3. R ESULTS
3.3. The nature of the earthworm gut microbiota
3.3.1. Fermentative responses of gut contents and soil
In unsupplemented microcosms, soil displayed marginal activity whereas gut contents produced diverse fermentation products such as H2, CO2, acetate, methylbutyrate, propionate, and succinate (Figure 60). In gut content treatments, the rapid formation of fermentation products appeared to occur without appreciable delay, suggesting that gut-associated fermenters were poised to respond to endogenous nutrients in gut contents. These findings illustrated qualitatively and quantitatively dissimilar fermentative activities in soil and gut content.
Fresh microbial cell lysate (a) simulates gizzard-disrupted biomass and (b) strongly enhanced fermentative activities of gut content-associated microbes (Section 3.2.1). Although unsupplemented soil treatments displayed only a negligible fermentative activity, soil and gut contents displayed nearly identical fermentative responses when both were challenged with the complex nutrients of cell lysate (Figure 61). Time-resolved analysis indicated that the initial fermentative response to supplemented cell lysate was more rapid in gut content treatments than in soil treatments (Figure 61). The formation of the dominant fermentation products in response to lysate was statistically significant (Table 50) and characteristic of those formed by fermentative facultative aerobes and fermentative obligate anaerobes (Buckel, 1999). Protein and RNA were evaluated for their potential to (a) stimulate fermentation in soil and thus (b) mimic the capacity of cell lysate to enhance fermentation. Although these biopolymers greatly enhanced gut content fermentations (Section 3.2.3), the fermentative response of soil to either protein or RNA was negligible compared to the strong response of soil to cell lysate (Table 51).
RESULTS 141
Figure 60. Fermentation product profiles of unsupplemented anoxic L. terrestris gut content and soil microcosms. Traces of lactate are not shown. Values are the arithmetic average of three replicate analyses, and error bars indicate the standard deviations. Some standard deviations are smaller than the size of the symbol and therefore not apparent. DW, dry weight. Figure modified and used with permission from Zeibich et al., 2019c.
Figure 61. Fermentation product profiles of anoxic L. terrestris gut content and soil microcosms supplemented with cell lysate. Traces of lactate are not shown. The amount of carbon derived from filter-sterilized lysate (5.0% dry weight) added per microcosm approximated 2 mmol. Filter filter-sterilized lysate alone did not display any fermentation activity. Values are the arithmetic average of three replicate analyses, and error bars indicate the standard deviations. Some standard deviations are smaller than the size of the symbol and therefore not apparent. DW, dry weight. Figure modified and used with permission from Zeibich et al., 2019c.
RESULTS 143 Table 50. P values of fermentation products in cell lysate-supplemented gut content (A) and soil (B) treatments, and P values of gut content versus soil treatments (C).a (A) Gut Contentb
Products
CO2 H2 Succinate
(15h)
Lactate
(6h)
Formate
(15h) Acetate Propionate Butyrate Methylbutyrate Isobutyrate
C L C L C L C L C L C L C L C L C L C L
Mean valuec 43 394 2.1 61 4.0 63 2.4 14 0.9 33 52 377 9.8 99 3.0 26 12 103 3.5 25 Variance 6.8 886 0.3 60 0.2 1.4 0.1 1.5 0.0 0.2 14 125 1.0 34 0.1 8.7 0.3 116 0.3 12
P value 0.002 0.006 0.000 0.004 0.000 0.000 0.001 0.006 0.005 0.008
(B) Soilb
Products
CO2 H2 Succinate
(15h)
Lactate
(6h)
Formate
(15h) Acetate Propionate Butyrate Methylbutyrate Isobutyrate
C L C L C L C L C L C L C L C L C L C L
Mean valuec 5.4 178 0.0 14 0.3 19 0.0 2.8 0.0 18 4.7 271 1.8 36 0.1 24 1.0 56 0.3 12 Variance 22 1747 0.0 33 0.3 2.3 0.0 0.3 0.0 1.8 67 314 0.1 44 0.0 33 3.2 16 0.2 0.0
P value 0.019 0.054 0.002 0.014 0.002 0.000 0.012 0.018 0.000 0.000
(C) Gut Content versus Soild
Products
CO2 H2 Succinate
(15h)
Lactate
(6h)
Formate
(15h)
Acetate Propionate Butyrate Methylbutyrate Isobutyrate
G S G S G S G S G S G S G S G S G S G S
Mean valuec 394 178 61 14 63 19 14 2.8 33 18 337 271 99 36 26 24 103 56 25 12
Variance 886 1747 60 33 1.4 2.3 1.5 0.3 0.2 1.8 125 314 34 44 8.7 33 116 16 12 0.0
P value 0.002 0.001 0.000 0.001 0.003 0.003 0.000 0.737 0.006 0.003 (15h)
aTable modified and used with permission from Zeibich et al., 2019c.
bP values (significant at P ≤ 0.05) were calculated by t-test with unequal variances and are based on the difference between the amount of products in control (C) and cell lysate (L) treatments. For transient products (i.e., formate, succinate, isobutyrate and lactate), the significance of differences of net amounts between control and supplemented treatments were tested for the time point of the highest concentration (shown in parentheses). See Figure 60 and Figure 61 for product profiles.
cMean values (n = 3) are in µmol/gDW. DW, dry weight.
dP values (significant at P ≤ 0.05) were calculated by t-test with unequal variances and are based on the difference between the amount of products in lysate-supplemented gut content (G) and lysate-supplemented soil (S) treatments. Unless otherwise indicated, the values are based on the amount of products at the end of the incubation.
