Methods
Summary
This project will investigate whether a bioengineered humic-binding peptide can enhance extracellular electron transfer in brackish tidal marsh sediments and reduce methane (CH₄) and hydrogen sulfide (H₂S) production. The experiment will use six replicated mesocosms (three controls and three treatments) constructed to mimic natural marsh conditions using intact sediment cores collected from Cattus Island County Park.
Each mesocosm will contain a gravel drainage layer, marsh sediment, brackish water, and an airtight lid supplied with nitrogen gas to maintain anoxic conditions. The treatment chambers will receive microorganisms expressing the engineered humic-binding peptide, while control chambers will remain untreated. Throughout the experiment, methane concentrations, porewater chemistry, pH, salinity, temperature, oxidation-reduction potential, and dissolved iron and sulfur species will be monitored through dedicated sampling ports.
The project combines techniques from microbial ecology, environmental chemistry, and bioengineering, including mesocosm construction, anaerobic incubation, methane monitoring, porewater sampling, and statistical comparison of replicated treatments and controls.
Challenges
- Maintaining stable anoxic conditions: Oxygen intrusion could alter microbial activity and affect methane production. To minimize this risk, the chambers will be sealed and continuously supplied with nitrogen gas.
- Biological variability: Natural marsh sediments contain complex microbial communities that can vary between samples. This will be addressed by using intact sediment cores and including three replicated chambers per treatment.
- Sensor and sampling reliability: Methane measurements and porewater analyses may be influenced by sampling error or equipment limitations. Repeated measurements over time and standardized sampling procedures will be used to improve consistency.
- Peptide effectiveness: The engineered peptide may not produce a measurable change in electron transfer or gas emissions. In that case, the project will still provide valuable information about the role of humic-mediated electron transfer in marsh sediments and help refine future bioengineering approaches.
Pre Analysis Plan
Hypothesis: Mesocosms receiving the engineered humic-binding peptide will exhibit lower methane and hydrogen sulfide production and higher indicators of iron reduction than untreated control mesocosms.
Primary outcomes:
Methane concentration over time
Hydrogen sulfide concentration
Dissolved Fe²⁺ concentration
Secondary outcomes:
Oxidation-reduction potential (ORP)
pH
Salinity
Temperature
Analysis approach:
Data will be collected repeatedly from each mesocosm throughout the experiment. Mean values and variability will be calculated for control and treatment groups. Differences between groups will be evaluated using statistical tests appropriate for replicated experiments (for example, t-tests or analysis of variance, depending on the data distribution and time points collected).
Because multiple variables are being measured, the primary interpretation will focus on methane concentration as the main outcome. Changes in iron reduction, sulfide production, and ORP will be examined as supporting evidence for altered electron transfer processes.
Replication (n = 3 per group) is included to estimate natural variance and increase confidence that observed differences are due to the treatment rather than random environmental variation.
Protocols
This project has not yet shared any protocols.
