The unconventional waste: organic material of potential use

This type of waste from agroindustrial, urban and industrial areas, despite being considered not optimal for the development of earthworms under natural conditions, have been effectively biostabilized by vermicomposting. However, in some cases it is necessary to condition them to allow the survival of worms (^{Vargas-Machuca et al., 2008}; ^{Moreno et al., 2014}). Within this type of waste are sludge generated by some industries, in which vermicomposting is a simple and low-cost alternative for recycling.

The transformation of these residues is important for the stabilization of sludge generated by the food industry, the product obtained presents a significant chemical improvement in nitrogen, phosphorus, sodium and potassium, besides the stabilization of pH, total organic carbon and the C:N, including fertilizer obtained can be improved if it is enriched with other wastes, particularly with cattle manure or equine, obtaining a homogenous humus smell, granulation and dark (^{Garg et al., 2012}; ^{Meiyan et al., 2012}). In industry sago (Maranatha auridinacea) has used the benefits of vermicomposting, mixing the waste industry with cow dung and poultry, the resulting product is a fertilizer quality with convenient reduction of the C:N and improved nutritional status for crop production (^{Subramanian et al., 2010}).

One advantage of the vermicomposting of the residual sludge and its use as fertilizer is the improvement of the biochemical and physicochemical properties of the soil, especially of the less fertile ones. Studies in clay and sandy soils has shown that improvement starts from the activation of microbiological processes through the gradual modification of the nutritional conditions that occur within the live cover because of earthworm activity. The improved soil structure is agronomically important as good physical properties favor water retention, diffusion of oxygen and nutrient availability, which when combined improve quality and soil fertility (^{Masciandaro et al., 2010}).

The pharmaceutical industry comprising the processing of raw materials for pharmaceutical use generates organic pollutants which derives for the most part from the production of the active ingredient. These wastes can not be ruled out as conventional waste without giving prior special treatment (^{Ramos, 2006}; ^{Sreekanth et al., 2014}). The vermicomposting of herbs after processing mixed with cow manure, causes significant changes in the characteristics of the residue as a reduction of organic carbon, C:P, and C:N ratio, and generating an increase in nitrogen, phosphorus and potassium. The fertilizer obtained has potential use in ecological restoration and programs soil fertility (^{Singh and Suthar, 2012a}; ^{Singh and Suthar, 2012b}).

The vermicomposting also has application in the tannery industry, which generates toxic wastes and organic materials of slow degradation. This process is a viable alternative for the reduction and biodegradation of these pollutants, observing a decrease in pathogen concentration, electrical conductivity and volatile solids, the concentration of nitrogen and phosphorus at the end of the process are similar to those found in fertilizers organic. The earthworm and microorganisms combination with cattle manure modify the physicochemical properties of waste, obtaining a product free of odors, darker and homogeneous, with a decrease in the C:N (^{Cardoso-Vigueros and Ramírez-Camperos, 2006}; ^{Ravindran et al., 2015}).

In the paper industry that generates large amounts of waste, use of vermicomposting technology has generated high expectations. Studies conducted degradation of paper mixed with sawdust, horse manure and earthworm Perionyx excavatus has generated good results, to get vermicompost good quality (^{Abd et al., 2009}). As waste paper is rich in carbon and deficient in nitrogen, it can be mixed with bovine manure, making it more suitable for earthworms. This process increases the concentration of nitrogen, phosphorus and potassium, and decreases the pH and C:N ratio at the end of the process (^{Basheer and Agrawal, 2013}).

The waste from the forestry and livestock industry represents a serious ecological problem if not managed properly. An alternative to transform these materials is vermicomposting, and used safely as soil improvers. These residues when mixed with cow, rabbit and sheep manures, generate a good quality product with a decrease in the C:N ratio and pH, the contents of nitrates and nitrogen increase, giving the final product valuable chemical and nutritional properties (^{Hernández-Rodríguez et al., 2012}).

The wine industry of great economic importance in several countries generates large quantities of waste, including grape bagasse, a by-product of the process of extracting must in wine preparation. The vermicomposting bagasse generates a mature and stable product, with an increase in concentration of nutrients for use as organic fertilizer on farmland (^{Martínez-Cordeiro et al., 2013}). The speed with which these changes occur in the process make a good system to study the role of earthworms and microflora in the degradation of grape pomace (^{Gómez-Brandón et al., 2010}). Another residue from the alcoholic beverage industry is bagasse, a potential substrate for vermicomposting. The resulting humus provides a high nutrient reservoir in nitrogen, phosphorus, potassium, calcium and magnesium that allows its use in agriculture (^{Rodríguez et al., 2010}).

