**4. Effects of earthworms on the structure and activity of microbial communities during vermicomposting**

Since vermicomposting is a biological process, microorganisms play a key role in the evolution of the organic materials and in the transformations they suffer from wastes to safe organic amendments or fertilizers (vermicompost). Therefore, the effects that earthworms have on the microorganisms must be established because if the earthworms were to stimulate or depress microbiota or modify the structure and activity of microbial communities, they would have different effects on the decomposition of organic matter, and in turn on the quality of the final product. To address these questions we performed three laboratory experiments, with the following objectives:


#### **4.1 How do earthworms affect microbial communities through the gut associated processes?**

To provide further light into the effect of gut transit on microbial communities, we carried out an experiment with microcosms filled with cow manure and inoculated with 25 mature individuals of the earthworm species *E. andrei*. The microcosms consisted of 250 mL plastic

Vermicomposting: Composting with Earthworms to Recycle Organic Wastes 35

microbial activity was determined by hydrolysis of fluorescein diacetate (FDA), a colourless compound that is hydrolysed by both free and membrane bound enzymes, to release a coloured end product (fluorescein) that can be measured by spectrophotometry (Adam &

Recent reports suggest that the digestion of organic material by epigeic earthworms has negative effects on microbial biomass (Aira et al., 2006, 2009; Monroy et al., 2009). The present data are consistent with these findings, since we found a reduction in the viable microbial biomass as a result of the passage of the fresh substrate through the gut of the earthworm species *E. andrei* (Figure 5). More specifically, the total content of PLFAs was 1.5 times higher in the control treatment (1868.11 ± 129.02 µg g-1 dw) than that in earthworm

Fig. 5. Changes in the viable microbial biomass, measured as total PLFAs, after the passage of cow manure through the gut of the earthworm species *Eisenia andrei*. Values are means ±

Epigeic earthworms possess a diverse pool of digestive enzymes which enables them to digest bacteria, protozoa, fungi and partly decomposed plant debris (Zhang et al., 2000). Indeed, bacterial populations decreased in cow manure after transit through the earthworm gut (Figure 6a). As occurred with microbial biomass, bacterial PLFAs were 1.5 times lower in cast samples relative to the control (Figure 6a). However, the passage of cow manure through the earthworm gut affected fungal populations to a lesser extent than bacteria

Animal manures are microbial-rich environments in which bacteria constitute the largest fraction (around 70% of the total microbial biomass as assessed by PLFA analysis), with fungi mainly present as spores (Domínguez et al., 2010). Thus, earthworm activity is expected to have a greater effect on bacteria than on fungi in these organic substrates. These contrasting short-term effects on bacterial and fungal populations with earthworm activity are thus expected to have important implications on decomposition pathways during vermicomposting, because there exist important differences between both microbial

SE. Control is the manure incubated without earthworms.

Duncan, 2001). The data were analysed by a one-way ANOVA test, at α = 0.05.

**4.1.1 Microbial biomass** 

casts (1249.87 ± 158.43 µg g-1 dw).

(Figure 6b).

containers filled to three quarters of their capacity with sieved, moistened vermiculite. A plastic mesh was placed over the surface of the vermiculite and 100 g (fresh weight, fw) of the substrate was placed on top of the mesh, to avoid mixing the substrate with the vermiculite bedding (Figure 4a). The microcosms were covered with perforated lids and stored in random positions in an incubation chamber, at 20 °C and 90% relative humidity, for three days (Figure 4b). Control microcosms consisted of each type of manure incubated without earthworms. Each treatment was replicated five times. In order to obtain cast samples, earthworms were removed from the microcosms, washed three times with distilled water and placed in Petri dishes on moistened filter paper (Figure 4b). Casts from the same Petri dish were then collected with a sterile spatula and pooled for analysis in 1.5 mL Eppendorf tubes (Figure 4b); the same amount of manure samples were also collected from the control microcosms. Viable microbial biomass was determined as the sum of all identified phospholipid fatty acids (PLFAs) (Zelles, 1999). The structure of microbial communities was assessed by PLFA analysis; some specific PLFAs were used as biomarkers to determine the presence and abundance of specific microbial groups (Zelles, 1997). The sum of PLFAs characteristic of Gram-positive (iso/anteiso branched-chain PLFAs), and Gram-negative bacteria (monounsaturated and cyclopropyl PLFAs) were chosen to represent bacterial PLFAs, and the PLFA 18:2ω6c was used as a fungal biomarker. Total

Fig. 4. Scheme of the (a) microcosm and (b) procedure for incubation of microcosms and collection of cast samples.

microbial activity was determined by hydrolysis of fluorescein diacetate (FDA), a colourless compound that is hydrolysed by both free and membrane bound enzymes, to release a coloured end product (fluorescein) that can be measured by spectrophotometry (Adam & Duncan, 2001). The data were analysed by a one-way ANOVA test, at α = 0.05.

