Emilio Benitez

VERMICOMPOSTING OF SLUDGES FROM PAPER MILL AND DAIRY INDUSTRIES WITH EISENIA ANDREI: A PILOT-SCALE STUDY

Abstract We studied vermicomposting with Eisenia andrei of sludges from a paper mill mixed with cattle manure in a six-month pilot-scale experiment. Initially, a small-scale laboratory experiment was carried out to determine the growth and reproduction rates of earthworms in the different substrates tested. In the pilot-scale experiment, the number of earthworms increased between 22- and 36-fold and total biomass increased between 2·2- and 3·9-fold. The vermicomposts were rich in nitrogen and phosphorus and had good structure, low levels of heavy metals, low conductivity, high humic acid contents and good stability and maturity. These sludges could be potentially useful raw substrates in larger commercial vermicomposting systems, and would reduce the costs related with the exclusive use of different types of farm wastes as feed for earthworms.

Enzyme activities as indicators of the stabilization of sewage sludges composting with Eisenia foetida

Evolution of earthworm (Eisenia foetida, Savigny) biomass and changes in enzyme activities during 18 weeks of sewage sludge vermicomposting were studied. With time, hydrolase (β-glucosidase, urease, BAA-hydrolysing protease and phosphatase) and dehydrogenase (DH-ase) activities decreased as available organic compounds (water-soluble carbon) decreased. A high correlation among all enzyme activities and between each activity and the water-soluble carbon (WSC) led to the conclusion that both hydrolytic and dehydrogenase activities could be feasible indicators of the state and evolution of the organic matter. The ‘potential metabolic index’ (DH-ase/WSC) enabled distinguishing between hydrolytic and maturation phases in a sewage sludge vermicomposting process.

Assessing the impact of composting and vermicomposting on bacterial community size and structure, and microbial functional diversity of an olive-mill waste

The aim of this study was to couple biochemical and molecular methodologies for evaluating the impact of two recycling technologies (composting and vermicomposting) on a toxic organic waste. To do this, six enzyme activities controlling the key metabolic pathways of the breakdown of organic matter, real-time PCR assays targeting 16S rRNA genes, and denaturing gradient gel electrophoresis (DGGE) profiling-sequence analysis of PCR-amplified 16S rRNA fragments have been used to determine the functional diversity, bacterial number, and bacterial community structure, respectively, in a mixture of olive waste and sheep manure, and in the derived compost and vermicompost. Both the recycling technologies were effective in activating the microbial parameters of the toxic waste, the vermicomposting being the best process to produce greater bacterial diversity, greater bacterial numbers and greater functional diversity. Although several identical populations were detected in the processed and non-processed materials, each technology modified the original microbial communities of the waste in a diverse way, indicating the different roles of each one in the bacterial selection.

Vermicomposting of Winery Wastes: A Laboratory Study

In Mediterranean countries, millions of tons of wastes from viticulture and winery industries are produced every year. This study describes the ability of the earthworm Eisenia andrei to compost different winery wastes (spent grape marc, vinasse biosolids, lees cakes, and vine shoots) into valuable agricultural products. The evolution of earthworm biomass and enzyme activities was tracked for 16 weeks of vermicomposting, on a laboratory scale. Increases in earthworm biomass for all winery wastes proved lower than in manure. Changes in hydrolytic enzymes and overall microbial activities during the vermicomposting process indicated the biodegradation of the winery wastes. Vermicomposting improved the agronomic value of the winery wastes by reducing the C:N ratio, conductivity and phytotoxicity, while increasing the humic materials, nutrient contents, and pH in all cases. Thus, winery wastes show potential as raw substrates in vermicomposting, although further research is needed to evaluate the feasibility of such wastes in large-scale vermicomposting systems.

