Host taxonomy determines the composition, structure, and diversity of the earthworm cast microbiome under homogenous feeding conditions.

Host evolutionary history is a key factor shaping the earthworm cast microbiome, although its effect can be shadowed by the earthworm's diet. To untangle dietary from taxon effects, we raised nine earthworm species on a uniform diet of cow manure and compared cast microbiome across species while controlling for diet. Our results showed that, under controlled laboratory conditions, earthworm microbiomes are species-specific, more diverse than that of the controlled diet, and mainly comprised of native bacteria (i.e. not acquired from the diet). Furthermore, diet has a medium to large convergence effect on microbiome composition since earthworms shared 16%-74% of their bacterial amplicon sequence variants (ASV). The interspecies core microbiome included 10 ASVs, while their intraspecies core microbiomes were larger and varied in ASV richness (24%-48%) and sequence abundance across earthworm species. This specificity in core microbiomes and variable degree of similarity in bacterial composition suggest that phylosymbiosis could determine earthworm microbiome assembly. However, lack of congruence between the earthworm phylogeny and the microbiome dendrogram suggests that a consistent diet fed over several generations may have weakened potential phylosymbiotic effects. Thus, cast microbiome assembly in earthworms seem to be the result of an interplay among host phylogeny and diet.

Composition, Structure and Diversity of Soil Bacterial Communities before, during and after Transit through the Gut of the Earthworm Aporrectodea caliginosa.

Earthworms heavily modify the soil microbiome as it passes throughout their guts. However, there are no detailed studies describing changes in the composition, structure and diversity of soil microbiomes during gut transit and once they are released back to the soil as casts. To address this knowledge gap, we used 16S rRNA next-generation sequencing to characterize the microbiomes of soil, gut and casts from the earthworm Aporrectodea caliginosa. We also studied whether these three microbiomes are clearly distinct in composition or can be merged into metacommunities. A large proportion of bacteria was unique to each microbiome-soil (82%), gut (89%) and casts (75%), which indicates that the soil microbiome is greatly modified during gut transit. The three microbiomes also differed in alpha diversity, which peaked during gut transit and decreased in casts. Furthermore, gut transit also modified the structure of the soil microbiome, which clustered away from those of the earthworm gut and cast samples. However, this clustering pattern was not supported by metacommunity analysis, which indicated that soil and gut samples make up one metacommunity and cast samples another. These results have important implications for understanding the dynamics of soil microbial communities and nutrient cycles.

Gut microbiome and telomere length in gull hatchlings.

In many animals, recent evidence indicates that the gut microbiome may be acquired during early development, with possible consequences on newborns' health. Thus, it has been hypothesized that a healthy microbiome protects telomeres and genomic integrity against cellular stress. However, the link between the early acquired microbiome and telomere dynamics has not hitherto been investigated. In birds, this link may also be potentially modulated by the transfer of maternal glucocorticoids, since these substances dysregulate microbiome composition during postnatal development. Here, we examined the effect of the interplay between the microbiome and stress hormones on the telomere length of yellow-legged gull hatchlings by using a field experiment in which we manipulated the corticosterone content in eggs. We found that the hatchling telomere length was related to microbiome composition, but this relationship was not affected by the corticosterone treatment. Hatchlings with a microbiome dominated by potential commensal bacteria (i.e. Catellicoccus and Cetobacterium) had larger telomeres, suggesting that an early establishment of the species-specific microbiome during development may have important consequences on offspring health and survival.

Earthworms drastically change fungal and bacterial communities during vermicomposting of sewage sludge.

Wastewater treatment plants produce hundreds of million tons of sewage sludge every year all over the world. Vermicomposting is well established worldwide and has been successful at processing sewage sludge, which can contribute to alleviate the severe environmental problems caused by its disposal. Here, we utilized 16S and ITS rRNA high-throughput sequencing to characterize bacterial and fungal community composition and structure during the gut- and cast-associated processes (GAP and CAP, respectively) of vermicomposting of sewage sludge. Bacterial and fungal communities of earthworm casts were mainly composed of microbial taxa not found in the sewage sludge; thus most of the bacterial (96%) and fungal (91%) taxa in the sewage sludge were eliminated during vermicomposting, mainly through the GAP. Upon completion of GAP and during CAP, modified microbial communities undergo a succession process leading to more diverse microbiotas than those found in sewage sludge. Consequently, bacterial and fungal community composition changed significantly during vermicomposting. Vermicomposting of sewage resulted in a stable and rich microbial community with potential biostimulant properties that may aid plant growth. Our results support the use of vermicompost derived from sewage sludge for sustainable agricultural practices, if heavy metals or other pollutants are under legislation limits or adequately treated.

