Effect of Tropical Algae as Additives on Rumen in Vitro Gas Production and Fermentation Characteristics

Baptiste Dubois, Nigel W. Tomkins, Robert D. Kinley, Mei Bai, Scott Seymour, Nicholas A. Paul, Rocky de Nys

AgroParisTech, 16 rue Claude Bernard, Paris, France.
CSIRO, Livestock Industries, Australian Tropical Sciences Innovation Pre- cinct, James Cook Drive, James Cook University, Townsville, Australia.
School of Marine and Tropical Biology, James Cook Uni- versity, Townsville, Australia.

DOI: 10.4236/ajps.2013.412A2005                

Abstract

Algae have become an area of intensive research in many fields of study. Areas of application are becoming increasingly diverse with the advent of technologies particularly in the mass production of algae biomass. Algae contain complex bioactive compounds and these are gaining importance in emerging technologies with nutritional and environmental applications. In this study, a preliminary investigation evaluated 15 species of algae from the major categories of marine and fresh water algae for their potential as inclusions in ruminant diets for management of greenhouse gas emissions. It was hypothesized that algae would positively affect rumen fermentation and gas production while reducing methane production. The hypothesis was tested using an Ankom automated gas monitoring system and rumen fluid from Bos indicus steers fed tropical forage diets. The results were variable between algae species with some showing a significant reduction in total gas and methane production, with others increasing gas and fermentation. The red and brown algae stand out as having potential for greenhouse gas mitigation with the brown alga Cystoseira having the most prominent effect. The effects observed on fermentation may be manipulated through dosage management and beneficial effects could be potentially maximized by preparing combinations of algal supplements. It has been demonstrated in this study that algae have the potential to assist in rumen fermentation management for improved gas production, and greenhouse gas abatement.

Keywords

Algae; Enteric Gas; Macroalgae; Methane; Rumen; Seaweed

1. Introduction

In northern Australia, cattle are managed under extensive pastoral conditions dominated by native grasses [1], and animal productivity is lower than in temperate regions where pasture improvement and intensive management are commonplace [2]. Implementing practices to reduce the environmental impact of livestock systems across northern Australia, while maintaining a viable level of productivity, is challenging [3]. However, when feed supplementation is incorporated to improve ruminant production then employing additives that have an effect on greenhouse gas production would be beneficial for individual producers and the industry. A number of feed additives such as halogenated analogues [4], monensin [5] and a range of plant compounds such as tannins [6], saponins [7], essential oils [8,9], and various secondary metabolites [10] have been demonstrated to have potential in reducing greenhouse gas emission from livestock production systems, but in most part it’s difficult to administer across large herds which are managed in extensive environments.

Algae exist in many forms and can be broadly classified on size (micro or macroalgae), and photosynthetic and accessory pigments (green, red or brown algae). Both marine and freshwater algae have been used in human nutrition, cosmetics, and pharmaceutical products and have the potential to be used as a supplement for livestock feeds [11]. Secondary metabolites with antimicrobial activity have been identified in green, red and brown algae [12- 14]. Other species tend to have anti-viral, antioxidant, or anti-inflammatory properties that may be used to manipulate livestock health and productivity. Specifically, Ascophyllum nodosum has received attention for application in ruminant diets and effects on greenhouse gas production [15], but few papers describe the potential in a range of species of algae. Further work is required to screen numerous algae species to study the relationship between composition and effective fermentation characteristics. In vitro gas production techniques have been improved and applied to study fermentation kinetics relative to feed composition [16-19]. Consequently in vitro techniques can allow for rapid screening of a large number of feed additives that may have effects on gas production.

The objective of this study was to first rank the capability of fifteen tropical species of algae to influence in vitro gas production when incubated with a tropical grass (Chloris gayana) as the primary substrate and then assess dose response effects of selected algae species on in vitro gas production and fermentation characteristics using pH and methane concentration as proxy indicators.

2. Materials and Methods

2.1. Selection and Preparation of Algae

The algal species used in this study (Table 1) were selected due to their natural abundance in local aquaculture systems or intertidal reefs around Townsville, QLD, Australia, and their potential to be cultured under controlled conditions.

Seven of the algae used in this study (Caulerpa lentillifera, C. taxifolia, Cladophora patentiramea, Ulva sp 3., Ulva ohnoi, Derbesia tenuissima and Oedogonium sp.) were maintained as isolated species at James Cook University, QLD, Australia. Tarong polyculture, a polyculture of freshwater microalgae, was sourced from MBD Energy Ltd., Townsville, QLD. The remaining five species of algae were collected from natural sources near Townsville, QLD.