Isolation by isoelectric focusing of humic-urease complexes from earthworm (Eisenia fetida)-processed sewage sludges

Abstract Vermicomposting is an eco-biotechnological process that transforms energy-rich and complex organic substances into a stabilized humus-like product. In a laboratory experiment, Eisenia fetida (Sav.) earthworms were employed to process putrescible sewage sludges into a high-value biofertilizer, very rich in urease activity and humic-urease complexes (stabilized extracellular enzymes). Extracellular humic-urease complexes were extracted by a single 24-h extraction at 37  °C using neutral pyrophosphate (0.1 M); then, the extracts were dialysed and characterized by means of an analytical isoelectric focusing technique. This technique gave a multiplicity of humic bands enzymatically active, with isoelectric points ranging from 4.8 to 5.6. The results demonstrated that, after an 18-week incubation period, sewage sludge had undergone a biochemical evolution, which caused a doubling of absolute urease activity and a six-fold increase in specific activity (activity with reference to the humic C fraction). The biochemical evolution of the vermicompost was evaluated also from the sharp decrease in pyrophosphate-extractable C and water-soluble C. Stabilization of organic C during vermicomposting and the activity of humic-urease complexes expressed at low pH values are of extreme importance when organic wastes are used in acid soils for biochemical restoration purposes.

Biosolids and biosolids-ash as sources of heavy metals in a plant-soil system

Generally, the potential for biosolids (digested or composted)to contribute heavy metals to the soil-plant system has beencompared with commercial fertilizers and other organic wastesbut not with biosolids-ash. An column study was conducted in agreenhouse to determine the availability, extractability andleachability of metals in a degraded, non-calcareous soilamended with different biosolids (200 Mg ha-1). Thebiosolids investigated were dewatered, anaerobically digestedbiosolids, composted biosolids and biosolids-ash. The columns(26 cm) were planted with wheat (Triticum aestivum L. cvMexa). The addition of digested biosolids decreased the drymatter yield of wheat. Treatments including organic biosolidsincreased Cu and Zn concentrations in wheat roots, straw andgrain, whereas the addition of biosolids-ash did not affect theconcentrations of these metals in wheat. Concentrations of Ni,Co, Pb, Cr and Cd in wheat were below reliable detection limits(0.06, 0.05, 0.1, 0.06 and 0.02 mg kg-1, respectively).After harvesting, total and AB-DTPA extractable Cu, Zn and Pbincreased in the upper layer of the soil amended with thedifferent biosolids studied, whereas levels of AB-DTPAextractable Ni and Co were affected only when the soil wasamended with digested or composted biosolids. Total chromiumincreased only in treatments including organic biosolids. TheAB-DTPA extractable Cu, Zn and Pb in the lower layer of thesoil in treatments including biosolids evidenced downwardmovement of these metals. However, absence of these metals incolumn leachates indicates that this movement was gradual.

Dynamic changes in bacterial community structure and in naphthalene dioxygenase expression in vermicompost-amended PAH-contaminated soils

The aim of the present study was to explore the potential for using vermicompost from olive-mill waste as an organic amendment for enhanced bioremediation of polycyclic aromatic hydrocarbons (PAHs)-contaminated soils. The focus was to analyse the genetic potential and the naphthalene dioxygenase (NDO) expression of the bacterial communities involved in the degradation of naphthalene, as chemical model for the degradation of PAH. The structure of the metabolically active bacterial population was evidenced in the RNA-based denaturing gradient gel electrophoresis (DGGE) profiles. The relative expression of NDO was determined with real-time PCR in both the soil and the vermicompost cDNA. Naphthalene changed the structure of the metabolically active bacterial community in the vermicompost when this was artificially contaminated. When used as amendment, naphthalene-free vermicompost modified the bacterial population in the PAH-contaminated soil, evidenced in the DGGE gels after 1 month of incubation. In the amended soil, the vermicompost enhanced the NDO enzyme expression with a concomitant biodegradation of naphthalene. The effect of the vermicompost was to induce the expression of biodegradation indicator genes in the autochthonous bacterial community and/or incorporate new bacterial species capable of degrading PAH. The results indicated that vermicompost from olive-mill wastes could be considered a suitable technology to be used in PAH bioremediation.

