Engineering topics

Ruhl, Ilona A.

Publications and source records attributed to Ruhl, Ilona A..

3 records

DOE OSTI2024

Feedstock variability impacts the bioconversion of sugar and lignin streams derived from corn stover by Clostridium tyrobutyricum and engineered Pseudomonas putida

Abstract Feedstock variability represents a challenge in lignocellulosic biorefineries, as it can influence both lignocellulose deconstruction and microbial conversion processes for biofuels and biochemicals production. The impact of feedstock variability on microbial performance remains underexplored, and predictive tools for microbial behaviour are needed to mitigate risks in biorefinery scale‐up. Here, twelve batches of corn stover were deconstructed via deacetylation, mechanical refining, and enzymatic hydrolysis to generate lignin‐rich and sugar streams. These batches and their derived streams were characterised to identify their chemical components, and the streams were used as substrates for producing muconate and butyrate by engineered Pseudomonas putida and wildtype Clostridium tyrobutyricum , respectively. Bacterial performance (growth, product titers, yields, and productivities) differed among the batches, but no strong correlations were identified between feedstock composition and performance. To provide metabolic insights into the origin of these differences, we evaluated the effect of twenty‐three isolated chemical components on these microbes, including three components in relevant bioprocess settings in bioreactors, and we found that growth‐inhibitory concentrations were outside the ranges observed in the streams. Overall, this study generates a foundational dataset on P. putida and C. tyrobutyricum performance to enable future predictive models and underscores their resilience in effectively converting fluctuating lignocellulose‐derived streams into bioproducts.

09 BIOMASS FUELS↗

DOE OSTI2022

GAL08, an Uncultivated Group of Acidobacteria, Is a Dominant Bacterial Clade in a Neutral Hot Spring

GAL08 are bacteria belonging to an uncultivated phylogenetic cluster within the phylum Acidobacteria . We detected a natural population of the GAL08 clade in sediment from a pH-neutral hot spring located in British Columbia, Canada. To shed light on the abundance and genomic potential of this clade, we collected and analyzed hot spring sediment samples over a temperature range of 24.2–79.8°C. Illumina sequencing of 16S rRNA gene amplicons and qPCR using a primer set developed specifically to detect the GAL08 16S rRNA gene revealed that absolute and relative abundances of GAL08 peaked at 65°C along three temperature gradients. Analysis of sediment collected over multiple years and locations revealed that the GAL08 group was consistently a dominant clade, comprising up to 29.2% of the microbial community based on relative read abundance and up to 4.7 × 10 5 16S rRNA gene copy numbers per gram of sediment based on qPCR. Using a medium quality threshold, 25 single amplified genomes (SAGs) representing these bacteria were generated from samples taken at 65 and 77°C, and seven metagenome-assembled genomes (MAGs) were reconstructed from samples collected at 45–77°C. Based on average nucleotide identity (ANI), these SAGs and MAGs represented three separate species, with an estimated average genome size of 3.17 Mb and GC content of 62.8%. Phylogenetic trees constructed from 16S rRNA gene sequences and a set of 56 concatenated phylogenetic marker genes both placed the three GAL08 bacteria as a distinct subgroup of the phylum Acidobacteria , representing a candidate order ( Ca. Frugalibacteriales) within the class Blastocatellia. Metabolic reconstructions from genome data predicted a heterotrophic metabolism, with potential capability for aerobic respiration, as well as incomplete denitrification and fermentation. In laboratory cultivation efforts, GAL08 counts based on qPCR declined rapidly under atmospheric levels of oxygen but increased slightly at 1% (v/v) O 2 , suggesting a microaerophilic lifestyle.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Dissecting the dominant hot spring microbial populations based on community-wide sampling at single-cell genomic resolution

With advances in DNA sequencing and miniaturized molecular biology workflows, rapid and affordable sequencing of single-cell genomes has become a reality. Compared to 16S rRNA gene surveys and shotgun metagenomics, large-scale application of single-cell genomics to whole microbial communities provides an integrated snapshot of community composition and function, directly links mobile elements to their hosts, and enables analysis of population heterogeneity of the dominant community members. To that end, we sequenced nearly 500 single-cell genomes from a low diversity hot spring sediment sample from Dewar Creek, British Columbia, and compared this approach to 16S rRNA gene amplicon and shotgun metagenomics applied to the same sample. We found that the broad taxonomic profiles were similar across the three sequencing approaches, though several lineages were missing from the 16S rRNA gene amplicon dataset, likely the result of primer mismatches. At the functional level, we detected a large array of mobile genetic elements present in the single-cell genomes but absent from the corresponding same species metagenome-assembled genomes. Moreover, we performed a single-cell population genomic analysis of the three most abundant community members, revealing differences in population structure based on mutation and recombination profiles. While the average pairwise nucleotide identities were similar across the dominant species-level lineages, we observed differences in the extent of recombination between these dominant populations. Most intriguingly, the creek's Hydrogenobacter sp. population appeared to be so recombinogenic that it more closely resembled a sexual species than a clonally evolving microbe. Together, this work demonstrates that a randomized single-cell approach can be useful for the exploration of previously uncultivated microbes from community composition to population structure.

59 BASIC BIOLOGICAL SCIENCES↗