The marine macroalgal biomass used in this study was initially washed in clean, fresh seawater for 2 minutes to minimize the amount of fouling organisms and silt and then rinsed thoroughly in dechlorinated freshwater for 1 minute to remove residual salt. Freshwater macroalgae were thoroughly rinsed in dechlorinated water immediately after collection. Washed algal biomass was placed in 100 μm mesh for excess water removal by centrifuge

Table 1. Chemical composition of the 15 species used this study (g/kg DM unless stated otherwise).

at 1000 rpm for 6 minutes in a commercial washing machine and then stored at −10.0˚C. Prior to the experiments algae were freeze dried in a SP Industries VirTis K bench top freeze-drier (Warminster, PA). Dried material was stored in sealed poly bags at −10.0˚C.

2.2. Donor Animals and Preparation of Rumen Fluid Inoculum

Rumen fluid was obtained from two rumen fistulated Brahman (Bos indicus) steers (505 ± 7 kg) preconditioned ad libitum for two weeks with Rhodes grass (Chloris gayana) hay typical of feeding in tropical QLD, Australia. The Rhodes grass contained on a g/kg dry matter (DM) basis: organic matter (OM), 920; crude protein (CP), 107; neutral detergent fibre (NDF), 672; and acid detergent fibre (ADF), 467. The animals were maintained according to the guidelines of the Australian Code of Practice for the care and use of Animals for Scientific Purposes [20].

Rumen digesta was collected (1.5 L from each steer) with a manual suction apparatus into insulated, preheated (39˚C) thermal flasks according to [21]. Fibrous material from the rumen mat from forward, posterior, and lateral regions was included at approximately 10% of the 3 L collection volume. The pooled rumen fluid was blended at high speed for 30 seconds to ensure solid phase associated bacteria were distributed throughout the inoculum [22] and filtered through a 1 mm sieve and maintained anaerobic under a stream of N₂.

2.3. Experimental Design

In experiment 1 four consecutive incubations were conducted to screen each species of algae for their effects on ruminal fermentation when 0.20 g (OM basis) of freeze dried algae of each species was incubated for 48 h. A substrate was prepared with 1.00 g OM of the previously described Rhodes grass. Each incubation set was completed with: 1) negative control containing only 1.00 g of Rhodes grass as substrate; 2) positive control (n = 2) containing 1.00 g of the substrate plus 0.02 g of soybean OM (OM 920, CP 558, and NDF 190 g/kg DM respectively), and also containing 0.18 g of sorghum (OM 987, CP 120, NDF 88.7 g/kg DM respectively). Blank incubations (n = 2) were also included.

For experiment 2 the gas production data obtained from experiment 1 combined with the composition and bioactive properties of the algae were used to select four species for assessment of dose response. For each of the selected algae (C. taxifolia, C. trinodis, Oedogonium sp. and Tarong polyculture) five doses (0, 20, 40, 80, 160 mg OM) were added to 1.00 g of the substrate assessed in 72 h incubations in quadruplicate in a Latin square design under conditions described for experiment 1. Each incubation also included blanks (n = 2) and the positive controls (n = 2) as described for experiment 1. Gas production, fermentation lag time, final pH, and final headspace CH₄ concentration were monitored.

2.4. In Vitro Incubation

All materials comprising the substrates were oven-dried and passed through a 1 mm sieve, and in vitro incubation was achieved using standard methodology [17,19,23]. Gas production, fermentation lag time, and final pH were monitored. Incubations and gas monitoring were completed with an ANKOM Technology (Macedon, NY) in vitro cumulative gas production system fitted with pressure transducers and gas sample collection ports. Filtered rumen inoculants were mixed with 39˚C anaerobic buffer solution (Kansas state buffer) [16,26] at 25 and 100 mL respectively, in pre-warmed (39˚C) 250 mL Schott (Mainz, Germany) bottles. The treatment incubations contained 1.0 g of substrate OM, and 0.20 g of algae additive and were prepared as previously described.

Bottles were sealed under a N₂ atmosphere and incubated at 39˚C in Ratek (Boronia, Australia) model OM11 orbital mixer-incubators. Gas pressure was measured every 60 seconds and cumulative pressure recorded every 20 minutes for 48 h in experiment 1, and 72 h in experiment 2.

2.5. Analytical Methods

Table 1 shows the chemical composition of the fifteen species of algae screened in this study. Dry matter of each species of algae and material used in the fermentation substrate was measured at 105˚C to constant weight. Organic matter was measured by loss on combustion at 550˚C for 8 h. Gross Energy (GE) content was determined using a Parr Instrument Company (Moline, IL) Model 1108 bomb calorimeter. Neutral detergent fibre (aNDFom) was measured using a Foss (Hilleroed, Denmark) FiberCap 2023 fibre analyzer. Crude protein and total nitrogen content was determined using a LECO (St. Joseph, MI) CHN628 series nitrogen analyzer. Methane concentration in headspace gas samples collected in 10 mL Labco Exetainer vials were measured by gas chromatography on a Shimadzu (Koyto, Japan) GC-2010, equipped with a Carbosphere 80/100 column and a Flame Ionization Detector (FID).

Conflicts of Interest

The authors declare no conflicts of interest.

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