#### **4.1.1 Microbial biomass**

34 Management of Organic Waste

containers filled to three quarters of their capacity with sieved, moistened vermiculite. A plastic mesh was placed over the surface of the vermiculite and 100 g (fresh weight, fw) of the substrate was placed on top of the mesh, to avoid mixing the substrate with the vermiculite bedding (Figure 4a). The microcosms were covered with perforated lids and stored in random positions in an incubation chamber, at 20 °C and 90% relative humidity, for three days (Figure 4b). Control microcosms consisted of each type of manure incubated without earthworms. Each treatment was replicated five times. In order to obtain cast samples, earthworms were removed from the microcosms, washed three times with distilled water and placed in Petri dishes on moistened filter paper (Figure 4b). Casts from the same Petri dish were then collected with a sterile spatula and pooled for analysis in 1.5 mL Eppendorf tubes (Figure 4b); the same amount of manure samples were also collected from the control microcosms. Viable microbial biomass was determined as the sum of all identified phospholipid fatty acids (PLFAs) (Zelles, 1999). The structure of microbial communities was assessed by PLFA analysis; some specific PLFAs were used as biomarkers to determine the presence and abundance of specific microbial groups (Zelles, 1997). The sum of PLFAs characteristic of Gram-positive (iso/anteiso branched-chain PLFAs), and Gram-negative bacteria (monounsaturated and cyclopropyl PLFAs) were chosen to represent bacterial PLFAs, and the PLFA 18:2ω6c was used as a fungal biomarker. Total

n = 5

*Sample collection*

Plastic mesh

24 h

Vermiculite bedding

Earthworm *E. andrei*

(25 mature individuals)

Fig. 4. Scheme of the (a) microcosm and (b) procedure for incubation of microcosms and

Cow manure (100 g)

a) **Microcosm**

*Microcosm incubation*

collection of cast samples.

20 °C

3 days

b)

Recent reports suggest that the digestion of organic material by epigeic earthworms has negative effects on microbial biomass (Aira et al., 2006, 2009; Monroy et al., 2009). The present data are consistent with these findings, since we found a reduction in the viable microbial biomass as a result of the passage of the fresh substrate through the gut of the earthworm species *E. andrei* (Figure 5). More specifically, the total content of PLFAs was 1.5 times higher in the control treatment (1868.11 ± 129.02 µg g-1 dw) than that in earthworm casts (1249.87 ± 158.43 µg g-1 dw).

Fig. 5. Changes in the viable microbial biomass, measured as total PLFAs, after the passage of cow manure through the gut of the earthworm species *Eisenia andrei*. Values are means ± SE. Control is the manure incubated without earthworms.

Epigeic earthworms possess a diverse pool of digestive enzymes which enables them to digest bacteria, protozoa, fungi and partly decomposed plant debris (Zhang et al., 2000). Indeed, bacterial populations decreased in cow manure after transit through the earthworm gut (Figure 6a). As occurred with microbial biomass, bacterial PLFAs were 1.5 times lower in cast samples relative to the control (Figure 6a). However, the passage of cow manure through the earthworm gut affected fungal populations to a lesser extent than bacteria (Figure 6b).

