Organomation General Manager David Oliva recently sat down with Dr. Ryland Giebelhaus to discuss groundbreaking research on fecal microbiota transplantation (FMT) that was published in two major studies. The conversation explored how advanced analytical chemistry techniques are helping researchers understand the complex world of gut microbiome therapeutics and the critical role that live bacteria play in treating recurrent Clostridioides difficile infections.
Watch the full interview here: https://www.youtube.com/watch?v=rAeobXdVXRs
Understanding FMT: A Life-Changing Therapy
FMT involves transplanting living bacteria from a donor with a healthy gut microbiome into a recipient suffering from recurrent C. difficile infection. These infections represent a devastating challenge for patients who have undergone major gastrointestinal surgery and develop recurring bacterial infections that don't respond to antibiotics. Patients endure severely diminished quality of life and persistent diarrhea that conventional treatments fail to resolve.
The therapy has demonstrated remarkable clinical efficacy, with success rates exceeding 80% in randomized controlled trials. Beyond treating C. difficile, researchers are now exploring FMT applications for various gastrointestinal diseases and even investigating connections between gut health and mental wellbeing.
The Power of GCxGC-TOFMS in Microbiome Research
Dr. Giebelhaus, a trained chromatographer specializing in metabolomics and multidimensional chromatography, explained why comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) proved essential for this research. Bacteria in fecal microbiome transplants produce numerous volatile compounds that are biologically relevant, including short-chain fatty acids (SCFAs) that have been linked to gut health and appear to mediate positive therapeutic effects.
The GC×GC-TOFMS platform enables researchers to detect thousands of individual analytes in a single separation, providing unprecedented insight into complex biological samples. This capability proves particularly valuable for untargeted studies where researchers seek to discover unknown metabolites that may contribute positively or negatively to clinical outcomes. The technique resolves analytes away from derivatization reagents in the second dimension, achieving lower limits of detection and purer mass spectra—critical advantages when analyzing complicated biological matrices.
SPME: Simplified Sample Preparation for Volatile Analysis
One of the most innovative aspects of Dr. Giebelhaus's methodology was the use of solid-phase microextraction (SPME) for analyzing short-chain fatty acids directly from FMT cultures. This approach offered several distinct advantages that streamlined the analytical workflow.
The SPME method involved minimal sample preparation: researchers simply added salt to aqueous FMT culture media, heated the samples, and exposed an SPME fiber to the headspace. Within minutes, volatile compounds sorbed onto the fiber, which was then placed directly onto the GC column. This technique eliminated the need for chemical derivatization, reducing the risk of losing short-chain fatty acids and avoiding potential false negatives.
Dr. Giebelhaus emphasized that the method was not only analytically sound but also environmentally responsible. The only reagent used was sodium chloride, contrasting sharply with derivatization reagents that can be environmentally toxic. This greener approach aligned with modern laboratory sustainability practices while maintaining analytical rigor.
Organomation Evaporators: Supporting Critical Research
When analyzing polar metabolites in the FMT samples, Dr. Giebelhaus's team needed to extract samples in methanol, dry them down, and then derivatize them before GC×GC-TOFMS analysis. This is where Organomation equipment played a crucial role in the research workflow.
Dr. Giebelhaus fondly mentioned using an Organomation nitrogen evaporator that, while "quite old" and having "seen some stuff," reliably performed the essential task of drying down extracted samples before derivatization. The equipment needed to remove solvent while keeping water out of the samples—a critical requirement for successful GC analysis.
The researcher's positive experience with the Organomation evaporator exemplifies the company's commitment to building durable, reliable laboratory equipment. When asked about sample preparation challenges, Dr. Giebelhaus noted there weren't many issues, describing it as "a pretty streamlined process"—a testament to both the methodology and the equipment supporting it.
Live Versus Dead Bacteria: Distinct Chemical Signatures
The research revealed fascinating differences between live and dead bacterial fractions in FMT preparations. Using fluorescence-activated cell sorting, the team separated viable and non-viable bacteria, then analyzed their community structures through 16S rRNA sequencing.
Live and dead bacterial fractions exhibited statistically significant differences in both alpha-diversity (Shannon indices) and beta-diversity (Bray-Curtis dissimilarity indices). At the phylum level, Actinobacteriota showed higher abundance in live cell fractions, while Firmicutes were more abundant in dead fractions. Specific genera like Blautia, Bifidobacterium, Dorea, and Faecalibacterium predominated in live fractions, whereas Fusicatenibacter, Anaerostipes, and members of Lachnospiraceae were more abundant among dead bacteria.
Storage Matters: Shelf Life and Clinical Efficacy
One of the most clinically relevant findings concerned how storage conditions affect FMT product quality and therapeutic outcomes. While bacterial viability remained fairly stable at 10-20% across various storage conditions (comparable to fresh FMT), storage duration significantly impacted bacterial diversity.