Bacterial Succession during Vermicomposting of Silver Wattle (Acacia dealbata Link).

Vermicomposting is the process of organic waste degradation through interactions between earthworms and microbes. A variety of organic wastes can be vermicomposted, producing a nutrient-rich final product that can be used as a soil biofertilizer. Giving the prolific invasive nature of the Australian silver wattle Acacia dealbata Link in Europe, it is important to find alternatives for its sustainable use. However, optimization of vermicomposting needs further comprehension of the fundamental microbial processes. Here, we characterized bacterial succession during the vermicomposting of silver wattle during 56 days using the earthworm species Eisenia andrei. We observed significant differences in α- and ß-diversity between fresh silver wattle (day 0) and days 14 and 28, while the bacterial community seemed more stable between days 28 and 56. Accordingly, during the first 28 days, a higher number of taxa experienced significant changes in relative abundance. A microbiome core composed of 10 amplicon sequence variants was identified during the vermicomposting of silver wattle (days 14 to 56). Finally, predicted functional profiles of genes involved in cellulose metabolism, nitrification, and salicylic acid also changed significantly during vermicomposting. This study, hence, provides detailed insights of the bacterial succession occurring during vermicomposting of the silver wattle and the characteristics of its final product as a sustainable plant biofertilizer.

Temporal Dynamics of Bacterial Communities in a Pilot-Scale Vermireactor Fed with Distilled Grape Marc.

Vermicomposting has been found as a profitable approach to dispose of and treat large quantities of raw grape marc. However, less information is available with regard to its efficiency for treating distillery winery byproducts, even though distillation has been widely used as a way to economically valorize grape marc. As such, we sought to characterize the compositional and functional changes in bacterial communities during vermicomposting of distilled grape marc by using 16S rRNA high-throughput sequencing. Samples were collected at the initiation of vermicomposting and at days 14, 21, 28, 35 and 42. There were significant changes (p < 0.0001) in the bacterial community composition of distilled grape marc after 14 days of vermicomposting that were accompanied by twofold increases in bacterial richness and diversity from a taxonomic and phylogenetic perspective. This was followed by significant increases in functional diversity of the bacterial community, including metabolic capacity, lignin and cellulose metabolism, and salicylic acid synthesis. These findings indicate that the most striking compositional and functional bacterial community changes took place during the active phase of the process. They also pinpoint functional attributes that may be related to the potential beneficial effects of distilled grape marc vermicompost when applied on soil and plants.

Rapid Bacterial Community Changes during Vermicomposting of Grape Marc Derived from Red Winemaking.

Previous studies dealing with changes in microbial communities during vermicomposting were mostly performed at lab-scale conditions and by using low-throughput techniques. Therefore, we sought to characterize the bacterial succession during the vermicomposting of grape marc over a period of 91 days in a pilot-scale vermireactor. Samples were taken at the initiation of vermicomposting, and days 14, 28, 42, and 91, representing both active and mature stages of vermicomposting. By using 16S rRNA high-throughput sequencing, significant changes in the bacterial community composition of grape marc were found after 14 days and throughout the process (p < 0.0001). There was also an increase in bacterial diversity, both taxonomic and phylogenetic, from day 14 until the end of the trial. We found the main core microbiome comprised of twelve bacterial taxa (~16.25% of the total sequences) known to be capable of nitrogen fixation and to confer plant-disease suppression. Accordingly, functional diversity included increases in specific genes related to nitrogen fixation and synthesis of plant hormones (salicylic acid) after 91 days. Together, the findings support the use of grape marc vermicompost for sustainable practices in the wine industry by disposing of this high-volume winery by-product and capturing its value to improve soil fertility.

Changes in the composition and function of bacterial communities during vermicomposting may explain beneficial properties of vermicompost.