Animal manures are microbial-rich environments in which bacteria constitute the largest fraction (around 70% of the total microbial biomass as assessed by PLFA analysis), with fungi mainly present as spores (Domínguez et al., 2010). Thus, earthworm activity is expected to have a greater effect on bacteria than on fungi in these organic substrates. These contrasting short-term effects on bacterial and fungal populations with earthworm activity are thus expected to have important implications on decomposition pathways during vermicomposting, because there exist important differences between both microbial

Vermicomposting: Composting with Earthworms to Recycle Organic Wastes 37

decreases in microbial activity were reported in casts of *Eu. eugeniae* and *E. fetida* fed on pig

Fig. 7. Changes in microbial activity assessed by fluorescein diacetate hydrolysis, after the passage of cow manure through the gut of the earthworm species *Eisenia andrei*. Values are

Earthworms of different functional groups, or even different species within the same functional group, have a particular mode of food selection, ingestion, digestion, assimilation and movement, thus their importance in mixing, decomposition or nutrient release, as well as in the structure and activity of microbial communities will vary both qualitatively and quantitatively (Curry & Schmidt, 2007). To determine how the earthworm species shape the relationships between earthworms and microorganisms during the active phase of vermicomposting, we performed an experiment with mesocosms filled with cow manure and inoculated with 10 mature individuals of the earthworm species *Eisenia andrei*, *Eisenia fetida* and *Perionyx excavatus*. The mesocosms consisted of 2 L plastic containers filled to three quarters of their capacity with sieved, moistened vermiculite. A plastic mesh was placed over the surface of the vermiculite and 200 g (fresh weight, fw) of the substrate was placed on top of the mesh, to avoid mixing the substrate with the vermiculite bedding. The mesocosms were covered with perforated lids and stored in random positions in an incubation chamber, at 20 °C and 90% relative humidity. Control mesocosms consisted of each type of manure incubated without earthworms. Each treatment was replicated three times. The length of the active phase depends greatly on the rates at which the earthworms ingest and process the substrate (Domínguez et al., 2010). The high rate of consumption, digestion and assimilation of organic matter by these earthworm species resulted in the substrates being completely processed by the earthworms in one month, as previously shown by Lores et al. (2006). After this time (i.e., active phase), the earthworms were removed from the mesocosms and the processed material was collected from the surface of the vermiculite. The same amount of sample was also collected from the control mesocosms.

means ± SE. Control is the manure incubated without earthworms.

**4.2 How does the earthworm species affect microbial communities?** 

and cow manures respectively (Aira et al., 2006; Aira & Domínguez, 2009).

Fig. 6. Changes in (a) bacterial biomass calculated as the sum of the bacterial PLFA markers: i14:0, i15:0, a15:0, i16:0, a17:0, 16:1ω7, 17:1ω7, 18:1ω7, cy17:0 and cy19:0, and (b) PLFA 18:2ω6c, a measure of fungal biomass, after the passage of cow manure through the gut of the earthworm species *Eisenia andrei*. Values are means ± SE. Control is the manure incubated without earthworms.

decomposers related to resource requirements and exploitation. This is based on the fact that bacteria are more competitive in the use of readily decomposable compounds and have a more exploitative nutrient use strategy by rapidly using newly produced labile substrates (Bardgett & Wardle, 2010); whereas fungi are more competitive with regard to the degradation of more slowly decomposable compounds such as cellulose, hemicellulose and lignin (de Boer et al., 2005).

#### **4.1.2 Microbial activity**

The transit of the organic material through the gut of the earthworm *E. andrei* reduced the microbial activity, measured as FDA hydrolysis, relative to the control (Figure 7). We found up to a 30% reduction in the microbial activity from the control treatment (524.8 ± 60.1 µg fluorescein g-1 dw h-1) to earthworm casts (208.0 ± 21.7 µg fluorescein g-1 dw h-1). Similar

Fig. 6. Changes in (a) bacterial biomass calculated as the sum of the bacterial PLFA markers: i14:0, i15:0, a15:0, i16:0, a17:0, 16:1ω7, 17:1ω7, 18:1ω7, cy17:0 and cy19:0, and (b) PLFA 18:2ω6c, a measure of fungal biomass, after the passage of cow manure through the gut of the earthworm species *Eisenia andrei*. Values are means ± SE. Control is the manure

decomposers related to resource requirements and exploitation. This is based on the fact that bacteria are more competitive in the use of readily decomposable compounds and have a more exploitative nutrient use strategy by rapidly using newly produced labile substrates (Bardgett & Wardle, 2010); whereas fungi are more competitive with regard to the degradation of more slowly decomposable compounds such as cellulose, hemicellulose and

The transit of the organic material through the gut of the earthworm *E. andrei* reduced the microbial activity, measured as FDA hydrolysis, relative to the control (Figure 7). We found up to a 30% reduction in the microbial activity from the control treatment (524.8 ± 60.1 µg fluorescein g-1 dw h-1) to earthworm casts (208.0 ± 21.7 µg fluorescein g-1 dw h-1). Similar

incubated without earthworms.

lignin (de Boer et al., 2005).