The sugar production is an important agricultural industry that generates solid waste such as bagasse, cachaza, clarification process sludge, ash and cane waste (^{Balakrishnan and Batra, 2011}). The transformation of this waste vermicompostings mixed with cattle manure and earthworm (Eisenia fetida), helps improve the physicochemical characteristics of the final product. The nutrients such as nitrogen, phosphorus and sodium increase favorably, with decrease of organic carbon and C:N ratio. In the transformation of these wastes they have been used as worms Eudrilus eugenia, Eisenia fetida and Perionyx excavates capable to convert a large part d and the matter organic waste humus in a relatively short period. The nutrients such as calcium, magnesium, sodium and potassium increase significantly and the pH is stable in the final product (^{Bhandarkar et al., 2014}). Experiments with sugar cane bagasse and rice straw have shown that vermicomposts obtained improve the growth of bean plants (Phaseolus vulgaris) and improvement significant in the soil quality (^{Adil and Jaikishun, 2010}).

Another residue of agro-industry is pineapple bagasse, which can represent up to 20% of total production. Part of this residue is used as cattle feed and the rest is discarded, becoming an environmental problem (^{Antonio et al., 2011}). Little is known about the processing capacity for the earthworms of pineapple residues. ^{Mainoo et al. (2009)}, consider that vermicomposting is promising as a management technology for this type of waste, it has been shown to vermicomposting with Eisenia eugeniae accelerate decomposition and loss of mass waste, an increase of nitrogen, phosphorus and potassium. However, the acidity initial of waste is a problem for worms, so a pre-composting improves pH conditions.

Biopesticide: a product of vermicomposting for sustainable organic production

A little known property of the vermicompost process is the presence of bioplagicidal agents in the vermicompost. Its potential has recently been investigated against plant pathogens, insects, mites and plant parasitic nematodes. When vermicompost is added to the soil, microbial diversity increases, with a broader range of microorganisms, these can act as controlling agents against different pests in plants, it has been suggested that the possible mechanisms of disease suppression is based on competition between microorganisms for nutrients and energy (^{Clive et al., 2006}; ^{Domínguez et al., 2010}; ^{Edwards et al., 2010a}; ^{Jack, 2012}).

The antifungal capacity of vermicompost leachate from cattle manure against the fungus Colletotrichum gloeosporioides has an inhibitory effect of 100% when applied without being sterilized. It has been found that the microorganisms present in the leachate compete against pathogenic fungus nutrient or are antagonistic towards the (^{Contreras-Blanca et al., 2014}). The application of vermicompost of cattle manure in tomato plants reduces the amount of parasitic nematode (Meloidogyne incognita) in root and soil (^{Castro et al., 2011}). The use of brushwood mature vermicompost coffee with cattle and goat manure on Tiquisque-system Pythium myriotylum, has a suppressive effect on the development of the disease caused by the fungus Pythium myriotylum in plants Tisquique (Xanthosoma sagittifolium) (^{Artavia et al., 2010}).

The suppression of aphids, mealybugs, mites and other pests is another use of vermicompost, studies in tomato and cucumber plants have shown that aqueous extracts of vermicompost suppress both pest establishment in the plant, and reproductive rates of green peach aphid, mealybugs and mites (spotted spider). The same effect has on beetles (Acalymna vittatum) cucumber and hornworms of snuff (Manduca sexta), which extracts significantly suppress the establishment of plant pests. Suppression is probably due to the fixing of phenolic compounds by plants and its rejection by pests (^{Edwards et al., 2010a}; ^{Edwards et al., 2010b}). The use of the vermicompost gives certain advantages to the soil and the plants, providing the necessary nutrients for the growth and production of the crop, and at the same time exerting a biological control against pests that affects the production.

Enzymatic activity: a measure of the biochemical activity of the vermicompost process

The enzymes are involved in most processes that take place in the soil, are responsible for the formation of organic molecules and particularly in the transformation of complex organic compounds to substances assimilable by plants. The enzymes in the soil may be extracellular, intracellular or be adsorbed to organic matter and colloidal system, suggesting that the soil can act as a temporary reservoir (^{Henríquez et al., 2014}).