Table 51. Effect of protein, RNA, and cell lysate on fermentation product profiles of anoxic soil treatments.a
Supplement
Products (µmol/gDW)
Time
(h) pH Total CO2 H2 Acetate Succinate Formate Propionate Butyrate
Methyl-butyrate
Iso-butyrate Protein 0 6.9 ± 0.0 1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.4 ± 0.0 0.0 ± 0.0 0.0 ± 0.4 0.9 ± 0.1 0.1 ± 0.0 0.0 ± 0.0 30 6.8 ± 0.0 18 4.0 ± 0.8 0.5 ± 0.1 5.0 ± 0.7 1.9 ± 1.2 6.2 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.0 0.0 ± 0.0 RNA 0 6.9 ± 0.0 5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.9 ± 0.3 2.5 ± 4.3 0.5 ± 2.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 30 6.7 ± 0.0 23 6.3 ± 1.1 3.0 ± 0.8 5.3 ± 1.1 3.3 ± 3.6 0.0 ± 0.0 0.0 ± 0.0 4.5 ± 5.5 0.0 ± 0.0 0.0 ± 0.0 Lysate 0 6.9 ± 0.0 1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.4 0.3 ± 0.0 0.0 ± 0.0 0.0 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 30 6.7 ± 0.0 507 136 ± 11 21 ± 7.2 230 ± 16 12 ± 9.0 0.0 ± 0.0 23 ± 12 20 ± 5.0 52 ± 1.8 13 ± 1.9
aThe amount of protein-, RNA-, and lysate-derived carbon added per microcosm approximated 2.4, 2.4, and 2.0 mmol, respectively. Values are the arithmetic average of three replicate analyses (± standard derivation). Lysate values obtained from Figure 61. DW, dry weight.Table modified and used with permission from Zeibich et al., 2019c.
RESULTS 145 Likewise, soil microcosms displays essentially no fermentative activity when they were challenged with glucose and other gut-associated saccharides that rapidly augment fermentation in gut contents (Meier et al., 2018). These findings indicates that the marked response of soil to complex nutrients, available in cell lysate, did not occur when saccharides, protein, or RNA were provided as ‘high quality’ substrates.
The pH approximated 7 and did not vary much in both soil and gut content microcosms during the incubation (Figure 61), reinforcing the likelihood that the complex nutrient input rather than a change in pH was an important factor for the observed fermentation activities. Based on the theoretical recoveries of lysate-derived carbon and reducing equivalents, approximately half of the lysate-derived organic carbon was recovered in the fermentation products of soil and gut content treatments (Table 52), demonstrating that the amount of available supplemental organic carbon was adequate for the observed fermentative responses to lysate. Formate, succinate, and isobutyrate were transient in both treatments (Figure 61). Previous findings indicated that succinate can be decarboxylated to CO2 and propionate, whereas formate can be converted to CO2 and H2 (Section 3.2.8). The continuous and strong production of CO2, H2, and propionate in soil and gut content treatments (a) reinforced the occurrence of these secondary processes and (b) illustrated that such processes were not exclusive for gut contents.
Table 52. Estimated recoveries of carbon and reducing equivalents (i.e., electrons) in yeast lysate-supplemented gut content or soil treatments.a
Gut Content Soil
Recoveries (%) Recoveries (%)
Main Products
Net amt.
(µmol) Carbon
Reducing equivalents
Net amt.
(µmol) Carbon
Reducing equivalents
CO2 158 7.7 na 131 6.4 na
H2 26 na 0.6 10 na 0.3
Acetate 145 14 14 204 20 20
Methylbutyrate 41 10 13 41 10 13
Propionate 41 6.1 7.0 27 4.0 4.6
Butyrate 10 2.0 2.5 18 3.6 4.5
Isobutyrate 11 2.2 2.7 9.8 1.9 2.4
Succinate 3.9 0.8 0.7 4.2 0.8 0.7
Formate 0.3 - - 0.1 - -
Total: 437 43 41 447 47 45
aSee Figure 60 and Figure 61 for product profiles of lysate treatments. Net amounts of products formed in the unsupplemented control were subtracted from those of supplemented treatments; recoveries are based on the amount of substrate provided. Values are based on the arithmetic average of three replicate analyses. Recoveries were calculated from the net amounts of products. amt., amount; -, no net increase of the product during the incubation; na, not applicable. Table modified and used with permission from Zeibich et al., 2019c.