Methane Reduction Potential of Two Pacific Coast Macroalgae During in vitro Ruminant Fermentation

With increasing interest in feed-based methane mitigation strategies and regional legal directives aimed at methane production from the agricultural sector, identifying local sources of biological feed additives will be critical for rendering these strategies affordable. In a recent study, the red alga Asparagopsis taxiformis harvested offshore Australia was identified as highly effective for reducing methane production from enteric fermentation. Due to potential difference in methane-reduction potential and the financial burden associated with transporting the harvested seaweed over long distances, we examined locally sourced red seaweed A. taxiformis and brown seaweed Zonaria farlowii for their ability to mitigate methane production when added to feed widely used in the Californian dairy industry. At a dose rate of 5% dry matter (DM), California-sourced A. taxiformis and Z. farlowii reduced methane production by up to 74% (p < 0.05) and 11% (p < 0.05) during in vitro rumen fermentation, respectively. No effect on CO2 production was observed for either seaweed. The measured decrease in methane production induced by A. taxiformis and Z. farlowii amendment, suggest that these local macroalgae are indeed promising candidates for biotic methane mitigation strategies in California, the largest milk producing state in the United States. To determine their real potential as methane mitigating feed supplements in the dairy industry, their effect in vivo will need to be investigated.


INTRODUCTION
Methane (CH 4 ), produced by human activity, accounts for more than 10% of the total greenhouse gases emitted in the United States (EPA, 2020) and enteric fermentation from ruminant animals accounts for approximately 25% of the total anthropogenic methane produced (NASEM, 2018). Thus, efficient strategies to lower enteric CH 4 production would enable a significantly reduced carbon footprint of agriculture and more specifically of animal production.
In vitro studies have demonstrated that some brown and red macroalgae can inhibit microbial methanogenesis (Machado et al., 2014;Roque et al., 2019) and they have been suggested as feed supplements to reduce methanogenesis during enteric fermentation (Wang et al., 2008;Dubois et al., 2013;Machado et al., 2016). Utilization of these macroalgae could also promote higher growth rates and feed conversion efficiencies in ruminants via the potential net energy yield from the redistribution of energy from the microbial methanogenesis pathway into more favorable pathways for the animal (i.e., volatile fatty acids) (Hansen et al., 2003;Marin et al., 2009). Therefore, macroalgae feed supplementation may be an effective strategy to simultaneously improve profitability and sustainability of beef and dairy operations.
In a recent study (Machado et al., 2014), the red macroalgae Asparagopsis taxiformis stood out as the most effective species from a panel of tested seaweed to reduce methane production. In this screening effort, the effect of a variety of macroalgal species including freshwater, green, red, and brown algae on CH 4 production during in vitro incubation were compared, and results showed that A. taxiformis amendment yielded the most significant reduction (∼98.9%) of CH 4 production.
A major challenge in the implementation of an A. taxiformisbased methane mitigation strategy is the availability of this bioactive seaweed, which has led to the exploration of alternative seaweed species. Given that California, which is the largest milk producing state in the United States, has a shoreline of 1,350 km, it is imperative that local seaweeds are tested for methane mitigation properties in order to reduce the financial burden associated with harvesting the seaweed at locations that require long storage and transportation. Besides the economical aspect, the identification of local seaweed is of particular importance since the key halogenated compound responsible for macroalgae's methane mitigation abilities, bromoform, differs in concentration across both location and season (Carpenter and Liss, 2000). Here, we tested the potential of two different species of subtidal macroalgae (A. taxiformis and the brown alga, Zonaria farlowii) collected from offshore Southern California for their ability to mitigate methane production during in vitro rumen fermentation.

Experimental Design
To determine the effect of two locally sourced macroalgae species on methane production during in vitro rumen fermentation, A. taxiformis and Z. farlowii were supplemented to an in vitro gas production system at a dose rate of 5% DM. This dose rate showed significant methane reduction during in vitro rumen fermentation for Australia-sourced A. taxiformis Roque et al., 2019). Rumen fluid was diluted threefold with artificial saliva buffer (Oeztuerk et al., 2005). After homogenization, 200 mL of the mixture was allocated to 300 mL vessels fitted with Ankom head units (Ankom Technology RF Gas Production System, Macedon, NY, United States). Each vessel received 2 g of rumen solids, and 2 g of a basic ration (Super Basic Ration; SBR; 70% alfalfa, 15% dried distillers' grain, and 15% rolled corn) commonly used in the dairy industry in California. Rumen solids and SBR were sealed in separate Ankom 5 cm × 5 cm concentrate feed bags and seaweed was included in the respective SBR feed bags (Ankom, Macedon, NY, United States). Control vessels were run in parallel with each seaweed treatment. Vessels were placed and incubated in a temperature controlled shaking water bath (39 • C; 40 rpm). Foil gas bags (Restek, United States) were connected to the Ankom head units to collect gas at 24 and 48 h, respectively. The experimental design of this study is summarized in Figure 1.

