Rapid Analytical Method for the Quantification of Bromoform in the Red Seaweeds Asparagopsis armata and Asparagopsis taxiformis Using Gas Chromatography–Mass Spectrometry

Abstract

The red seaweed Asparagopsis is rich in bromoform (CHBr3) which effectively inhibits the production of methane in ruminants, and ongoing global research is investigating the potential to utilize Asparagopsis as a feed additive to mitigate livestock methane emissions. Due to its volatility, the concentration of bromoform in the harvested seaweed is affected by processing and storage conditions, and its quantification is thus not trivial. The accurate determination of bromoform in biomass is required for correct inclusion-response calculations and minimum effective feed inclusion rates, for interstudy comparisons, and for optimizing processing protocols. However, no such standardized protocol, with appropriate quality control and quality assurance in place, exists. Here, we present a single-laboratory validation of a simple and rapid GC-MS method for quantifying the concentration of bromoform in seaweeds with a lower limit of quantitation of 0.26 mg/g in freeze-dried material. The method is employed to assess the concentrations of bromoform in frozen and dried Asparagopsis armata and Asparagopsis taxiformis biomass. The linearity, sensitivity, precision, accuracy, and robustness of the assay are presented. In addition, the assay was implemented in two independent laboratories, and the results were compared. The assay has been accredited by the International Accreditation New Zealand (IANZ) as suitable for assessing the concentration of bromoform of frozen and freeze-dried seaweed. Furthermore, the effects of sample storage conditions and freeze-drying of seaweed were investigated, with results highlighting the need of controlled seaweed handling with implications for downstream processing into a potential commercial feed additive.

Effect of brown and green seaweeds on diet digestibility, ruminal fermentation patterns and enteric methane emissions using the rumen simulation technique

“…2 experiment 2. 3 Phlorotannins (mg/g DM) analysed using the method ofLopes et al (2012).4 Bromoform (mg/g DM) analysed using the method ofRomanazzi et al (2021) for ASP1 andRoque et al (2019) for ASP2.…”

Identification of reproductive sex‐biased gene expression in Asparagopsis taxiformis (lineage 6) gametophytes

“…Bromoform is a volatile halogenated compound and is the most abundant bioactive in Asparagopsis spp. (Romanazzi et al, 2021). Yet, bromoform concentration in the seaweed is variable and depends on growth temperature, spatial origin of the isolates, biomass storage conditions, and detection method (Zanolla, Romanazzi, et al, 2022), which makes comparisons challenging.…”

“…From an omics standpoint, recent advances in complete genome assemblies (Thapa et al., 2020; Zhao et al., 2022) and proteomics (Patwary et al., 2023) could facilitate further research of interest, such as the selective breeding of A. taxiformis with high bromoform content if bromoform content is heritable. Bromoform, a halogenated secondary metabolite, is the most abundant, antimethanogenic compound in Asparagopsis (Romanazzi et al., 2021). Recent research has pinpointed the marine bromoform biosynthesis locus ( mbb locus) as the location of key genes involved in the biosynthesis of bromoform (Thapa et al., 2020); however, there is no information regarding whether there is any sex‐bias in gene expression that relates to reported differences in bromoform concentration between females and males (Vergés et al., 2008).…”

Asparagopsis Genus: What We Really Know About Its Biological Activities and Chemical Composition

“…The halomethane-type compounds are most often responsible for the effect of inhibiting ruminant microbial methanogenesis [ 171 ]. Taking into account that the main compound responsible for this activity seems to be bromoform, a standardized protocol for the quantification of this secondary metabolite in biomass was developed by Romanazzi et al [ 172 ]. However, it cannot be ruled out that other secondary metabolites, such as tannins and flavonoids, may exert this anti-methanogenic effect [ 173 ].…”