**4.1.2 Microbial activity** 

decreases in microbial activity were reported in casts of *Eu. eugeniae* and *E. fetida* fed on pig and cow manures respectively (Aira et al., 2006; Aira & Domínguez, 2009).

Fig. 7. Changes in microbial activity assessed by fluorescein diacetate hydrolysis, after the passage of cow manure through the gut of the earthworm species *Eisenia andrei*. Values are means ± SE. Control is the manure incubated without earthworms.

#### **4.2 How does the earthworm species affect microbial communities?**

Earthworms of different functional groups, or even different species within the same functional group, have a particular mode of food selection, ingestion, digestion, assimilation and movement, thus their importance in mixing, decomposition or nutrient release, as well as in the structure and activity of microbial communities will vary both qualitatively and quantitatively (Curry & Schmidt, 2007). To determine how the earthworm species shape the relationships between earthworms and microorganisms during the active phase of vermicomposting, we performed an experiment with mesocosms filled with cow manure and inoculated with 10 mature individuals of the earthworm species *Eisenia andrei*, *Eisenia fetida* and *Perionyx excavatus*. The mesocosms consisted of 2 L plastic containers filled to three quarters of their capacity with sieved, moistened vermiculite. A plastic mesh was placed over the surface of the vermiculite and 200 g (fresh weight, fw) of the substrate was placed on top of the mesh, to avoid mixing the substrate with the vermiculite bedding. The mesocosms were covered with perforated lids and stored in random positions in an incubation chamber, at 20 °C and 90% relative humidity. Control mesocosms consisted of each type of manure incubated without earthworms. Each treatment was replicated three times. The length of the active phase depends greatly on the rates at which the earthworms ingest and process the substrate (Domínguez et al., 2010). The high rate of consumption, digestion and assimilation of organic matter by these earthworm species resulted in the substrates being completely processed by the earthworms in one month, as previously shown by Lores et al. (2006). After this time (i.e., active phase), the earthworms were removed from the mesocosms and the processed material was collected from the surface of the vermiculite. The same amount of sample was also collected from the control mesocosms.

Vermicomposting: Composting with Earthworms to Recycle Organic Wastes 39

Fig. 9. Changes in (a) bacterial biomass calculated as the sum of the bacterial PLFA markers: i14:0, i15:0, a15:0, i16:0, i17:0, a17:0, 10Me16:0, 10Me17:0, 10Me18:0, 16:1ω7, 18:1ω7, cy17:0 and cy19:0, and (b) PLFA 18:2ω6c, a measure of fungal biomass, of cow manure after being processed by the epigeic earthworm species *Eisenia andrei*, *Eisenia fetida* and *Perionyx* 

*excavatus* during the active phase of vermicomposting. Values are means ± SE. Control is the

*E. andrei* reduced the bacterial growth rate by approximately 1.5 times relative to the control without earthworms after the active phase of vermicomposting (Figure 10a); no significant differences were detected with *E. fetida* and *P. excavatus* (Figure 10a). Despite the consistent effects on bacterial growth, earthworm activity did not affect the fungal growth rate (data not shown). Microbial activity in cow manure followed the same pattern as the bacterial

manure incubated without earthworms.

**4.2.2 Microbial activity** 

The viable microbial biomass was assessed as the sum of all identified PLFAs and certain PLFAs were used as biomarkers to determine the presence and abundance of specific microbial groups. Microbial community function was determined by measuring the bacterial and fungal growth rates. Bacterial growth was estimated by the incorporation of radioactively labelled leucine into proteins (Bååth, 1994), as modified by Bååth et al. (2001); fungal growth was estimated by the incorporation of radioactively labelled acetate into the fungal-specific lipid ergosterol Newell & Fallon (1991), with modifications by Bååth (2001). Total microbial activity was also assessed by measuring the rate of evolution of CO2. The data were analyzed by a one-way ANOVA test. Post hoc comparisons of means were performed by a Tukey HSD test, at α = 0.05.