Rumen Fluid Collection
All animal procedures were performed in accordance with the Institution of Animal Care and Use Committee (IACUC) at the University of California, Davis, under protocol number 19263. Rumen content was collected from a rumen fistulated Holstein cow, housed at the UC Davis Dairy Research and Teaching Facility Unit. The rumen content donor was fed a dry cow total mixed ration (50% wheat hay, 25% alfalfa hay/manger cleanings, 21.4% almond hulls, and 3.6% mineral pellet). Two liters of rumen fluid and 30 g of rumen solids were collected 90 min after morning feeding. Rumen content was collected via transphonation using a perforated PVC pipe, 500 mL syringe, and Tygon tubing (Saint-Gobain North America, PA, United States). Fluid was strained through a colander and four layers of cheesecloth into a 4 L pre-warmed, vacuum insulated container and transported to the laboratory. Rumen fluid and solids were collected on May 29, 2018 and June 19, 2018 for the in vitro trial to determine methane-mitigation potential of A. taxiformis and Z. Farlowii, respectively. Trials were started on the day of rumen fluid collection.

Greenhouse Gas Analysis
Methane and CO 2 were measured from gas bags using an SRI Gas Chromatograph (8610C, SRI, Torrance, CA, United States) fitted with a 3 × 1/8 stainless steel Haysep D column and a flame ionization detector (FID) with a methanizer. The oven temperature was held at 90 • C for 5 min. Carrier gas was high purity hydrogen at a flow rate of 30 mL/min. The FID was held at 300 • C. A 1 mL sample was injected directly onto the column. Calibration curves were developed with Airgas certified CH 4 and CO 2 standard (Airgas, United States). FIGURE 1 | Experimental design. Rumen content was collected from a rumen fistulated Holstein cow and rumen fluid was diluted threefold with saliva buffer after rumen solids and fluid were separated. Fluid/buffer mixture was amended with rumen solids and Super Basic Ration. To test effect of Asparagopsis taxiformis and Zonaria farlowii, fermentation vessels were supplemented with 5% dry matter (DM) of the corresponding seaweed. Carbon dioxide (CO 2 ), methane (CH 4 ) production and volatile acid profiles were determined after 24 and 48 h.

Statistical Analysis
Differences in CH 4 and CO 2 production between sampling time points for each seaweed were determined using unpaired parametric t-tests with Welch's correction conducted in Graphpad Prism 7 (Graphpad Software Inc., La Jolla, CA, United States). Significant differences among treatments were declared at p < 0.05.

Biochemical Analysis of Asparagopsis taxiformis and Zonaria farlowii
Biochemical analysis of the two Pacific Coast seaweeds revealed a stark difference in their bromoform composition (2,305 µg g −1 dry weight for A. taxiformis and 35 µg g −1 dry weight for Z. farlowii) as well as in their nutritional composition (Table 1). Additionally, crude protein and starch content was significantly (1.9-and 8-fold, respectively) higher, whereas crude fat and nonfiber carbohydrates were (2.2-and 2.6-fold, respectively) lower in A. taxiformis compared to Z. farlowii. Concentrations of iron were almost twice as high in Z. farlowii (1,765 ppm) compared to A. taxiformis (939 ppm).

