Co‑cultivation of anaerobic fungi with Clostridium acetobutylicum bolsters butyrate and butanol production from cellulose and lignocellulose

Abstract

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clostridium acetobutylicum. Co-cultures were grown simultaneously (e.g., 'one pot'), and compared to cultures where bacteria were cultured in fungal hydrolysate sequentially. Fungal hydrolysis of lignocellulose resulted in 7-11 mM amounts of glucose and xylose, as well as acetate, formate, ethanol, and lactate to support clostridial growth. Under these conditions, one-stage simultaneous co-culture of anaerobic fungi with C. acetobutylicum promoted the production of butyrate up to 30 mM. Alternatively, two-stage growth slightly promoted solventogenesis and elevated butanol levels (∼4-9 mM). Transcriptional regulation in the two-stage growth condition indicated that this cultivation method may decrease the time required to reach solventogenesis and induce the expression of cellulose-degrading genes in C. acetobutylicum due to relieved carbon-catabolite repression. Overall, this study demonstrates a proof of concept for biobutanol and bio-butyrate production from lignocellulose using an anaerobic fungal-bacterial co-culture system.

Keywords: Anaerobic fungi; Biofuel; Clostridia; Consortia; RNA-Seq.

Fig. 1

Metabolic map of major fermentation products and potential for lactate cross-feeding in an anaerobic co-culture system composed of anaerobic fungi and anaerobic bacterium C. acetobutylicum. The breakdown of plant biomass (lignocellulose or cellulose) is carried out by enzymes secreted by anaerobic fungi, enabling conversion of released glucose by the fungus and bacteria. Major metabolic steps in glucose utilization are shown for the anaerobic fungus A. robustus as well as the anaerobic bacterium C. acetobutylicum. Red text is used to denote primary products produced by C. acetobutylicum under solventogenesis conditions. Blue text denotes primary products produced by C. acetobutylicum under acidogenesis conditions. Lactate (circled) is produced by both C. acetobutylicum and anaerobic fungi and it is hypothesized that C. acetobutylicum can crossfeed lactate via a mechanism for lactate metabolism based on the lactate oxidation pathway in Acetobacterium woodii. This mechanism couples a flavin adenine dinucleotide (FAD)-dependent lactate dehydrogenase with an electron flavoprotein complex to convert a reduced ferredoxin, lactate, and two oxidized nicotinamide adenine dinucleotides (NAD) to an oxidized ferredoxin, pyruvate, and two reduced nicotinamide adenine dinucleotides (NADH) (Detman et al., ; Schwalm et al., 2019). Bold arrows denote that the TPM count of at least one gene associated with the conversion is equal to or exceeds the median TPM count (491.85) for all genes expressed in the pathways shown via RNA-Seq. TPM counts for individual genes can be found in the Supplementary material. Annotations were obtained from Crown, et al., and Dash et al., ; genes associated with lactate formation were obtained from KEGG (Crown et al., ; Dash et al., ; Ogata et al., 1999). Genes associated with lactate formation are also in agreement with i802 C. acetobutylicum model (Dash et al., 2014). Image made using Biorender.

Fig. 2

(A) Schematic of the two-stage anaerobic cultivation experiment to produce butanol and butyrate from cellulose or lignocellulose. The anaerobic fungus (either C. churrovis, N. californiae, or A. robustus) was inoculated into a culture containing reed canary grass and allowed to grow for 22 days. After 22 days of fungal growth, anaerobic bacterium C. acetobutylicum was inoculated directly into fungal supernatant with the reed canary grass substrate still remaining in it and grown for 10 days. Analogous short-term experiments were also conducted, whereby anaerobic fungus A. robustus was inoculated into a culture containing filter paper and allowed to grow for 8 days to release fermentation products. After 8 days of fungal growth, anaerobic bacterium C. acetobutylicum was inoculated directly into sterile-filtered spent fungal cultures and grown for another 56 hr. (B) Schematic of a one-stage simultaneous anaerobic cultivation experiment to produce butanol and butyrate from cellulose or lignocellulose. Anaerobic fungal strains C. churrovis, A. robustus, or N. californiae were inoculated into a culture containing reed canary grass as well as C. acetobutylicum to facilitate simultaneous metabolic cross-feeding and produce butanol and butyrate over a period of 29 days. Analogous short-term experiments were also conducted, whereby anaerobic fungus A. robustus was inoculated into a culture vessel containing filter paper and allowed to grow for 24 hr prior to inoculation with C. acetobutylicum for simultaneous growth and release of fermentation products. Image made using Biorender.

Fig. 3

Cumulative pressure data for long-term experiments indicate increased pressure production in the one-stage co-cultivation condition. An arrow is used to indicate when C. acetobutylicum was added to cultures for all two-stage conditions. One-stage fermentation with both C. acetobutylicum and the indicated fungal strains led to synergistic growth and greater pressure production relative to the two-stage condition for each fungal strain tested. This provides further evidence, in addition to the HPLC data, that suggests that C. acetobutylicum cross-feeds lactate from the anaerobic fungi and benefits fungal growth. The mean value is plotted for each set of replicates and error bars indicate standard deviation.

Fig. 4

Released fermentable sugars in fungal monocultures versus fungal-bacterial co-cultures grown in M2 on reed canary grass. Sugar concentrations were measured after 29 days of microbial growth for the one-stage co-cultivation condition or 10 days of C. acetobutylicum growth for the two-stage co-cultivation condition grown in spent fungal supernatant that the fungi had grown for 22 days previously. Solid fill indicates glucose measured and patterned fill indicates xylose measured. Colors correspond to a particular fungal strain used in the monoculture or co-culture as provided in the legend. Higher levels of glucose and xylose were released compared to sucrose and arabinose (graphs for sucrose and arabinose included in the Supplementary material). With the exception of sucrose, sugars released by the fungi were significantly depleted in all experimental cultures containing C. acetobutylicum, indicating that sugars released by the fungus can sustain growth of C. acetobutylicum. The mean value is plotted for each set of replicates and error bars indicate standard deviation.

Fig. 5

Production of butyrate, lactate, and butanol in cultures grown in M2 on reed canary grass. Concentrations were measured after 29 days of microbial growth for the one-stage co-cultivation condition or 10 days of C. acetobutylicum growth in the two-stage co-cultivation condition grown in spent fungal supernatant that the fungi had grown in for 22 days previously. Lactate cross-feeding occurs in both experimental conditions. Butanol was produced in the long-term cultivation condition, in contrast to the short-term cultivation condition. Butyrate levels were significantly increased for all experimental conditions relative to controls. The mean value is plotted for each set of replicates and error bars indicate standard deviation.

Fig. 6

(A) Timecourse graph of lactate and butyrate production for C. acetobutylicum cultivated in anaerobic fungal supernatant (short-term co-cultivation) and C. acetobutylicum monoculture controls grown in Medium B the fungi had not grown in previously. Sugar release was also measured but did not exceed the limit of detection (0.1 g/l). (B) Timecourse graphs (short-term co-cultivation) of lactate and butyrate production for C. acetobutylicum co-cultured with the anaerobic fungal strain A. robustus and C. acetobutylicum monoculture controls. Sugars released were also measured but did not exceed the limit of detection in any of the cultures (0.1 g/l). Significantly higher levels of lactate were detected in the cultures in which C. acetobutylicum was co-cultivated with actively growing A. robustus, even though fungal monoculture controls did not grow enough to produce fungal metabolite levels above the limit of detection. The mean value is plotted for each set of replicates and error bars indicate standard deviation.