#### **4.2.1 Microbial biomass**

The viable microbial biomass was about 3.8 times lower in the presence of *E. andrei* than that in the control (Figure 8), while no such pronounced decrease was detected in relation to the activity of *E. fetida* and *P. excavatus* (Figure 8). Similarly, the activity of *E. andrei* drastically reduced the bacterial and fungal biomass in cow manure, relative to the control (3.7 and 5.3 times, respectively), after the active phase of vermicomposting (Figure 9).

In the present study, the earthworm species *E. andrei* could have reduced the abundance of these microbial groups directly through ingestion, digestion and assimilation in the gut, and/or indirectly by accelerating the depletion of resources for the microbes, since greater losses of carbon were found as a result of earthworm activity after the active phase of vermicomposting (data not shown). However, the second explanation seems more likely to justify the reduction in fungal populations, since no significant changes were found in this microbial group after the passage through the gut of *E. andrei* (see experiment 1).

Fig. 8. Changes in the viable microbial biomass, measured as total PLFAs, of cow manure after being processed by the epigeic earthworm species *Eisenia andrei*, *Eisenia fetida* and *Perionyx excavatus* during the active phase of vermicomposting. Values are means ± SE. Control is the manure incubated without earthworms.

The viable microbial biomass was assessed as the sum of all identified PLFAs and certain PLFAs were used as biomarkers to determine the presence and abundance of specific microbial groups. Microbial community function was determined by measuring the bacterial and fungal growth rates. Bacterial growth was estimated by the incorporation of radioactively labelled leucine into proteins (Bååth, 1994), as modified by Bååth et al. (2001); fungal growth was estimated by the incorporation of radioactively labelled acetate into the fungal-specific lipid ergosterol Newell & Fallon (1991), with modifications by Bååth (2001). Total microbial activity was also assessed by measuring the rate of evolution of CO2. The data were analyzed by a one-way ANOVA test. Post hoc comparisons of means were

The viable microbial biomass was about 3.8 times lower in the presence of *E. andrei* than that in the control (Figure 8), while no such pronounced decrease was detected in relation to the activity of *E. fetida* and *P. excavatus* (Figure 8). Similarly, the activity of *E. andrei* drastically reduced the bacterial and fungal biomass in cow manure, relative to the control (3.7 and 5.3

In the present study, the earthworm species *E. andrei* could have reduced the abundance of these microbial groups directly through ingestion, digestion and assimilation in the gut, and/or indirectly by accelerating the depletion of resources for the microbes, since greater losses of carbon were found as a result of earthworm activity after the active phase of vermicomposting (data not shown). However, the second explanation seems more likely to justify the reduction in fungal populations, since no significant changes were found in this

times, respectively), after the active phase of vermicomposting (Figure 9).

microbial group after the passage through the gut of *E. andrei* (see experiment 1).

Fig. 8. Changes in the viable microbial biomass, measured as total PLFAs, of cow manure after being processed by the epigeic earthworm species *Eisenia andrei*, *Eisenia fetida* and *Perionyx excavatus* during the active phase of vermicomposting. Values are means ± SE.

Control is the manure incubated without earthworms.

performed by a Tukey HSD test, at α = 0.05.

**4.2.1 Microbial biomass** 

Fig. 9. Changes in (a) bacterial biomass calculated as the sum of the bacterial PLFA markers: i14:0, i15:0, a15:0, i16:0, i17:0, a17:0, 10Me16:0, 10Me17:0, 10Me18:0, 16:1ω7, 18:1ω7, cy17:0 and cy19:0, and (b) PLFA 18:2ω6c, a measure of fungal biomass, of cow manure after being processed by the epigeic earthworm species *Eisenia andrei*, *Eisenia fetida* and *Perionyx excavatus* during the active phase of vermicomposting. Values are means ± SE. Control is the manure incubated without earthworms.

#### **4.2.2 Microbial activity**

*E. andrei* reduced the bacterial growth rate by approximately 1.5 times relative to the control without earthworms after the active phase of vermicomposting (Figure 10a); no significant differences were detected with *E. fetida* and *P. excavatus* (Figure 10a). Despite the consistent effects on bacterial growth, earthworm activity did not affect the fungal growth rate (data not shown). Microbial activity in cow manure followed the same pattern as the bacterial

Vermicomposting: Composting with Earthworms to Recycle Organic Wastes 41

occurred in this experiment, indicating that it is a more extreme r strategist than *E. fetida*, as

