14.69 mM. Klebsiella sp. strain BRL6-2 has 1,107 genes classified as protein coding genes connected to transporters, and these transporters are likley the source of resistance to high ionic strenght, as was also observed in E. lignolyticus SCF1 [42].

Conclusion

Klebsiella sp. strain BRL6-2 is an “Enterobacterales” in the order Gammaproteobacteria, originally isolated based on its ability to grow on lignin as sole carbon source under anaerobic conditions. Its ability to degrade lignin likely has origins in its full protocatechuate pathway, six putative peroxidase genes, two putative lactate dehydrogenase genes, and two putative catalase genes. It also has multiple cytochrome oxidase genes, suggesting the possibility of dissimilatory as well as assimilatory lignin degradation pathways. We also observed high tolerance of ionic strenght conditions, likely facilitated by its many transporter classified genes. Future experiments with Klebsiella sp. strain BRL6-2 should assess its growth kinetics on purified lignin compounds aerobically and anaerobically to determine the extent of its lignin-degrading potential. However, its fast growth, facultative lifestyle, and tolerance to high ionic strength conditions make it an attractive microbial host to bioengineer for industrial lignocellulose degradation and consolidated bioprocessing of biofuels.

Declarations

Acknowledgements

The work conducted in part by the U.S. Department of Energy Joint Genome Institute and in part by the Joint BioEnergy Institute, and is supported by the Office of Science of the U.S. Department of Energy Under Contract No. DE-AC02-05CH11231.

Authors’ Affiliations

(1)
Microbial Communities Group, Deconstruction Division, Joint BioEnergy Institute
(2)
Physical Biosciences Division, Lawrence Berkeley National Laboratory
(3)
Department of Civil & Environmental Engineering, The University of Tennessee
(4)
Department of Microbiology, The University of Tennessee
(5)
Department of Earth & Planetary Sciences, The University of Tennessee
(6)
Sandia National Lab
(7)
Los Alamos National Laboratory
(8)
Department of Energy Joint Genome Institute
(9)
Biological Sciences Division, Pacific Northwest National Laboratory
(10)
Lawrence Berkeley National Laboratory
(11)
Microbiology Department, University of Massachusetts

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