Enzyme activities and chemical changes in wet olive cake after treatment with Pleurotus ostreatus or Eisenia fetida

A laboratory experiment was conducted to evaluate the enzyme activities and chemical changes recorded in a recalcitrant phenolic-rich waste after treatment with Pleurotus ostreatus or Eisenia fetida. The waste used was wet olive cake (alperujo in Spanish), a waste produced in huge amounts by the olive oil industry. Both P. ostreatus and E. fetida were very effective in removing phenolic compounds, the initial concentration in the wet olive cake being reduced in both cases by around 90%. Laccase and manganese peroxidase activities were measured in the growth medium of P. ostreatus, and catechol 2,3 dioxygenase activity was only detected in the waste treated with Eisenia; these could be the main factors responsible for the oxidation of phenolic compounds. Increases of dehydrogenase and β-glucosidase activities were detected in the degraded wet olive cake by fungi or earthworms. In comparison with the natural wet olive cake, the degraded products had lower total organic carbon and humic acid contents but were rich in nitrogen and other nutrients, having lower C:N ratios. In addition, the toxicity of the wet olive cake against the seeds of Lepidium sativum significantly decreased after degradation. The low toxicity as well as moderate stability and maturity recorded in the wet olive cake treated with P. ostreatus or E. fetida imply that these products could be used as soil amendments.

Feasibility of vermicomposting dairy biosolids using a modified system to avoid earthworm mortality

A laboratory study was conducted to examine the feasibility of vermicomposting dairy biosolids (dairy sludge), either alone or with either of the bulking agents-cereal straw or wood shavings, using the epigeic earthworm-Eisinea andrei. Earthworms added directly to these three substrates died within 48 hours. A system was developed to overcome the toxic effect of unprocessed dairy biosolids. The substrates were placed over a layer of vermicomposted sheep manure into which the earthworms were inoculated. Within two weeks, all earthworms were within the upper layer of substrate. Compared to sheep manure which is a favourable substrate for vermicomposting, the three substrates containing dairy biosolids were more effective in supporting earthworm growth and reproduction. The final products obtained after 63 days of vermicomposting had 39-53% less organic carbon than the initial substrates. Organic fractionation indicated that vermicomposting increased the stability of the materials to biological decomposition. The vermicomposts obtained from the three substrates with dairy biosolids had low heavy metal contents and electrical conductivities, and did not inhibit plant growth when compared with a commercial vermicompost in a bioassay.

Restoring biochemical activity and bacterial diversity in a trichloroethylene-contaminated soil: the reclamation effect of vermicomposted olive wastes.

