Isolation and characterization of cellulose-mineralizing haloalkaliphilic bacteria from Siberian soda lakes

Abstract

Soda lakes are unique double-extreme habitats characterized by high salinity and soluble carbonate alkalinity, yet harboring rich prokaryotic life. Despite intensive microbiology studies, little is known about the identity of the soda lake hydrolytic bacteria responsible for the primary degradation of the biomass organic matter, in particular cellulose. In this study, aerobic and anaerobic enrichment cultures with three forms of native insoluble cellulose inoculated with sediments from five soda lakes in south-western Siberia resulted in the isolation of four cellulotrophic haloalkaliphilic bacteria and their four saccharolytic satellites. The final aerobic enrichment included a cellulotrophic bacteroidetes (strain ABcell3) related to Sporocytophaga accompanied by a hemicellulolytic Marinimicrobium strain ABcell2. The anaerobic enrichments resolved in three primary cellulotrophic bacteria and their three saccharolytic bacteroidetes satellites. The culture selected on amorphous cellulose (ac) included a new cellulotrophic member of the Chitinispirillaceae (Fibrobacterota)-strain ANBcel5, and two different saccharolytic satellites from the Marinilabiliales and Balneolales orders. The final enrichment selected on Sigma 101 cellulose consisted of an endospore-forming cellulotrophic strain ANBcel31 belonging to the genus Herbivorax (Acetivibrionales) and its saccharolytic satellite from the Balneolales order. The anaerobic enrichment on a filter paper yielded a binary consortium with the cellulotrophic endospore-forming Halanaerobiales strain ANBcel28 in obligate syntrophy with a cellobiose-utilizing Natronincola. A functional genome analysis of the cellulotrophic isolates confirmed the presence of a large repertoire of genes encoding excreted cellulases, mostly from the GH9 and GH5 families, and indicated that in the endospore-forming anaerobic strains, ANBcel28 and ANBcel31 most of their endo-glucanases are assembled in cellulosomes. Overall, this study showed that cellulose can be mineralized in soda lakes at moderately saline and highly alkaline conditions either by aerobic or fermentative haloalkaliphilic bacteria.

Keywords: cellulase; cellulose; cellulotrophic bacteria; haloalkaliphilic; soda lakes.

Figure 1

Enrichment and isolation scheme of soda lake bacteria involved in cellulose mineralization.

Figure 2

Cell morphology of aerobic hemicellulolytic strain ABcell2 (A,B) grown on glucomannan and macro-(C,D) and micro (E–G) morphology of aerobic cellulolytic strain ABcell3 grown on cellulose. A,B,E,F – phase contrast and B,G – electron microscopy microphotographs. (C) Dynamic of filter paper degradation in the culture of strain ABcell3; (D) Spot-colonies of strain ABcell3 forming clearance zones on soft-agar-shake plates with amorphous cellulose. (E) Colonization of amorphous cellulose particle by cells of strain ABcell3; (F,G) Cyst-like circular cells formed by strain ABcell3 during growth on amorphous cellulose.

Figure 3

Cell and colony morphology of haloalkaliphilic anaerobic cellulotrophic bacteria isolated from soda lakes. (A,B) Endospore-forming Clostridiales strain ANBcel31: (A) colonizing Sigma cellulose 101 fragments and (B) free cells growing on cellobiose. (C,D) Fibrobacterota strain ABcell5: (C) young cells colonizing cellulose particles and (D) massive lipid cyst formation during growth on amorphous cellulose. (E,F) endospore-forming Halanaerobiales strain ANBcel28: (E) colonization of a cellulose fibril fragment; (F) soft agar colonies with amorphous cellulose hydrolysis zones.

Figure 4

Phylogenetic position of haloalkaliphilic bacteria from soda lakes involved in cellulose mineralization based on concatenated amino acid sequences of 120 bacterial single-copy conserved marker proteins. (A) anaerobic cellulotrophic strain ANBcel28 within the order Halanaerobiales (class Halanaerobiia); (B) fermentative saccharolytic satellites of anaerobic cellulotrophs strains ANBcel1 and ANBcel3 within the order Balneolales (class Rhodothermia); (C) anaerobic cellulotrophic strain ANBcel5 within the class Chitinivibrionia (phylum Fibribacterota); (D) aerobic cellulotrophic strain ABcell3 within the family Cytophagaceae (class Bacteroidia); (E) aerobic hemicellulolytic satellite of cellulotrophic strain ABcell2 within the family Cellvibrionaceae (class Gammaproteobacteria); (F) anaerobic cellulotrophic strain ANBcel2 within the family Marinilabiliaceae (class Bacteroidia); and (G) anaerobic cellulotrophic strain ANBcel31 within the order Acetivibrionaceae (class Clostrodia). Taxonomic designations correspond to the Genome Taxonomy DataBase Release 09-RS220. The length of the alignment is 22,097 aa. Consensus branch support values from UFBoot and SH-like aLRT are shown at the nodes. Bars, 0.1 change per position.