DISCUSSION
With increasing interest in feed-based methane mitigation strategies, fueled by legal directives aimed at reducing methane production from the agricultural sector, identification of local biotic feed-supplements will be critical to render large-scale methane mitigation strategies economical.
Our data indicate that subtidal macroalgae from Santa Catalina Island, Southern California reduced the in vitro production of CH 4 when added to rumen content from California dairy cattle. The addition of A. taxiformis reduced microbial methane production by up to ∼78%, which is slightly lower but similar to the reduction (∼95%) of enteric methane FIGURE 3 | Methane and CO 2 production during in vitro fermentation of rumen fluid amended with A. taxiformis. Production of CH 4 mL (g DM) −1 and CO 2 mL (g DM) −1 from vessels without (n = 4) and with 5% (n = 4) A. taxiformis as additive. Methane and CO 2 were measured at 24 h (A,B, respectively) and 48 h (C,D, respectively). " * " indicates significant difference (p-value < 0.05), "ns" indicates not significant. Error bars represent the standard error from the mean.
we reported previously when the effect of Australian sourced A. taxiformis on 96 h of in vitro rumen fermentation was determined (Roque et al., 2019). Taking the variations in microbiome composition and function as well as potential experimental variations into consideration a direct comparison of these two different seaweeds would be certainly be of great value to evaluate the real and explicit potential of these promising feed amendments. However, since in vitro assays are only a rough proxy of the in vivo response, such comparison might be better suited for an in vivo follow-up experiment. The effects of adding Pacific Z. farlowii on methane production was significantly less prominent: a reduction of only 11% was measured when Z. farlowii was added at 5% DM. This difference in methane mitigation is not surprising when considering the amount of bromoform concentration associated with the different seaweeds we tested (2,305 µg g −1 dry weight) for A. taxiformis and 35 µg g −1 dry weight for Z. farlowii). In fact, bromoform concentrations for A. taxiformis were maintained throughout our harvesting and sample preparation process at levels similar to the highest values that had been reported for the Australian A. taxiformis (Vucko et al., 2017). Organobromine compounds such as bromomethane (methyl bromide; CH 3 Br) and bromoform (CHBr 3 ) are abundantly found in marine algae (Gribble, 2000) and have been identified as inhibiting microbial methanogenesis (Tomkins et al., 2009). It is not surprising that California-sourced seaweed, specifically A. taxiformis with its elevated level of bromoform, reduced methanogenesis during in vitro rumen fermentation. Based on these findings, we therefore hypothesize that California-sourced seaweeds might represent a viable option for use in feed-based methane mitigation strategies, especially when considering the significantly reduced carbon footprint associated with utilizing local natural resources.
In addition to demonstrating the potential of the local A. taxiformis for methane mitigation during enteric fermentation, we were also able to demonstrate methane reduction with no obvious impact on total gas and CO 2 production (Figures 3A,B, 4A,B) by the brown alga Z. farlowii, a species of seaweed commonly found along the Southern California Bight.
Released CO 2 can be used as a highly sensitive proxy capable of detecting changes in the overall metabolic carbon respiration and growth of a microbial community within an ecosystem (Kaschuk et al., 2010;Zheng et al., 2019). Changes in CO 2 profiles would therefore indicate significant perturbation and dysbiosis of the healthy rumen microbiome with potentially significant changes in the overall function of the rumen ecosystem as observed for example during acidosis when animals are transition to quickly to feed that contains high level of rapidly digestible carbohydrate such as barley and other cereals and which poses a life-threatening condition to the ruminant animal (Navarre et al., 2012;Eger et al., 2018). The lack of changes of observed CO 2 profiles suggest that addition of neither A. taxiformis nor Z. farlowii had an effect on overall microbiome metabolism FIGURE 4 | Methane and CO 2 production during in vitro fermentation of rumen fluid amended with Z. farlowii. Production of CH 4 mL (g DM) −1 and CO 2 mL (g DM) −1 from vessels without (n = 3) and with 5% (n = 3) Z. farlowii as additive. Methane and CO 2 were measured at 24 h (A,B, respectively) and 48 h (C,D, respectively). " * " indicates significant difference (p-value < 0.05), "ns" indicates not significant. Error bars represent the standard error from the mean. and healthy function was maintained. To get a more detailed understanding of potential metabolic changes at the submicrobiome level, which might not be detected by measuring CO 2 production and release, more detailed microbiome analyses should be performed before these seaweeds are applied at large scale to ruminant animals.
The effectiveness of macroalgae in reducing methane production during rumen incubation has been linked to the concentration of halogenated bioactives including bromoform and di-bromochloromethane . In contrast to A. taxiformis, which has been shown to produce several halomethane compounds, Z. farlowii only caused a modest reduction of methanogenesis and consequently higher amounts of emitted enteric methane during in vitro rumen fermentation. These findings suggest that either the bioactives in Z. farlowii are more bioavailable but less effective or concentrated, or methane reduction mediated by Z. farlowii amendment occurs via a different compound or mode of action. Z. farlowii is commonly found along the Southern California Bight, which makes it a potential candidate for non-terrestrial farming operations along the Southern California Coast. In order to understand the molecular mechanism by which Z. farlowii reduces methane production from the rumen microbiota, as well as the nutritional value of Z. farlowii more detailed, follow-up in vitro and in vivo studies involving Z. farlowii as feed additive are required. Only with these data in hand will it be possible to determine Z. farlowii's value for future methane mitigation strategies and to determine its potential for use in dairy operations.

CONCLUSION
Asparagopsis taxiformis and Z. farlowii collected off Santa Catalina Island were evaluated for their ability to reduce methane production from dairy cattle fed a mixed ration widely utilized in California operations. The methane reducing effect of A. taxiformis and Z. farlowii described in this study make these macroalgae promising candidates for biotic methane mitigation strategies in the largest milk producing state in the United States, though further follow-up studies will be required to both substantiate methane mitigation ability and elucidate the molecular mechanism by which these macroalgae reduce methane production in livestock. With expected growth in livestock production, it is necessary to investigate and confirm the effect of these macroalgae in vivo in order to ensure that farmers have sufficient incentive to implement such strategies.

DATA AVAILABILITY STATEMENT
The datasets generated for this study are available on request to the corresponding author.

ETHICS STATEMENT
The animal study was reviewed and approved by all animal procedures were performed in accordance with the Institution of Animal Care and Use Committee (IACUC) at the University of California, Davis, under protocol number 19263.

AUTHOR CONTRIBUTIONS
CB, BR, and MH conceived and designed the experiment. DG, MCH, and SN collected seaweed. CB, BR, CS, NN, MG, AP, VD, and MH performed experiments and analyzed data. CB, BR, and MH wrote major part of the manuscript. All authors were involved in writing the final version of the manuscript.

FUNDING
Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s). This work was supported through funds from the College of Agricultural and Environmental Sciences at the University of California, Davis, by Elm Innovations and by the Hellman Foundation. MCH was supported by ARPA-E (Contract# 1726-1513) and SN was supported by a Wyatt Foundation gift.