Background, aim, and scopeIn this work, the potential for using olive-mill solid waste as an organic amendment for biochemical and biological restoration of a trichloroethylene-contaminated soil, which has previously been stabilized through vermicomposting processes, has been explored.Materials and methodsTrichloroethylene-contaminated water was pumped into soil columns with a layer of vermicompost at 10-cm depth (biobarrier system). The impacts of the trichloroethylene on the microbial community were evaluated by determining: (1) the overall microbial activity (estimated as dehydrogenase activity) and enzyme activities related to the main nutrient cycles (β-glucosidase, o-diphenoloxidase, phosphatase, urease, and arylsulphatase activities). In addition, isoelectric focusing of the soil extracellular humic-β-glucosidase complexes was performed to study the enzymatically active humic matter related to the soil carbon cycle. (2) The soil bacterial diversity and the molecular mechanisms for the bacterial resistance to organic solvents were also determined. For this, polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) was used to detect changes in bacterial community structure and PCR-single-strand conformational polymorphism (SSCP) was developed and optimised for detection and discrimination of the resistance-nodulation-division (RND) genes amplified from the contaminated soils.ResultsVermicompost reduced, with respect to the unamended soil, about 30% of the trichloroethylene leaching during the first month of the experiment. Trichloroethylene had a marked negative effect on soil dehydrogenase, β-glucosidase, urease, phosphatase, and arylsulphatase activities. Nevertheless, the vermicompost tended to avoid this toxic effect. Vermicompost also displays stable humic-β-glucosidase complexes that increased the extracellular activity related to C-cycle in the contaminated soils. The isoelectric focusing technique showed a more biochemically active humic matter in the soil sampled under the vermicompost. The behaviour of the three main phyla of bacteria isolated from the DGGE bands was quite different. Bands corresponding to Actinobacteria disappeared, whereas those affiliated with Proteobacteria remained after the trichloroethylene contamination. The disappeared Actinobacteria became visible in the soil amended with the vermicompost. Bands corresponding to Bacteriodetes appeared only in columns of contaminated soils. In this study, six types of RND proteins were detected by PCR-SSCP in the natural soil, three in the trichloroethylene-contaminated soil and 7/5 in trichloroethylene-contaminated soil above/below the vermicompost in the biobarrier columns. Trichloroethylene tended to reduce or eliminate all the clones detected in the uncontaminated soil, whereas new efflux pumps appeared in the biobarrier columns.DiscussionAlthough enzymes incorporated into the humic substances of vermicomposted olive wastes are quite stable, trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity in the amended soils. The decrease was less severe in the biobarrier system, but in any case, no relation was found between the levels of trichloroethylene in soil and extracellular β-glucosidase activity, or between the latter and the quantity of humic carbon in soils. The isoelectric focusing technique was carried out in the humic fraction to determine whether the loss of activity occurred in overall extracellular β-glucosidase or in that linked to stable humic substances (humic–enzyme complexes). The contaminated soils showed the lower enzyme activities, whereas contaminated and amended soils presented greater quantity of focalised (and therefore stable) humic carbon and spectra heterogeneity: very different bands with higher enzyme activities. No clear relationship between trichloroethylene concentration in soil and diversity of the bacterial population was noted. Similar patterns could be found when the community structures of bacteria and microbial activity were considered. Since the use of the dehydrogenase assay has been recognised as a useful indicator of the overall measure of the intensity of microbial metabolism, these results could be attributed to PCR-DGGE methodology, since the method reveals the presence of dominant populations regardless of their metabolic state. Trichloroethylene maintained or even increased the number of clones with the DNA encoding for RND proteins, except for the contaminated soil located above the vermicompost. However, the main effect of trichloroethylene was to modify the structure of the community in contaminated soils, considering the type of efflux pumps encoded by the DNA extracted from soil bacteria.ConclusionsTrichloroethylene inhibited specific functions in soil and had a clear influence on the structure of the autochthonous bacterial community. The organic matter released by the vermicomposted olive waste tended to avoid the toxic effect of the contaminant. Trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity, even when vermicompost was present. In this case, the effect of the vermicompost was to provide and/or to stimulate the humic-β-glucosidase complexes located in the soil humic fraction >104, increasing the resistance of the enzyme to the inhibition. The bacterial community from the soil presented significantly different mechanisms to resistance to solvents (RND proteins) under trichloroethylene conditions. The effect of the vermicompost was to induce these mechanisms in the autochthonous bacterial community and/or incorporated new bacterial species, able to grow in a trichloroethylene-contaminated ambient. Coupled biochemical and molecular methodologies are therefore helpful approaches in assessing the effect of an organic amendment on the biochemical and biological restoration of a trichloroethylene-contaminated soil.Recommendations and perspectivesSince the main biochemical and biological effects of the organic amendment on the contaminated soil seem to be the incorporation of biochemically active humic matter, as well as new bacterial species able to grow in a trichloroethylene-contaminated ambient, isoelectric focusing and PCR-SSCP methodologies should be considered as parts of an integrated approach to determine the success of a restoration scheme.