Vermicomposting is the process by which organic waste is broken down through the synergistic actions of earthworms and microbial communities. Although vermicomposting has been shown to effectively reduce organic biomass and generate high-quality fertilizer for plants, little is known about the bacterial communities that are involved in this decomposition process. Since optimization of vermicomposting for commercial use necessitates additional knowledge of the underlying biological processes, this study sought to characterize the bacterial succession involved in the vermicomposting of Scotch broom (Cytisus scoparius), a leguminous shrub that has become invasive around the world with consequences for the dynamics and productivity of the ecosystems they occupy. Scotch broom was processed in a pilot-scale vermireactor for 91 days with the earthworm species Eisenia andrei. Samples were taken at the initiation of vermicomposting, and days 14, 42 and 91, representing both active and mature stages of vermicomposting. Significant changes (P < 0.0001) in the bacterial community composition (richness and evenness) were observed throughout the process. Increases in taxonomic diversity were accompanied by increases in functional diversity of the bacterial community, including metabolic capacity, streptomycin and salicylic acid synthesis, and nitrification. These results highlight the role of bacterial succession during the vermicomposting process and provide evidence of microbial functions that may explain the beneficial effects of vermicompost on soil and plants.

Bacterial succession and functional diversity during vermicomposting of the white grape marc Vitis vinifera v. Albariño.

Winemaking produces millions of tons of grape marc, a byproduct of grape pressing, each year. Grape marc is made up of the skins, stalks, and seeds remaining after pressing. Raw grape marc can be hazardous to the environment due to its low pH and high polyphenol content, but previous work has shown that grape marc can be stabilized via vermicomposting to produce organic fertilizer. Here, we utilize 16S rRNA high-throughput sequencing to characterize the bacterial community composition, diversity and metabolic function during vermicomposting of the white grape marc Vitis vinifera v. Albariño for 91 days. Large, significant changes in the bacterial community composition of grape marc vermicompost were observed by day 7 of vermicomposting and throughout the duration of the experiment until day 91. Similarly, taxonomic and phylogenetic α-diversity increased throughout the experiment and estimates of ß-diversity differed significantly between time points. Functional diversity also changed during vermicomposting, including increases in cellulose metabolism, plant hormone synthesis, and antibiotic synthesis. Thus, vermicomposting of white grape marc resulted in a rich, stable bacterial community with functional properties that may aid plant growth. These results support the use of grape marc vermicompost for sustainable agricultural practices in the wine industry.

Multigene phylogeny reveals a new Iranian earthworm genus (Lumbricidae: Philomontanus) with three new species.

Lumbricidae taxonomy is vastly restricted by the morphological simplicity of earthworms and their lack of complex appendices. This has led to confusing results in the Lumbricidae classifications, which in turn, has hindered our ability to identify and assign new and cryptic species to the family. Here we propose the addition of a new Lumbricidae genus from the Zagros and Elburz Mountains of Iran, i.e. Philomontanus gen. nov, including three new species. Our taxonomic inferences were based on the phylogenetic analysis of two nuclear gene regions (28S rDNA and 18S rDNA) and 11 mitochondrial gene regions (16S rDNA, 12S rDNA, NADH dehydrogenase I, cytochrome oxidase subunits I and II and tRNAs Asn, Asp, Val, Leu, Ala and Ser). Philomontanus gen. nov comprises the earthworm species Philomontanus sarii sp. nov., Philomontanus mahmoudi sp. nov. and Philomontanus baloutchi sp. nov. These three species are morphologically similar to each other with only a few characters separating them (e.g. size, pigmentation and position of clitellum). Our findings support the adoption of an integrative approach including molecular information (e.g., DNA sequences) to aid earthworm classification and develop a robust taxonomy.

Glucocorticoids modulate gastrointestinal microbiome in a wild bird.

It has recently been hypothesized that stress exposure (e.g. via glucocorticoid secretion) may dysregulate the bacterial gut microbiome, a crucial 'organ' in animal health. However, whether stress exposure (e.g. via glucocorticoid secretion) affects the bacterial gut microbiome of natural populations is unknown. We have experimentally altered the basal glucocorticoid level (corticosterone implants) in a wild avian species, the yellow-legged gull Larus michahellis, to assess its effects on the gastrointestinal microbiota. Our results suggest underrepresentation of several microbial taxa in the corticosterone-implanted birds. Importantly, such reduction included potentially pathogenic avian bacteria (e.g. Mycoplasma and Microvirga) and also some commensal taxa that may be beneficial for birds (e.g. Firmicutes). Our findings clearly demonstrate a close link between microbiome communities and glucocorticoid levels in natural populations. Furthermore, they suggest a beneficial effect of stress in reducing the risk of infection that should be explored in future studies.