**4.3 How do earthworms affect microbial communities of a plant residue in the short-**

In this study we evaluated the effectiveness of the active phase of vermicomposting for the short-term stabilization of grape marc, a lignocellulosic enriched residue that consists of the stalks, skin, pulp and seeds remaining after the grape crushing and pressing stages in wine production (Flavel et al., 2005). This by-product is a valuable resource as a soil fertilizer with high contents of macro- and micronutrients for crop growth (Bertran et al., 2004). However, the overproduction of grape marc – more than 750,000 ton per year in Spain (Fernández-Bayo et al., 2007) – has become a problem that requires strategies for its disposal and/or management. Whilst composting has been widely used for the treatment of winery wastes (Bertran et al., 2004; Marhuenda-Egea et al., 2007; Fernández et al., 2008; Bustamante et al., 2009; Paradelo et al., 2010), there are still very few studies on the application of vermicomposting as a methodological alternative to recycling such wastes (Nogales et al.,

The vermicomposting of grape marc was performed in mesocosms that consisted of plastic containers (2 L), which were filled to three quarters of the capacity with moistened (80% moisture content) and mature vermicompost in order to ensure the survival of the earthworms. Five hundred juvenile and adult specimens of the epigeic earthworm species *Eisenia andrei* were placed on the surface of the vermicompost. One kilogram (fresh weight) of grape marc was placed on a mesh on the surface of the vermicompost and was rewetted by spraying it with 20mL of tap water. The mesocosms were covered with perforated lids and stored in random positions in an incubation chamber, at 20 °C and 90% relative humidity. Control mesocosms consisted of the grape marc incubated without earthworms. Each treatment was replicated five times. The high density of earthworms used and the relatively rapid gut transit time of the epigeic earthworm species *E. andrei*, around 2.5–7 h, resulted in the grape marc being completely processed by the earthworms in 15 days. After this time (i.e., active phase), the earthworms were removed from the mesocosms and the processed material was collected from the surface of the vermicompost bedding. The same amount of sample was also collected from the control mesocosms. The viable microbial biomass was assessed as the sum of all identified PLFAs and certain PLFAs were used as biomarkers to determine the presence and abundance of specific microbial groups. Microbial community function was determined by measuring the total microbial activity assessed by basal respiration, and by determining the activity of enzymes involved in C and

Earthworm activity reduced the viable microbial biomass measured as total PLFAs relative to the control without earthworms (96.90 ± 1.04 µg mL -1 and 113.60 ± 1.04 µg mL -1 for treatments with and without earthworms). Similarly, the presence of earthworms also reduced the abundance of both bacteria and fungi after the active phase of vermicomposting

shown by more rapid growth and reproduction (Domínguez et al., 2005).

**term?** 

2005; Romero et al., 2007, 2010).

N cycles, i.e. protease and cellulase activities.

**4.3.1 Microbial biomass** 

of grape marc (Figure 11).

growth rate (Figure 10b). As mentioned before, bacteria constitute the largest fraction of the microbiota in animal manures, and they are therefore expected to contribute greatly to the respiration rate.

Fig. 10. Changes in (a) bacterial growth rate, estimated as leucine uptake and (b) microbial activity, measured as basal respiration, of cow manure after being processed by the epigeic earthworm species *Eisenia andrei*, *Eisenia fetida* and *Perionyx excavatus* during the active phase of vermicomposting. Values are means ± SE. Control is the manure incubated without earthworms.

The above-mentioned results highlight the potential of *E. andrei* for biodegrading organic substrates. The species *E. andrei* and *E. fetida* are closely related, although *E. andrei* predominates in mixed cultures, especially when there is no substrate limitation, as

growth rate (Figure 10b). As mentioned before, bacteria constitute the largest fraction of the microbiota in animal manures, and they are therefore expected to contribute greatly to the

Fig. 10. Changes in (a) bacterial growth rate, estimated as leucine uptake and (b) microbial activity, measured as basal respiration, of cow manure after being processed by the epigeic earthworm species *Eisenia andrei*, *Eisenia fetida* and *Perionyx excavatus* during the active phase of vermicomposting. Values are means ± SE. Control is the manure incubated without

The above-mentioned results highlight the potential of *E. andrei* for biodegrading organic substrates. The species *E. andrei* and *E. fetida* are closely related, although *E. andrei* predominates in mixed cultures, especially when there is no substrate limitation, as

respiration rate.

earthworms.

occurred in this experiment, indicating that it is a more extreme r strategist than *E. fetida*, as shown by more rapid growth and reproduction (Domínguez et al., 2005).

