Utilization of lignocellulosic biofuel conversion residue by diverse microorganisms

Abstract

Background: Lignocellulosic conversion residue (LCR) is the material remaining after deconstructed lignocellulosic biomass is subjected to microbial fermentation and treated to remove the biofuel. Technoeconomic analyses of biofuel refineries have shown that further microbial processing of this LCR into other bioproducts may help offset the costs of biofuel generation. Identifying organisms able to metabolize LCR is an important first step for harnessing the full chemical and economic potential of this material. In this study, we investigated the aerobic LCR utilization capabilities of 71 Streptomyces and 163 yeast species that could be engineered to produce valuable bioproducts. The LCR utilization by these individual microbes was compared to that of an aerobic mixed microbial consortium derived from a wastewater treatment plant as representative of a consortium with the highest potential for degrading the LCR components and a source of genetic material for future engineering efforts.

Results: We analyzed several batches of a model LCR by chemical oxygen demand (COD) and chromatography-based assays and determined that the major components of LCR were oligomeric and monomeric sugars and other organic compounds. Many of the Streptomyces and yeast species tested were able to grow in LCR, with some individual microbes capable of utilizing over 40% of the soluble COD. For comparison, the maximum total soluble COD utilized by the mixed microbial consortium was about 70%. This represents an upper limit on how much of the LCR could be valorized by engineered Streptomyces or yeasts into bioproducts. To investigate the utilization of specific components in LCR and have a defined media for future experiments, we developed a synthetic conversion residue (SynCR) to mimic our model LCR and used it to show lignocellulose-derived inhibitors (LDIs) had little effect on the ability of the Streptomyces species to metabolize SynCR.

Conclusions: We found that LCR is rich in carbon sources for microbial utilization and has vitamins, minerals, amino acids and other trace metabolites necessary to support growth. Testing diverse collections of Streptomyces and yeast species confirmed that these microorganisms were capable of growth on LCR and revealed a phylogenetic correlation between those able to best utilize LCR. Identification and quantification of the components of LCR enabled us to develop a synthetic LCR (SynCR) that will be a useful tool for examining how individual components of LCR contribute to microbial growth and as a substrate for future engineering efforts to use these microorganisms to generate valuable bioproducts.

Keywords: Biofuel; Conversion residue; Lignocellulose; Streptomyces; Valorization; Yeasts.

Fig. 1

Composition of lignocellulosic conversion residue. Lignocellulosic conversion residue (LCR) was generated from “Cave in Rock” switchgrass harvested in 2016, subjected to AFEX and enzyme pretreatments, fermented by Zymomonas mobilis, and finally distilled to remove ethanol. The total potential chemical energy of the remaining LCR was determined by COD analysis while the remaining carbon sources, Zymomonas waste products, and other compounds that might affect future microbial metabolism were quantified via a combination of different separation techniques combined with HPLC and GC–MS analysis then converted to g/L COD to calculate the percent composition of the LCR. The most abundant components of this LCR were oligomeric sugars shown in shades of green (54.2%), monomeric sugars in shades of blue (14.7%), C1–C4 metabolites in shades of purple (10.8%) and acetamide in red (6.4%). These numbers are the average of five batches of LCR

Fig. 2

Phylogenetic trees and growth on lignocellulosic conversion residue. Phylogenetic trees of select Streptomyces (A) and yeast species (B) show the diversity within the tested strains. The bar graphs depict growth of these microorganisms in LCR as the average dry cell weight (mg/mL) of at least 2 mLs of culture from at least two biological replicates with the average dry cell weight (mg/mL) of the microbial consortium at the bottom of each panel. Streptomyces strains capable of moderate (≥ 5 mg/mL DCW) and high (≥ 10 mg/mL DCW) growth after seven days at 28 °C with shaking formed distinct phylogenetic groupings indicated as clade 1 (blue), 2 (red), and 3 (yellow) above. Similarly, the highest growing yeast species after four days rolling at room temperature were from two distinct clades: the Dipodascaceae/Trichomonascaceae clade containing the Blastobotrys or the CUG-Ser1 clade containing the Debaryomyces. The number (n) of species in condensed yeast clades is indicated, and the reported values are the mean and standard deviation of values for all species in that clade. Full growth data are available in Additional file 2: Table S2. Clade, species, and strain designations are available in Additional file 5: Table S7

Fig. 3

Utilization of lignocellulosic conversion residue by Streptomyces, yeasts, and mixed microbial consortium. Microbes were incubated in LCR then subjected to COD assays and metabolite analyses via HPLC. Streptomyces are shown in orange, yeasts in purple, and the mixed microbial consortium (MMC) in black on each panel. Percent of soluble COD utilized after incubation of indicated microbes in LCR is calculated relative to a media control. Characterized metabolites include C1–C6 compounds formate, acetate, ethanol, succinate, pyruvate, propionate, lactate, glycerol, xylitol, xylose, and glucose, as well as the glucose dimer cellobiose. The uncharacterized fraction includes all other soluble components such as oligomeric sugars, monolignols from the plant matter, AFEX pretreatment residues, cell debris, or other metabolic byproducts. Values reported are the average of at least two biological replicates with standard deviation denoted by error bars

Fig. 4

Lignocellulosic conversion residue metabolite utilization by Streptomyces, yeasts, and mixed microbial consortium. Patterns of indicated characterized metabolites present in LCR after incubation with microbes are shown relative to media controls. Analysis of at least two biological replicates was averaged. Metabolites that were present in lower levels than the media control are shown in red, metabolites that were present in higher levels than the media control are shown in blue, and white indicates no change relative to the media control. This gives a pattern of characterized metabolite consumption (red) and generation (blue) for these LCR degrading microbes

Fig. 5

Utilization of synthetic conversion residue by Streptomyces and mixed microbial consortium. Microbes were incubated aerobically in synthetic conversion residue (SynCR) with crystalline cellulose and with or without lignocellulose-derived inhibitors (LDIs) for seven days then subjected to COD assays and metabolite analyses via HPLC. The bars labeled SynCR show the metabolite levels in the uninoculated media controls while the remaining bars indicate the amounts of the those metabolites present in spent SynCR after 7 days of incubation with either the mixed microbial consortium (MMC) or the indicated Streptomyces strains. Values reported are the average amounts of metabolites remaining after incubation from 3 biological replicates

Fig. 6

Distribution of bacterial taxa in the mixed microbial consortium on different growth media. Bacterial taxa were identified within the initial inoculum source and following a 7-day incubation period in LCR or SynCR with crystalline cellulose and with and without LDIs. Individual OTUs were clustered to the highest taxonomic level (c, class; o, order; g, genus), with clusters greater than 1% total relative abundance shown above, organized by phylum (Pa., Patescibacteria; Sa., Saccharibacteria; Bacter., Bacteroidota; Actino., Actinobacteriota). Taxa with distinct differences in abundance between the microbial consortia grown on different types of CR are indicated in bold