Diversity, structure and sources of bacterial communities in earthworm cocoons.

Animals start interactions with the bacteria that will constitute their microbiomes at embryonic stage. After mating, earthworms produce cocoons externally which will be colonized with bacteria from their parents and the environment. Due to the key role bacterial symbionts play on earthworm fitness, it is important to study bacterial colonization during cocoon formation. Here we describe the cocoon microbiome of the earthworms Eisenia andrei and E. fetida, which included 275 and 176 bacterial species, respectively. They were dominated by three vertically-transmitted symbionts, Microbacteriaceae, Verminephrobacter and Ca. Nephrothrix, which accounted for 88% and 66% of the sequences respectively. Verminephrobacter and Ca. Nephrothrix showed a high rate of sequence variation, suggesting that they could be biparentally acquired during mating. The other bacterial species inhabiting the cocoons came from the bedding, where they accounted for a small fraction of the diversity (27% and 7% of bacterial species for E. andrei and E. fetida bedding). Hence, earthworm cocoon microbiome includes a large fraction of the vertically-transmitted symbionts and a minor fraction, but more diverse, horizontally and non-randomly acquired from the environment. These data suggest that horizontally-transmitted bacteria to cocoons may play an important role in the adaptation of earthworms to new environments or diets.

Tracheal Microbiota in Patients With a Tracheostomy Before, During and After an Acute Respiratory Infection.

Examining tracheal microbiota before, during and after acute respiratory infection in patients with a tracheostomy demonstrated large baseline intrapatient microbiota variability and a significant bloom of Haemophilus and Moraxella on day 1 of acute respiratory infection symptoms. The tracheal microbiota community composition changed significantly from baseline to 1 month after acute respiratory infection.

The temporal dynamics of the tracheal microbiome in tracheostomised patients with and without lower respiratory infections.

BACKGROUND: Airway microbiota dynamics during lower respiratory infection (LRI) are still poorly understood due, in part, to insufficient longitudinal studies and lack of uncontaminated lower airways samples. Furthermore, the similarity between upper and lower airway microbiomes is still under debate. Here we compare the diversity and temporal dynamics of microbiotas directly sampled from the trachea via tracheostomy in patients with (YLRI) and without (NLRI) lower respiratory infections. METHODS: We prospectively collected 127 tracheal aspirates across four consecutive meteorological seasons (quarters) from 40 patients, of whom 20 developed LRIs and 20 remained healthy. All aspirates were collected when patients had no LRI. We generated 16S rRNA-based microbial profiles (~250 bp) in a MiSeq platform and analyzed them using Mothur and the SILVAv123 database. Differences in microbial diversity and taxon normalized (via negative binomial distribution) abundances were assessed using linear mixed effects models and multivariate analysis of variance. RESULTS AND DISCUSSION: Alpha-diversity (ACE, Fisher and phylogenetic diversity) and beta-diversity (Bray-Curtis, Jaccard and Unifrac distances) indices varied significantly (P<0.05) between NLRI and YLRI microbiotas from tracheostomised patients. Additionally, Haemophilus was significantly (P = 0.009) more abundant in YLRI patients than in NLRI patients, while Acinetobacter, Corynebacterium and Pseudomonas (P<0.05) showed the inverse relationship. We did not detect significant differences in diversity and bacterial abundance among seasons. This result disagrees with previous evidence suggesting seasonal variation in airway microbiotas. Further study is needed to address the interaction between microbes and LRI during times of health and disease.

Feeding on microbiomes: effects of detritivory on the taxonomic and phylogenetic bacterial composition of animal manures.