To measure the metabolic activity of the microorganisms, a variety of enzymatic assays can be used, through tests to determine specific enzymes (^{Atlas and Bartha, 2001}). The knowledge of the evolution of enzymatic activities during vermicomposting is important, especially the enzymatic activities of the hydrolases type, which are activities involved in the cycles of the biophilic elements such as carbohydrases, chitinase, β-glucosidase and β-galactosidase of the C, the P of cycle phosphatase, urease and proteases arylsulfatase cycle N and S of the cycle, which are regarded as specific parameters (^{García et al., 2003}). In the microbial processes that are generated in the soil or other substrate, the quantification of the dehydrogenase and catalase are some of the most used methods and are considered as general tests (^{Acosta and Paolini, 2005}).

The understanding of enzyme activity is important to handle the biochemical processes that occur during the vermicomposting (^{Quintero-Lizaola et al., 2005}). Most of the enzymatic activities studied, after an increase during the early stages of the process, tend to decrease as the biodegradation of organic waste progresses through the action of earthworms and microorganisms. Therefore, the vermicompost obtained has a lower activity than the fresh organic residues. However, it should be noted that the vermicompost, having stabilized its organic matter, will achieve greater efficiency in the construction of a “stable”enzymatic pool; that is, “enzyme-humus” complexes capable of resisting the denaturation of the enzymes (^{Vargas-Machuca, 2010}).

Among the enzymatic biotransformation processes, it has been found that the presence of earthworm stimulates the activity of the enzymes amylase, cellulase, lipase, invertase, protease, amidase, urease, nitrogenases, acid and alkaline phosphatase, phosphomonoesterases, arylsufatase and dehydrogenase. The activity of these enzymes is greater in the residues with worms than in those where they are absent, and decreases in direct relation with the maturity of the compost. The increase of this activity is related to the interaction of three important factors: presence and absence of worms, substrate size and sampling time.

The knowledge about the enzymatic dynamics of the vermicomposting process can be used to manage in a more sustainable way and even accelerate the biotransformation process of the residues (^{Quintero, 2014}). During vermicomposting the increase in dehydrogenase activity may be related to the amount of humus applied to the soil. ^{Contreras et al. (2013)}, indicate that when the vermicompost is used at the rate of 30% as fertilizer for the soil, the enzyme dehydrogenase increases its activity during the first 60 days, afterwards this activity decreases. This decrease may be due to the fact that the microorganisms have depleted the readily degradable carbon source, which is why the production of this intracellular action enzyme decreases.

Studies with the earthworm Amynthas hupeiensis show that their presence increases the fibrinolytic enzyme activity during the vermicomposting process when the corn stover is used. The activities of cellulase, avicelasa, β-glucosidase, endo-β-1,4-xylanase, acetyl esterase, and manganese peroxidase are significantly increased. Due to this activity, the content of cellulose, hemicellulose and lignin in the vermicompost process is reduced in the presence of earthworms for the first 30 days or so. Although microorganisms are the main agents responsible for the decomposition of cellulose, hemicellulose and lignin, earthworms play a key role in the decomposition of plant residues and is one of the reasons for the promotion of enzymatic activity during vermicomposting to stimulate microbial growth (^{Yuxiang et al., 2015}).

The increase in enzymatic activity is also related to the presence of fresh organic material present at the beginning of the transformation process. Comparative studies between traditional composting and vermicomposting have shown that worms significantly increase enzyme activity during the initial stages of the process and then gradually decrease. The activity of the enzyme urease, phosphatase and dehydrogenase during vermicomposting with Eudrilus eugeniae, using paper sludge as a conditioner and cow manure has increased at the beginning of the process.

This increase is related to the availability of substrate for extracellular enzyme activity, and decreased this activity may be due to the reduction of the microbial population in the final stages of composting processes and vermicomposting (^{Ponmani and Udayasoorian, 2014}). ^{Rama et al. (2014)}, consider that enzymatic activities are high in composting and vermicomposting. However, vermicomposting exhibits the highest activity for the enzymes during the first 30 days, decreasing at the end of the process. This may be due to the availability of readily degradable substances for microbial activities in the initial stages.

Studies with Eisenia fetida have confirmed that the enzyme activity β-glucosidase, cellulase and phosphatase increase between 1.2 and 2.7 times when low rates of slurry (liquid manure in the pig sector, composed of manure, wash water and leftover food) applies, but not the protease activity that increases in high rates of pig slurry applied. In both purines rates applied, the presence of earthworms in younger layers stimulates microbial growth, which decreases once the worms leave these layers (^{Aira et al., 2007}).

