Comparative metabolite fingerprinting of the rumen system during colonisation of three forage grass (Lolium perenne L.) varieties

Abstract

The rumen microbiota enable ruminants to degrade complex ligno-cellulosic compounds to produce high quality protein for human consumption. However, enteric fermentation by domestic ruminants generates negative by-products: greenhouse gases (methane) and environmental nitrogen pollution. The current lack of cultured isolates representative of the totality of rumen microbial species creates an information gap about the in vivo function of the rumen microbiota and limits our ability to apply predictive biology for improvement of feed for ruminants. In this work we took a whole ecosystem approach to understanding how the metabolism of the microbial population responds to introduction of its substrate. Fourier Transform Infra Red (FTIR) spectroscopy-based metabolite fingerprinting was used to discriminate differences in the plant-microbial interactome of the rumen when using three forage grass varieties (Lolium perenne L. cv AberDart, AberMagic and Premium) as substrates for microbial colonisation and fermentation. Specific examination of spectral regions associated with fatty acids, amides, sugars and alkanes indicated that although the three forages were apparently similar by traditional nutritional analysis, patterns of metabolite flux within the plant-microbial interactome were distinct and plant genotype dependent. Thus, the utilisation pattern of forage nutrients by the rumen microbiota can be influenced by subtleties determined by forage genotypes. These data suggest that our interactomic approach represents an important means to improve forages and ultimately the livestock environment.

Figure 1. Metabolite fingerprinting of rumen interactome fractions using Fourier Transform Infra-Red spectroscopy.

Fourier Transform Infra-Red (FTIR) spectra of fractions of rumen microbe-plant fermentation mixes fractionated into (A) residue (plant residue including colonising micro-organisms), (B) pellet (bacterial pellet recovered from planktonic phase) and (C) footprint (cell free planktonic phase liquor). Areas of the spectra corresponding to discrete chemical groups are highlighted in grey; sugars (wavenumbers 900-950 cm^-1), amides (wavenumbers 1680-1655 cm^-1 [Amide I] and 1530-1550cm^-1 [Amide II]) and fatty acids (wavenumbers 3100-2800 cm^-1). Principal Component Analyses (PCA) of FTIR spectra from (D) residue, (E) footprint and (F) pellet samples. Discriminant Function Analyses (DFA) based on 3 PCs from FTIR spectra for (G) residue, (H) footprint and (I) pellet samples. Total explained variance (TEV) by 3 PCs for each DFA model is indicated. Green/ P = Premium; blue/ D = AberDart; red/ M = AberMagic. 0, 2, 4, 6 12 and 24 refers to the corresponding hours after addition of rumen microbes to plant genotypes.

Figure 2. Metabolite fingerprinting of the residue fraction of the rumen microbe-plant genotype interactome.

Principal Component Analyses (PCA) of FTIR spectra from (A) Premium, (B) AberDart and (C) AberMagic. Discriminant Function Analyses (DFA) based on 3 PCs for (D) Premium, (E) AberDart and (F) AberMagic. Total explained variance (TEV) by 3 PCs for each DFA model is indicated. P = Premium; D = AberDart; M = AberMagic. 0 (blue circles), 2 (green cross), 4 (black cross), 6 (blue down triangle), 12 (red square) and 24 (cyan triangle) refers to the corresponding hours after addition of rumen microbes to plant genotypes. The loading vectors indicating major sources of variation within DF1 in (G) Premium, (H) AberDart and (I) AberMagic. The horizontal red lines (G, H, I) indicate the points where wavenumbers are making no contribution to DF1. Areas of the spectra corresponding to discrete chemical groups are highlighted in grey; sugars (wavenumbers 900-950 cm^-1), amides (wavenumbers 1680-1655 cm^-1 [Amide I] and 1530-1550cm^-1 [Amide II]), fatty acids (wavenumbers 3100-2800 cm^-1) and substituted alkanes (wavenumbers 1750-1800 cm^-1).

Figure 3. Metabolite fingerprinting of the footprint fraction of the rumen microbe-plant genotype interactome.

Principal Component Analyses (PCA) of FTIR spectra from (A) Premium, (B) AberDart and (C) AberMagic. Discriminant Function Analyses (DFA) based on 3 PCs for (D) Premium, (E) AberDart and (F) AberMagic. Total explained variance (TEV) by 3 PCs for each DFA model is indicated. P = Premium; D = AberDart; M = AberMagic. 0 (blue circles), 2 (green cross), 4 (black cross), 6 (blue down triangle), 12 (red square) and 24 (cyan triangle) refers to the corresponding hours after addition of rumen microbes to plant genotypes. The loading vectors indicating major sources of variation within DF1 in (G) Premium, (H) AberDart and (I) AberMagic. The horizontal red lines (G, H, I) indicate the points were wavenumbers are making no contribution to DF1. Areas of the spectra corresponding to discrete chemical groups are highlighted in grey; sugars (wavenumbers 900-950 cm^-1), amides (wavenumbers 1680-1655 cm^-1 [Amide I] and 1530-1550cm^-1 [Amide II]), fatty acids (wavenumbers 3100-2800 cm^-1) and substituted alkanes (wavenumbers 1750-1800 cm^-1).

Figure 4. Metabolite fingerprinting of the pellet fraction of the rumen microbe-plant genotype interactome.

Principal Component Analyses (PCA) of FTIR spectra from (A) Premium, (B) Aber Dart and (C) AberMagic. Discriminant Function Analyses (DFA) based on 3 PCs for (D) Premium, (E) AberDart and (F) AberMagic. Total explained variance (TEV) by 3 PCs for each DFA model is indicated. P = Premium; D = AberDart; M = AberMagic. 0 (blue circles), 2 (green cross), 4 (black cross), 6 (blue down triangle), 12 (red square) and 24 (cyan triangle) refers to the corresponding hours after addition of rumen microbes to plant genotypes. The loading vectors indicating major sources of variation within DF1 in (G) Premium, (H) AberDart and (I) AberMagic. The horizontal red lines (G, H, I) indicate the points were wavenumbers are making no contribution to DF1. Areas of the spectra corresponding to discrete chemical groups are highlighted in grey; sugars (wavenumbers 900-950 cm^-1), amides (wavenumbers 1680-1655 cm^-1 [Amide I] and 1530-1550cm^-1 [Amide II]), fatty acids (wavenumber 3100-2800 cm^-1) and substituted alkanes (wavenumbers 1750-1800 cm^-1).

Figure 5. Changes in metabolite groups in rumen interactome fractions as revealed using Fourier Transform Infra-Red spectroscopy.

Absorbancies of areas of the spectra corresponding to discrete chemical groups are highlighted in grey; sugars (wavenumbers 900-950 cm^-1), amides (wavenumbers 1680-1655 cm^-1 [Amide I] and 1530-1550cm^-1 [Amide II]), fatty acids (wavenumbers 3100-2800 cm^-1) and substituted alkanes (wavenumbers 1750-1800 cm^-1) were extracted for spectra of residue, pellet and footprint fractions of rumen microbe-plant fermentation mixes “residue” (plant residue including colonising micro-organisms). Mean values +/- SE (n = 6) are plotted for each interactome based on input of plant genotypes: Premium (blue diamond), AberMagic (green triangle) and AberDart (red square).