Metagenomics and cultivation of a member of the Group 4 Aigarchaeota from the thermophilic lignocellulosic consortia

Lizett J. Gonzalez, California State University - San Bernardino

Abstract

Aigarchaeota is a thermophilic lineage within in the domain Archaea that does not have any cultivated representatives. Based on phylogenetic inferences, Aigarchaeota might represent a phylum or a deeply branching member of the Thaumarchaeota within TACK super phylum, which has been proposed to be a sister lineage to the progenitor of eukaryotes. Group 4 Aigarchaeota (Aig G4), one of nine genus level groups that have been detected so far, was previously found to be enriched in lignocellulose (corn stover) in situ enrichments at an ~85 °C site in Great Boiling Spring (GBS), NV. To attempt to cultivate Aig G4 in the laboratory, twelve anaerobic enrichment media conditions were designed and inoculated using Aig G4 corn stover in situ enrichments in the field. Maintenance of Aig G4 in enrichment cultures was assessed by quantitative polymerase chain reaction using primers specific for a region of the Aig G4 16S rRNA gene. Aig G4 was successfully maintained at 1-5% of total 16S rRNA gene copies and 0.5–2x106 16S rRNA gene copies/mL in two enrichment cultures. These cultures were grown in anaerobic conditions at 80 °C on semi-synthetic media containing GBS spring water with corn stover, keratin, and xyloglucan as major growth substrates and either HEPES or bicarbonate as buffers. Further experiments determined that corn stover, but neither keratin nor xyloglucan, could serve as a sole substrate to support growth of Aig G4. No growth of Aig G4 was observed under microaerophilic conditions, suggesting that it may be strictly anaerobic. No growth was observed in the presence of 0.1 mM thiosulfate and nitrate, suggesting that one or both of these compounds are toxic to Aig G4. Aig G4 were not maintained in synthetic medium without spring water or supplemented with a bulk organic extract derived from GBS spring water, suggesting that one or more inorganic components of GBS water may be required for growth. Fluorescence in situ hybridization (FISH) was also used to visualize the presence and morphology of Aig G4 in the in situ corn stover enrichments. These laboratory enrichment cultures will serve as a basis for testing substrate utilization of Aig G4 using stable isotope labeling experiments and FISH-nanoSIMS. Furthermore, studying the growth of Aig G4 and other thermophilic microbes on corn stover will help us better understand the degradation of cellulosic material at high temperatures.

This work was supported by NSF grant DEB-1557042.

 

Metagenomics and cultivation of a member of the Group 4 Aigarchaeota from the thermophilic lignocellulosic consortia

Aigarchaeota is a thermophilic lineage within in the domain Archaea that does not have any cultivated representatives. Based on phylogenetic inferences, Aigarchaeota might represent a phylum or a deeply branching member of the Thaumarchaeota within TACK super phylum, which has been proposed to be a sister lineage to the progenitor of eukaryotes. Group 4 Aigarchaeota (Aig G4), one of nine genus level groups that have been detected so far, was previously found to be enriched in lignocellulose (corn stover) in situ enrichments at an ~85 °C site in Great Boiling Spring (GBS), NV. To attempt to cultivate Aig G4 in the laboratory, twelve anaerobic enrichment media conditions were designed and inoculated using Aig G4 corn stover in situ enrichments in the field. Maintenance of Aig G4 in enrichment cultures was assessed by quantitative polymerase chain reaction using primers specific for a region of the Aig G4 16S rRNA gene. Aig G4 was successfully maintained at 1-5% of total 16S rRNA gene copies and 0.5–2x106 16S rRNA gene copies/mL in two enrichment cultures. These cultures were grown in anaerobic conditions at 80 °C on semi-synthetic media containing GBS spring water with corn stover, keratin, and xyloglucan as major growth substrates and either HEPES or bicarbonate as buffers. Further experiments determined that corn stover, but neither keratin nor xyloglucan, could serve as a sole substrate to support growth of Aig G4. No growth of Aig G4 was observed under microaerophilic conditions, suggesting that it may be strictly anaerobic. No growth was observed in the presence of 0.1 mM thiosulfate and nitrate, suggesting that one or both of these compounds are toxic to Aig G4. Aig G4 were not maintained in synthetic medium without spring water or supplemented with a bulk organic extract derived from GBS spring water, suggesting that one or more inorganic components of GBS water may be required for growth. Fluorescence in situ hybridization (FISH) was also used to visualize the presence and morphology of Aig G4 in the in situ corn stover enrichments. These laboratory enrichment cultures will serve as a basis for testing substrate utilization of Aig G4 using stable isotope labeling experiments and FISH-nanoSIMS. Furthermore, studying the growth of Aig G4 and other thermophilic microbes on corn stover will help us better understand the degradation of cellulosic material at high temperatures.

This work was supported by NSF grant DEB-1557042.