Practical Guide to Measuring Wetland Carbon Pools and Fluxes

Wetlands cover a small portion of the world, but have disproportionate influence on global carbon (C) sequestration, carbon dioxide and methane emissions, and aquatic C fluxes. However, the underlying biogeochemical processes that affect wetland C pools and fluxes are complex and dynamic, making measurements of wetland C challenging. Over decades of research, many observational, experimental, and analytical approaches have been developed to understand and quantify pools and fluxes of wetland C. Sampling approaches range in their representation of wetland C from short to long timeframes and local to landscape spatial scales. This review summarizes common and cutting-edge methodological approaches for quantifying wetland C pools and fluxes. We first define each of the major C pools and fluxes and provide rationale for their importance to wetland C dynamics. For each approach, we clarify what component of wetland C is measured and its spatial and temporal representativeness and constraints. We describe practical considerations for each approach, such as where and when an approach is typically used, who can conduct the measurements (expertise, training requirements), and how approaches are conducted, including considerations on equipment complexity and costs. Finally, we review key covariates and ancillary measurements that enhance the interpretation of findings and facilitate model development. The protocols that we describe to measure soil, water, vegetation, and gases are also relevant for related disciplines such as ecology. Improved quality and consistency of data collection and reporting across studies will help reduce global uncertainties and develop management strategies to use wetlands as nature-based climate solutions. Supplementary Information The online version contains supplementary material available at 10.1007/s13157-023-01722-2.

and Kok 1963) to separate volatile organic compounds from water or air by a thin membrane and has been employed in on-line and real-time analyses in industrial processes (e.g., fermentation, water chlorination) and environmental monitoring (e.g., urban air plumes, municipal tap water) (Ketola et al. 2002).Based on our knowledge, the first application of MIMS to wetland samples for determination of carbon dioxide (CO2) and methane (CH4) concentrations was conducted by Lloyd et al. (1986).Since then, MIMS has been used in the study of greenhouse gases (GHGs) in marine sediments (Bell et al. 2012), peat cores (Benstead and Lloyd 1996;Beckmann et al. 2004), wetland soils (Askaer et al. 2010;Elberling et al. 2011), terrestrial ecosystems, andgrassland systems (Sheppard andLloyd 2002).
This approach typically uses a semi-permeable polymer to enrich certain analytes from gaseous or liquid samples.As solutions tangentially cross the membrane, analytes are partitioned across the membrane while the bulk of the matrix is rejected.Analytes pass through the membrane at rates that depend on their solution concentration, their solubility in the membrane, and their diffusivity in the membrane.Analyte concentration is at maximum on the high-pressure side (sample side) of the membrane and falls to a minimal value on the vacuum side.These separated analytes are then directly transferred as mixtures (often using a helium carrier gas acceptor phase) to a mass spectrometer for their subsequent resolution and measurements.
The MIMS device consists of a vacuum inlet fitted with a permeable silicone tube.The inlet allows gas to pass into the vacuum system, where it is routed through a cold trap (typically dry ice) and into a quadrapole mass spectrometer.Water from samples or a standard is pumped through the membrane using a peristaltic pump.Partial pressure data are acquired on the data acquisition system in multiple ion-monitoring mode and can be processed using standard spreadsheet software.
Typically, a long stainless steel gas inlet capillary probe (1.56 mm outside diameter, 0.5 mm inside diameter) with a 50 µm diameter orifice near the tip, is used to insert into the soil core (Sheppard and Lloyd 2002).The advantage of MIMS is that it can be used to quantify a number of gas species, continuously and simultaneously, and it can record spatial and temporal variations in subsurface gas concentrations as low as 1 µM (Lloyd and Scott 1985;Lloyd and James 1987).
Based on the mass-to-charge ratio (m/Z) of characteristic positive ions of gases, a variety of gases can be monitored (e.g., m/z = 15: CH4, m/z = 32: oxygen (O2), m/z= 44: CO2).This technique enables the direct measurement of multiple gas species throughout soil cores with minimal disturbance.The MIMS device is also a field portable instrument (Etzkorn et al. 2009).
Perhaps the only disadvantage is the high operating cost for purchasing and maintaining the instrument.Although the instrument is considered portable, the gas chromatography-mass spectrometry power requirement access to remote areas is still a difficult task.

