Effect of Padina gymnospora biowaste inclusion on in vitro methane production, feed fermentation, and microbial diversity

Abstract

In vitro studies were undertaken aiming to study the methane (CH₄) mitigation potential of biowaste (BW) of Padina gymnospora at the graded inclusion of 0% (C), 2% (A₂), 5% (A₅), and 10% (A₁₀) of the diet composed of straw and concentrate in 40:60 ratio. The chemical composition analysis revealed that the BW contained higher crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and ether extract (EE) than the PF (fresh seaweed, P. gymnospora). The concentration of cinnamic acid, sinapic acid, kaempferol, fisetin p-coumaric acid, ellagic acid, and luteolin in BW was 1.5-6-folds less than the PF. Inclusion of BW decreased (P < 0.0001) CH₄ production by 34%, 38%, and 45% in A₂, A₅, and A₁₀ treatments, respectively. A decrease (P < 0.0001) of 7.5%-8% in dry matter (DM) and organic matter (OM) digestibility was also recorded with the BW supplementation. The BW inclusion also decreased the numbers of total (P = 0.007), Entodinomorphs (P = 0.011), and Holotrichs (P = 0.004) protozoa. Metagenome data revealed the dominance of Bacteroidetes, Proteobacteria, Firmicutes, Actinobacteria, and Fibrobacter microbial phyla. At the phylum level, Euryarchaeota dominated the archaeal community, whereas Methanobrevibacter was most abundant at the genus level. It can be concluded that the inclusion of BW in straw and concentrate based diet by affecting rumen fermentation, protozoal numbers, and compositional shift in the archaeal community significantly decreased CH₄ production. Utilization of biowaste of P. gymnospora as a CH₄ mitigating agent will ensure its efficient utilization rather than dumping, which shall cause environmental pollution and health hazards.

Keywords: Padina gymnospora; archaea; biowaste; metagenome; methane; seaweeds.

FIGURE 1

(a) Fresh brown seaweed Padina gymnospora-PF and (b) dried biowaste of P. gymnospora-BW obtained after the supercritical fluid extraction.

FIGURE 2

Relative concentration of bioactive compounds, i.e., phenolic acids (A), flavonoids (B), sugars (C), organic acids (D), and carotenoids (E) in the biowaste (BW) of Padina gymnospora. The folds increase or decrease in the concentration of bioactive compounds in BW is relative to the concentration in PF (seaweed fresh).

FIGURE 3

Comparative CH₄ production from the biowaste of Padina gymnospora and fresh seaweed (P. gymnospora). C, PF, and BW represent control (without seaweed/biowaste, gray bars), fresh seaweed (blue bars), and seaweed biowaste (green bars), respectively. A₂, A₅, and A₁₀ represent 2%, 5%, and 10% inclusion of BW or PF in the diet, respectively. Each bar in C, BW, and PF category represented the mean value of six observations (N = 6 replicates) for the corresponding treatments C, A₂, A₅, and A₁₀. Overall categories represent the mean values of average CH₄ production in A₂, A₅, and A₁₀ in C, BW, or PF. CH₄, methane; ml, milliliter; g, gram; DM, dry matter. P values were calculated using unpaired parametric t-test at 95% confidence level and the significance was ascertained at P < 0.05.

FIGURE 4

Effect of seaweed biowaste (BW) inclusion levels on CH₄ production kinetics. C, A₂, A₅, and A₁₀ represented the treatments with variable levels of BW inclusion of 0%, 2%, 5%, and 10% in the diet, respectively. CH₄, methane; C, control; ml, milliliter; g, gram; DM, dry matter; hrs, hours of incubation. Each data point in line plot represents the mean value of the six observations (N = 6 replicates per treatment) at different incubation hours.

FIGURE 5

Alpha diversity in different treatments (C, A₂, A₅, and A₁₀) represented by Shannon index (A), beta diversity index among the treatments (B). C was control (without seaweed biowaste), whereas A₂, A₅, and A₁₀ treatments represented the inclusion of BW at the corresponding levels of 2%, 5%, and 10% in the diet. Individual mean value in each bar was based on six observations (N = 6 replicates).

FIGURE 6

Rumen microbiota at different taxonomic ranks. The stacked bar graphs illustrate the relative percent abundances of rumen microbiota in different treatment as influenced by the biowaste inclusion levels at the (A) phylum, (B) order, and (C) genus levels. The stacked bar graphs represent the top 20 microbes at each taxonomic ranks. C was control (without seaweed biowaste), whereas A₂, A₅, and A₁₀ treatments represent the inclusion of BW at the corresponding levels of 2%, 5%, and 10% in the diet. Each mean value was based on six observations (N = 6 replicates) for the corresponding treatments C, A₂, A₅, and A₁₀.

FIGURE 7

Rumen archaea at the different taxonomic ranks. The stacked bar graphs illustrate the relative abundances of archaea in different treatment as influenced by the biowaste inclusion levels at the (A) phylum, (B) order, and (C) genus levels. The stacked bar graphs represent the top 20 microbes at genus level. C was control (without seaweed biowaste), whereas A₂, A₅, and A₁₀ treatments represent the inclusion of BW at the corresponding levels of 2%, 5%, and 10% in the diet. Each mean value is based on six observations (N = 6 replicates) for the corresponding treatments C, A₂, A₅, and A₁₀.