Earthworms play a key role in nutrient cycling by interacting with microorganisms thus accelerating organic matter turnover in soil systems. As detritivores, some earthworm types ingest and digest a mixture of dead organic matter and microorganisms, like animal manures (i.e. animal gut microbiomes). Here we described the earthworm cast microbiome and the role ingested bacteria play on its composition. We fed Eisenia andrei with cow, horse and pig manures and determined the taxonomic and phylogenetic composition of the these manures before and after passage through the earthworm gut. Earthworm cast microbiomes showed a smaller diversity than the manure they fed on. Manures strongly differed in their taxonomic and phylogenetic composition, but these differences were markedly reduced once transformed into earthworm cast microbiomes after passage through the earthworm gut. The core earthworm cast microbiome comprised 30 OTUs (2.6% of OTUs from cast samples), of which 10 are possibly native to the earthworm gut. Most of the core cast microbiome OTUs belonged to phyla Actinobacteria and Proteobacteria, as opposed to already described animal core gut microbiomes, which are composed mainly of Firmicutes and Bacteroidetes. Our results suggest that earthworms build up their cast microbiome by selecting from the pool of ingested bacteria.

Underground evolution: new roots for the old tree of lumbricid earthworms.

Earthworms belonging to the family Lumbricidae are extremely abundant in terrestrial temperate regions. They affect soil properties and nutrient cycling, thus shaping plant community composition and aboveground food webs. Some lumbricids are also model organisms in ecology and toxicology. Despite the intense research efforts dedicated to lumbricids over the last 130years, the evolutionary relationships and taxonomic classification of these organisms are still subject to great debate. Resolution of their systematics is hampered by the structural simplicity of the earthworm body plan and the existence of cryptic species. We sampled 160 earthworm specimens belonging to 84 lumbricid species (28 genera) and 22 Lumbricoidea outgroups, sequenced two nuclear genes, four mitochondrial genes and seven mitochondrial tRNAs and examined 22 morphological characters. We then applied a combination of phylogenetic methods to generate the first robust genus-level phylogeny of the Lumbricidae. Our results show that the current Lumbricidae classification and the underlying hypotheses of character evolution must be revised. Our chronogram suggests that lumbricids emerged in the Lower Cretaceous in the holarctic region and that their diversification has been driven by tectonic processes (e.g. Laurasia split) and geographical isolation. Our chronogram and character reconstruction analysis reveal that spermathecae number does not follow a gradual pattern of reduction and that parthenogenesis arose from sexual relatives multiple times in the group; the same analysis also indicates that both epigeic and anecic earthworms evolved from endogeic ancestors. These findings emphasize the strong and multiple changes to which morphological and ecological characters are subjected, challenging the hypothesis of character stasis in Lumbricidae.

An earthworm riddle: systematics and phylogeography of the Spanish lumbricid Postandrilus.

BACKGROUND: As currently defined, the genus Postandrilus Qui and Bouché, 1998, (Lumbricidae) includes six earthworm species, five occurring in Majorca (Baleares Islands, western Mediterranean) and another in Galicia (NW Spain). This disjunct and restricted distribution raises some interesting phylogeographic questions: (1) Is Postandrilus distribution the result of the separation of the Baleares-Kabylies (BK) microplate from the proto-Iberian Peninsula in the Late Oligocene (30-28 Mya)--vicariant hypothesis? (2) Did Postandrilus diversify in Spain and then colonize the Baleares during the Messinian salinity crisis (MSC) 5.96-5.33 Mya--dispersal hypothesis? (3) Is the distribution the result of a two-step process--vicariance with subsequent dispersal? METHODOLOGY/PRINCIPAL FINDINGS: To answer these questions and assess Postandrilus evolutionary relationships and systematics, we collected all of the six Postandrilus species (46 specimens - 16 locations) and used Aporrectodea morenoe and three Prosellodrilus and two Cataladrilus species as the outgroup. Regions of the nuclear 28S rDNA and mitochondrial 16S rDNA, 12S rDNA, ND1, COII and tRNA genes (4,666 bp) were sequenced and analyzed using maximum likelihood and Bayesian methods of phylogenetic and divergence time estimation. The resulting trees revealed six new Postandrilus species in Majorca that clustered with the other five species already described. This Majorcan clade was sister to an Iberian clade including A. morenoe (outgroup) and Postandrilus bertae. Our phylogeny and divergence time estimates indicated that the split between the Iberian and Majorcan Postandrilus clades took place 30.1 Mya, in concordance with the break of the BK microplate from the proto-Iberian Peninsula, and that the present Majorcan clade diversified 5.7 Mya, during the MSC. CONCLUSIONS: Postandrilus is highly diverse including multiple cryptic species in Majorca. The genus is not monophyletic and invalid as currently defined. Postandrilus is of vicariant origin and its radiation began in the Late Oligocene.