The metabolomic analysis revealed important functional differences between frozen and lyophilized formulations. When researchers performed in vitro fiber fermentation followed by SPME-GC×GC-TOFMS analysis, they found that lyophilized formulations produced more short-chain fatty acids than frozen formulations. All linear SCFAs except propanoic acid correlated with lyophilized FMT in principal component analysis.
Most significantly, clinical outcome data from 537 patients demonstrated that storage duration negatively impacted treatment success, with the effect more pronounced for lyophilized than frozen formulations. Frozen FMT maintained greater than 75% success rates when stored up to 250 days, while lyophilized FMT achieved this threshold only up to 140 days. These findings provide empirical evidence for establishing product shelf life guidelines to optimize treatment outcomes.
Live Microbes: The Essential Therapeutic Component
A complementary study published in The Lancet Gastroenterology & Hepatology provided crucial evidence about what makes FMT effective. This multicentre, randomized, double-blinded trial compared lyophilized sterile faecal filtrate (LSFF)—free of live bacteria—with standard lyophilized donor stool (LFMT).
The trial enrolled 138 patients with recurrent C. difficile infection who were randomly assigned to receive either LSFF or LFMT. At the 8-week primary endpoint, only 65% of LSFF recipients remained recurrence-free compared to 88% of LFMT recipients—a 23% difference that exceeded the pre-specified non-inferiority margin. The study was terminated early at the recommendation of the data safety monitoring board due to inefficacy of the bacteria-free filtrate.
Dr. Giebelhaus's GC×GC-TOFMS analysis contributed to understanding these differences. While both preparations contained metabolites and cellular components, the absence of live bacteria in LSFF proved therapeutically critical. Longitudinal microbiome analysis showed that successful LFMT treatment led to recovery of depleted commensal bacterial groups like Ruminococcaceae, Oscillospiraceae, and Lachnospiraceae to healthy donor levels. This recovery was less pronounced or absent with LSFF treatment.
Implications for Synthetic Microbial Therapeutics
The research findings carry significant implications for developing next-generation microbial therapeutics. Dr. Giebelhaus suggested that GC×GC-TOFMS metabolomic profiling could guide the design of synthetic or defined microbial consortia by revealing what small molecules are present in effective FMT products.
Understanding both the volatile and non-volatile metabolome helps ensure that synthetic products preserve the beneficial small molecules found in natural FMT preparations. Researchers developing synthetic therapeutics need to consider not only which bacterial strains to include but also whether those bacteria produce relevant metabolites like short-chain fatty acids. This metabolite-informed approach may help maintain high clinical efficacy as the field moves toward standardized, manufactured microbial products.
The Future: Comprehensive Multidimensional Separations
Dr. Giebelhaus expressed enthusiasm about the expanding role of multidimensional chromatography in metabolomics research. Having recently started as a professor at the University of Victoria in Canada, he is working to establish comprehensive two-dimensional liquid chromatography mass spectrometry (LC×LC-MS) capabilities for metabolomics.
Just as GC×GC has become powerful for analyzing volatile and semi-volatile compounds, LC×LC-MS promises to cover more of the metabolome in single analyses. This technology will enable researchers to identify clinically relevant compounds, discover new metabolites, and generate valuable biological insights from fewer samples. Dr. Giebelhaus emphasized his excitement about collaborating with researchers studying unique problems with small molecules and addressing challenges using the infrastructure he's developing.
Advice for Undergraduate Researchers
When asked what undergraduate chemistry students should know about this research area, Dr. Giebelhaus emphasized the critical importance of data analysis skills. Working with metabolomics data requires strong competencies in statistics, coding, and proficient use of tools like Excel.
Untargeted metabolomics and compound discovery involve multivariate statistics that go well beyond simple t-tests. Dr. Giebelhaus noted that many undergraduate chemistry programs don't adequately teach these skills, leaving students to pursue computer science minors or learn independently. He advised students to develop strong backgrounds in data science alongside their physical science training, as this combination opens numerous opportunities in graduate research and creates employable skill sets that remain valuable even as technology evolves.
Conclusion
This research demonstrates how sophisticated analytical chemistry techniques like GC×GC-TOFMS and SPME are essential for advancing our understanding of microbiome-based therapeutics. The work confirms that live bacteria are critical for FMT efficacy and provides quantitative methods for monitoring product quality and predicting clinical outcomes.
Throughout the analytical workflow, reliable equipment like Organomation's nitrogen evaporators plays a supporting role that enables researchers to focus on scientific questions rather than technical challenges. As Dr. Giebelhaus continues pushing the boundaries of metabolomics and multidimensional chromatography, the insights gained will help develop more effective, safer microbial therapeutics for patients suffering from C. difficile infections and other dysbiosis-associated conditions.
Watch the full interview here: https://www.youtube.com/watch?v=rAeobXdVXRs