#### **4.3 How do earthworms affect microbial communities of a plant residue in the shortterm?**

In this study we evaluated the effectiveness of the active phase of vermicomposting for the short-term stabilization of grape marc, a lignocellulosic enriched residue that consists of the stalks, skin, pulp and seeds remaining after the grape crushing and pressing stages in wine production (Flavel et al., 2005). This by-product is a valuable resource as a soil fertilizer with high contents of macro- and micronutrients for crop growth (Bertran et al., 2004). However, the overproduction of grape marc – more than 750,000 ton per year in Spain (Fernández-Bayo et al., 2007) – has become a problem that requires strategies for its disposal and/or management. Whilst composting has been widely used for the treatment of winery wastes (Bertran et al., 2004; Marhuenda-Egea et al., 2007; Fernández et al., 2008; Bustamante et al., 2009; Paradelo et al., 2010), there are still very few studies on the application of vermicomposting as a methodological alternative to recycling such wastes (Nogales et al., 2005; Romero et al., 2007, 2010).

The vermicomposting of grape marc was performed in mesocosms that consisted of plastic containers (2 L), which were filled to three quarters of the capacity with moistened (80% moisture content) and mature vermicompost in order to ensure the survival of the earthworms. Five hundred juvenile and adult specimens of the epigeic earthworm species *Eisenia andrei* were placed on the surface of the vermicompost. One kilogram (fresh weight) of grape marc was placed on a mesh on the surface of the vermicompost and was rewetted by spraying it with 20mL of tap water. The mesocosms were covered with perforated lids and stored in random positions in an incubation chamber, at 20 °C and 90% relative humidity. Control mesocosms consisted of the grape marc incubated without earthworms. Each treatment was replicated five times. The high density of earthworms used and the relatively rapid gut transit time of the epigeic earthworm species *E. andrei*, around 2.5–7 h, resulted in the grape marc being completely processed by the earthworms in 15 days. After this time (i.e., active phase), the earthworms were removed from the mesocosms and the processed material was collected from the surface of the vermicompost bedding. The same amount of sample was also collected from the control mesocosms. The viable microbial biomass was assessed as the sum of all identified PLFAs and certain PLFAs were used as biomarkers to determine the presence and abundance of specific microbial groups. Microbial community function was determined by measuring the total microbial activity assessed by basal respiration, and by determining the activity of enzymes involved in C and N cycles, i.e. protease and cellulase activities.

#### **4.3.1 Microbial biomass**

Earthworm activity reduced the viable microbial biomass measured as total PLFAs relative to the control without earthworms (96.90 ± 1.04 µg mL -1 and 113.60 ± 1.04 µg mL -1 for treatments with and without earthworms). Similarly, the presence of earthworms also reduced the abundance of both bacteria and fungi after the active phase of vermicomposting of grape marc (Figure 11).

Vermicomposting: Composting with Earthworms to Recycle Organic Wastes 43

both vermicomposting treatments (vermicomposting and a combination of composting and vermicomposting) produced more stabilized substrates than the active phase of composting

Fig. 12. Changes in microbial activity assessed by basal respiration of grape marc after being

The study of enzyme activities has been shown to be a reliable tool for characterizing the state and evolution of the organic matter during vermicomposting (Benítez et al., 2005), as they are implicated in the biological and biochemical processes that transform organic wastes into stabilized products. In the present study, earthworm activity greatly reduced the activities of the protease (Figure 13a) and cellulase enzymes (Figure 13b) in comparison with the control. These findings are in agreement with microbial activity data, which reinforces that a higher degree of stability was reached after the active phase of vermicomposting. Similarly, Lazcano et al. (2008) reported lower values of protease activity, relative to the control, after vermicomposting and composting with subsequent vermicomposting (3 and 4.4 times lower, respectively). However, they did not find any differences in relation to this enzyme activity after the active phase of composting, indicating that the vermicomposted

processed by the epigeic earthworm species *Eisenia andrei* during the active phase of vermicomposting. Values are means ± SE. Control is the grape marc incubated without

materials were significantly more stabilized than the compost.

in terms of microbial activity.

earthworms.