Microorganisms: study of their abundance and diversity in the vermicompost process

Despite the knowledge about the close relationship between earthworms and microorganisms during biotransformation of organic waste, it is necessary to know which microorganisms participate in the process and what role they play. Because of the complexity and heterogeneity of the vermicomposting process, knowledge about biodiversity, microbial dynamics and functionality is very limited, and the results often are contradictory (^{Vargas-Machuca, 2010}; ^{Moreno et al., 2014}).

The study of microbial ecology focuses on two important aspects: biodiversity of microorganisms including isolation, identification, quantification and activity in their habitat (^{Madigan et al., 2009}). There are specific methods in the literature that allow to know the structure and functionality of the microbial populations involved in vermicomposting, ranging from traditional culture media to the use of techniques based on molecular markers.

The classical methods of detecting microorganisms are to provide the viable microbial cells on a solid or liquid medium. The classical method is effective to detect microorganisms when they are in a significant proportion (^{Atlas and Bartha, 2001}). The determination of specific groups using selective media can provide reliable data. However, the counting of all cells in a sample using a single medium and a single set of growth conditions may result in a low estimate, known as anomaly count in solid medium (^{Madigan et al., 2009}).

Due to the simplicity of culture in solid medium, it has been commonly used to quantify changes in the number of microorganisms. The use of differential culture media allows the growth of microorganisms with specific physiological capabilities, this methodology has been used to study the diversity of heterotrophic bacteria involved in different types of composting. The traditional culture media have been used to characterize microbial populations during the vermicomposting of organic residues, grouping them into three taxonomic categories (bacteria, actinomycetes and fungi) (^{Durán and Henríquez, 2007}). The use of means of selective cultures usually are used to quantify bacteria, fungi and actinomycetes in the transformation of waste, such as horse manure, coffee pulp and straw vermicomposting oats (^{Raphael and Velmourougane, 2011}; ^{Polo et al., 2012}; ^{Quintero, 2014}).

Although traditional culture methods provide relevant information on the abundance of many microorganisms involved in vermicomposting of different types of residues, it probably underestimates the true density of the microbial population, since many microorganisms are unable to develop in the media of conventional crops. With techniques of enumeration of viable organisms, no more than 10% and up to less than 1% of these values are obtained (^{Alexander, 1980}; ^{Campbell, 2001}).

In recent years, the development of new techniques in microbiology has allowed the study of microorganisms present in various types of biological samples without the need for cultivation. One of these techniques are molecular markers. New techniques for analyzing microorganisms based on the analysis of DNA patterns, fatty acid patterns and carbonate substrates are useful because they provide information on the structure and diversity of microbial communities, allowing the comparison between initial organic residues and vermicomposted (^{Vargas-Machuca, 2010}). With the growth of genetic research and classification bacterial, molecular markers are an important tool to identify bacterial species. The analysis of the DNA contained in a sample, allows to study the biodiversity of all active, dormant or sporulated microorganisms, which places it as one of the most commonly used techniques (^{Weilong et al., 2012}; ^{Moreno et al., 2014}).

Many studies on microbial biodiversity do not need to isolate microorganisms to quantify or identify them. Instead, these molecular markers are used to measure biodiversity. Recent literature reports a significant number of molecular, alternative, sensitive and selective techniques for the detection, enumeration and identification of microorganisms in different environmental samples including vermicomposting (^{Palomino-Camargo and Gonzáles-Muños, 2014}). One of the techniques mostly used to investigate microbial communities associated with vermicomposting is PCR-DGGE. It has been used to differentiate bacteria based on Sequence differences in his 16S rDNA and be used to identify new microorganisms which are rare or noncultivable or to monitor variation in microbial structure on the abundance of kinds of bacteria and fungi, with which one can identify specific taxa bacteria as Gammaproteobacteria, Betaproteobacteria, Actinobacteria and Alphaproteobacteria during vermicomposting (^{Hong et al., 2011}; ^{Castillo et al., 2013}).

The use of PCR-DGGE techniques has revolutionized the knowledge about the microbial ecology of the degradation processes of organic compounds in the soil and other types of environmental samples. It allows monitoring specific microbial populations and their activities without resorting to cultivation in selective media. Using PCR-DGGE found that taxa of bacteria Actinobacteria, Betaproteobacteria, Gammaproteobacteria, Alphaproteobacteria, Firmicutes, Cianobacterias, Bacteroidetes, Actinomycetes and Sordariomycetes, Ascomycotina, Basidiomycota and Zygomycota fungi are the dominant organisms during the process of vermicomposting plant waste cabbage, lettuce, potato peels, residues of fresh fruits and vegetables (^{Huang et al., 2013}; ^{Huang et al., 2014}).