Macroalgae: Nutritional values and chemical analysis
Marine macroalgae (seaweeds, kelp) are an economically valued, renewable resource of food, biofuel, and biofertilizer.Food consumption of brown, red, and green macroalgae can be largely attributed to its nutrition properties, which also make it sought-after for fodder, fertilizer, cosmetics, and medicines (Robledo and Freile Pelegrín 1997;Dawes 1998;McHugh 2003;Banerjee et al. 2020).In terms of human consumption and nutrition, macroalgae are excellent sources of proteins, lipids, carbohydrates, minerals, vitamins, antioxidants, and phytochemicals, and thus provide numerous health benefits (Table S1; Parekh and Chauhan 1982;Kumari et al. 2010;Holdt and Kraan 2011;van Ginneken et al. 2011;Banerjee et al. 2020;Ganesan et al. 2020;Lozano Muñoz and Díaz 2022).Globally, it is estimated that around 8 million tons of macroalgae are harvested annually to support its many uses (McHugh 2003).The exploitation of marine algae for nutritional purposes is primarily based on its biochemical constituents (Parekh and Chauhan 1982).Macroalgae show great variation in nutrient content based on species, level of maturity, geographical distribution, and environmental conditions like seawater temperature, salinity, light, and nutrients (Dawes 1998).

Protein, carbohydrate, lipid, and astaxanthin chemical analyses
Protein analysis following the method originally from Lowry et al. (1951).About 0.1 g of powered macroalgae is extracted with trisodium phosphate (Na3PO4) (buffer pH = 0.7) and centrifuged.An aliquot of sample extract is added to a reagent of sodium carbonate (Na2CO3) and another reagent of copper(II) sulfate (CuSO4).Then, Folin-Ciocalteu phenol reagent (2:1) is added and left undisturbed for 30 minutes for color development.The intensity of the color is measured at 660 nm.For quantifying the protein content of the sample, a standard curve is prepared with a known concentration of bovine serum albumin as standard.The value is expressed in percentage.For additional information regarding protein measurement using the Folin phenol reagent (Lowry et al. 1951) see reviews by Peterson (1979) and Singleton et al. (1999), an application by Ledoux and Lamy (1986), and an assessment of the Folin-Ciocalteu reagent by Everette et al. (2010).
Carbohydrate content can be estimated by using the procedure of Sadasivam and Manickam (2007).Dried macroalgae powder (0.1 g) is extracted with 80% methanol and centrifuged.This extraction is repeated twice, and the pooled supernatant is evaporated until the methanol is removed.The sample extract is then combined with anthrone reagent and the absorbance is measured at 630 nm using a spectrophotometer.The value is expressed as mg g −1 (dry weight) or percentage using glucose as standard.
The lipid contents of dried macroalgal samples can be determined by continuous extraction in a lipid extractor (Soxhlet Apparatus, Folch et al. 1957) for 3 hours using petroleum ether as a solvent.Astaxanthin content can be estimated using the procedure of (Banerjee et al. 2009).Dried powdered seaweed is extracted with dimethyl sulfoxide and centrifuged until the extract is colorless.Absorbance is measured at 471 to 477 nm.
Macroalgal biomass often varies seasonally and can be affected by several abiotic and biotic factors such as salinity, temperature, pH, and nutrient concentrations (Banerjee et al. 2009).Thus, it is important to collect key covariates and ancillary variables when sampling macroalgae (Fig. S1).

Fig. S1
Fig. S1 Macroalgal (seaweed) growth on rocky surfaces along the coast of India.Student Prajna Paramita Mohapatra (Banerjee lab) collecting macroalgae (seaweeds) by hand scraping biomass from within a sample quadrat from Vishakhapatnam coast of Andhra Pradesh in western Bay of Bengal, India.Images with permission from Banerjee and Mohapatra