Epigeic earthworms exert a bottleneck effect on microbial communities through gut associated processes.

BACKGROUND: Earthworms play a critical role in organic matter decomposition because of the interactions they establish with microorganisms. The ingestion, digestion, assimilation of organic material in the gut and then casting is the first step in earthworm-microorganism interactions. The current knowledge of these direct effects is still limited for epigeic earthworm species, mainly those living in man-made environments. Here we tested whether and to what extent the earthworm Eisenia andrei is capable of altering the microbiological properties of fresh organic matter through gut associated processes; and if these direct effects are related to the earthworm diet. METHODOLOGY: To address these questions we determined the microbial community structure (phospholipid fatty acid profiles) and microbial activity (fluorescein diacetate hydrolysis) in the earthworm casts derived from three types of animal manure (cow, horse and pig manure), which differed in microbial composition. PRINCIPAL FINDINGS: The passage of the organic material through the gut of E. andrei reduced the total microbial biomass irrespective of the type of manure, and resulted in a decrease in bacterial biomass in all the manures; whilst leaving the fungi unaffected in the egested materials. However, unlike the microbial biomass, no such reduction was detected in the total microbial activity of cast samples derived from the pig manure. Moreover, no differences were found between cast samples derived from the different types of manure with regards to microbial community structure, which provides strong evidence for a bottleneck effect of worm digestion on microbial populations of the original material consumed. CONCLUSIONS/SIGNIFICANCE: Our data reveal that earthworm gut is a major shaper of microbial communities, thereby favouring the existence of a reduced but more active microbial population in the egested materials, which is of great importance to understand how biotic interactions within the decomposer food web influence on nutrient cycling.

Selective reduction of the pathogenic load of cow manure in an industrial-scale continuous-feeding vermireactor.

Vermicomposting is a suitable technology for processing different wastes, to produce a valuable end product (vermicompost). However, the pathogenic load of the waste must be greatly reduced in order to prevent risks to human health. Although Eisenia andrei may reduce the levels of several pathogens, the feasibility of vermicomposting, with regard to pathogen reduction, has not been tested on an industrial scale. This work studied whether vermicomposting in a continuous feeding vermireactor, is able to reduce the pathogenic load of cow manure. The effect of E. andrei on pathogens depended on the type of pathogen; thus, levels of Clostridium, total coliforms and Enterobacteria were not modified, but levels of faecal enterococci, faecal coliforms and Escherichia coli were reduced to acceptable levels. Pathogens could have maintained their levels in continuous feeding vermireactors, as fresh layers of manure are added to the top, which allows the vertical spread of pathogens through leaching.

Earthworm effects without earthworms: inoculation of raw organic matter with worm-worked substrates alters microbial community functioning.

BACKGROUND: Earthworms are key organisms in organic matter decomposition because of the interactions they establish with soil microorganisms. They enhance decomposition rates through the joint action of direct effects (i.e. effects due to direct earthworm activity such as digestion, burrowing, etc) and indirect effects (i.e. effects derived from earthworm activities such as cast ageing). Here we test whether indirect earthworm effects affect microbial community functioning in the substrate, as when earthworms are present (i. e., direct effects). METHODOLOGY/PRINCIPAL FINDINGS: To address these questions we inoculated fresh organic matter (pig manure) with worm-worked substrates (vermicompost) produced by three different earthworm species. Two doses of each vermicompost were used (2.5 and 10%). We hypothesized that the presence of worm-worked material in the fresh organic matter will result in an inoculum of different microorganisms and nutrients. This inoculum should interact with microbial communities in fresh organic matter, thus promoting modifications similar to those found when earthworms are present. Inoculation of worm-worked substrates provoked significant increases in microbial biomass and enzyme activities (ß-glucosidase, cellulase, phosphatase and protease). These indirect effects were similar to, although lower than, those obtained in pig manure with earthworms (direct and indirect earthworm effects). In general, the effects were not dose-dependent, suggesting the existence of a threshold at which they were triggered. CONCLUSION/SIGNIFICANCE: Our data reveal that the relationships between earthworms and microorganisms are far from being understood, and suggest the existence of several positive feedbacks during earthworm activity as a result of the interactions between direct and indirect effects, since their combination produces stronger modifications to microbial biomass and enzyme activity.