Fig. 11. Changes in (a) bacterial biomass calculated as the sum of the bacterial PLFA markers: i14:0, i15:0, a15:0, i16:0, i17:0, a17:0, 16:1ω7, 17:1ω7, cy17:0 and cy19:0, and (b) PLFAs 18:1ω9c and 18:2ω6c, a measure of fungal biomass, of grape marc after being processed by the epigeic earthworm species *Eisenia andrei* during the active phase of vermicomposting. Values are means ± SE. Control is the grape marc incubated without earthworms.

#### **4.3.2 Microbial activity**

As occurred in the two previous experiments, the total microbial activity measured as basal respiration was about 1.7 times lower in the presence of *E. andrei* than that in the control without earthworms (Figure 12). This suggests that the presence of earthworms favoured the stabilization of the residue, as shown by Lazcano et al. (2008). These authors found that

Fig. 11. Changes in (a) bacterial biomass calculated as the sum of the bacterial PLFA markers: i14:0, i15:0, a15:0, i16:0, i17:0, a17:0, 16:1ω7, 17:1ω7, cy17:0 and cy19:0, and (b) PLFAs 18:1ω9c and 18:2ω6c, a measure of fungal biomass, of grape marc after being processed by the epigeic earthworm species *Eisenia andrei* during the active phase of vermicomposting. Values are means ± SE. Control is the grape marc incubated without

As occurred in the two previous experiments, the total microbial activity measured as basal respiration was about 1.7 times lower in the presence of *E. andrei* than that in the control without earthworms (Figure 12). This suggests that the presence of earthworms favoured the stabilization of the residue, as shown by Lazcano et al. (2008). These authors found that

earthworms.

**4.3.2 Microbial activity** 

both vermicomposting treatments (vermicomposting and a combination of composting and vermicomposting) produced more stabilized substrates than the active phase of composting in terms of microbial activity.

Fig. 12. Changes in microbial activity assessed by basal respiration of grape marc after being processed by the epigeic earthworm species *Eisenia andrei* during the active phase of vermicomposting. Values are means ± SE. Control is the grape marc incubated without earthworms.

The study of enzyme activities has been shown to be a reliable tool for characterizing the state and evolution of the organic matter during vermicomposting (Benítez et al., 2005), as they are implicated in the biological and biochemical processes that transform organic wastes into stabilized products. In the present study, earthworm activity greatly reduced the activities of the protease (Figure 13a) and cellulase enzymes (Figure 13b) in comparison with the control. These findings are in agreement with microbial activity data, which reinforces that a higher degree of stability was reached after the active phase of vermicomposting. Similarly, Lazcano et al. (2008) reported lower values of protease activity, relative to the control, after vermicomposting and composting with subsequent vermicomposting (3 and 4.4 times lower, respectively). However, they did not find any differences in relation to this enzyme activity after the active phase of composting, indicating that the vermicomposted materials were significantly more stabilized than the compost.

Vermicomposting: Composting with Earthworms to Recycle Organic Wastes 45

gut of epigeic earthworms reduced the viable microbial biomass and affected the abundance of bacteria to a greater extent than fungi. Microbial activity also decreased after transit of the microorganisms through the earthworm gut. Accordingly, the presence of earthworms reduced microbial biomass and activity after the active phase of vermicomposting, although this effect depended on the earthworm species involved. The bacterial growth rate also decreased in the substrate, whereas the fungal growth rate was not affected after one month. The speed at which these transformations occurred made the active phase of vermicomposting a suitable stage for studying the relationships between earthworms and microorganisms and permitted us to understand the chemical and biological consequences of earthworm activities. Ultimately, these findings provide valuable information for the understanding of the transformations that organic matter undergoes during vermicomposting and, in addition constitute a powerful tool for the development of strategies leading to a more efficient process for the disposal and/or management of organic

This research was financially supported by the Spanish Ministerio de Ciencia e Innovación (CTM2009-08477). María Gómez Brandón is financially supported by a postdoctoral

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**6. Acknowledgments** 

**7. References** 

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research grant from Fundación Alfonso Martín Escudero.

Fig. 13. Changes in (a) protease activity, and (b) cellulase activity of grape marc after being processed by the epigeic earthworm species *Eisenia andrei* during the active phase of vermicomposting. Values are means ± SE. Control is the grape marc incubated without earthworms.
