3.1. Constraints set by resource

Any biomass crop that provides an extended harvest season has an advantage because most biorefineries want to operate as many hours per year as possible. Continuous operation yields the maximum product per unit of capital investment. Herbaceous crops cannot be harvested during the growing season. Thus, storage is always a component in a single­feedstock logistics system design.

Two examples are discussed from opposite ends of the length-of-harvest-season spectrum. In the Southeastern United States, wood is harvested year-round. This is referred to as "stored on the stump." Weather conditions in the Southeast are such that few harvest days are lost in the winter due to ice and snow. On the other end of the spectrum, consider the harvest of corn stover in the Upper Midwest of the United States. The grain harvest is completed in the fall, and then the stover is collected. In some years, there are less than 15 days between the completion of the grain harvest and the time when the fields are covered with snow, and no stover can be collected. All feedstock required for year-round operation of a bioenergy plant, if it uses only corn stover, must be harvested in a three — to five-week period. This is a significant challenge for a logistics system.

3.2. Constraints set by purchaser of raw biomass

Biorefinery operators are interested in the cost of the feedstock as it enters their plant. The plant can burn the biomass directly to produce heat and power, or it can use the biomass as a feedstock for some more complex conversion processes to produce a high-value product. The term "feedstock" is used to refer to any raw biomass before its chemical structure is modified by a conversion process, which can be direct combustion, thermo-chemical, or biological.

Feedstock cost ($/dry ton) is defined as the cost of the stream of size-reduced material entering the reactor at the Bioenergy plant for 24/7 operation. The reactor is defined as the unit operation where an initial chemical change in the feedstock occurs. The reason for choosing this reference point for the biomass logistics system is two-fold.

1. The plants that can operate continuously, 24/7, have an advantage. Maximum production (tons/y) per unit of capital investment gives a competitive advantage in the market place — cost to produce the product is lower.

2. Some logistics systems do size reduction with the harvesting machine, while some size reduce at a transfer point between in-field hauling machines and highway hauling machines (perhaps as a pre-requisite to a densification step), and some size reduce at the entrance to the processing plant. In order to compare the several systems, it is necessary to have a consistent end point for the system analysis.

The readers should be aware that many studies in the literature select a different analysis endpoint than used here. A typical endpoint is the cost of feedstock when a truckload of raw biomass enters the plant gate. This reference point is favorable as many agricultural and forest industries pay the producer when the raw material is delivered to the plant.

During the process of ozonation, complex chemical reactions occurred between ozone and organics. Pre-ozonation can change the biodegradability of organics in water, so generally BDOC is used as an index to analyze the water after ozonation. Fig. 7. shows the variation tendency of DOC, BDOC and BDOC/DOC in raw water after ozonation. The result indicates that after ozonation, DOC in raw water is seldom removed while BDOC increases obviously, among which the ratio of BDOC/DOC increases from

11.6 % to 26.4 %. After ozonation, the biodegradability of organics in water is highly improved, which enhance the biodegradation of BAC filter bed in the following procedure.

(i) hydrolysis (slow decomposition of polymeric substances to easy biodegradable substances, (ii) substrates assimilation by microorganisms correspondingly with Monod

equation and (iii) growth and decay of microorganisms, what can be written in form:

(5)

where: f(S) — substrate concentration related function, X — biomass concentration, gdm/m3,

Kd — biomass decay constant, 1/d.

Organic substrates in the wastewater are in the form of: suspended solids, colloids and soluble matter. In overall form it can be described as C18H19O9N. Due to their different forms (easy degradable, slowly degradable and not biodegradable fractions) they can be oxidized, assimilated or not biologically decomposed.

identified correspondingly to commonly used methodology [29-31]. Typical wastewater consists 10-27% of soluble easy biodegradable substances (Ss), 1-10% of not biodegradable soluble substances (Si), 37-60% of slowly biodegradable suspended solids (Xs) and 5-15% of very slowly biodegradable suspended solids (Xi) [31-34].

Easy biodegradable organic substances are an energy sources for denitrifying and phosphorus accumulating bacteria (PAB) and its concentrations have direct impact on the nitrogen and phosphorus removal.

Nitrogen in wastewater appear usually in form of soluble non-organic forms (mainly ammonium nitrogen, seldom nitrites and nitrates), organic soluble (degradable and not degradable) and as the suspended solids (slowly degradable, not degradable and as a biomass). Nitrogen compounds transformations are carried by autotrophic and heterotrophic bacteria and elementary processes need to preserve adequate technological conditions for these microorganisms.

The polymeric substances hydrolysis is catalysed by extracellular proteolytic enzymes to transform into simple monomers, which can be assimilated by microorganisms.

This process and ammonia nitrogen assimilation is related to fraction of nitrogen in biomass (5-12%). The nitrogen assimilation rate depends on the C/N ratio.

Another process of nitrogen transformation is nitrification — oxidation of ammonium nitrogen to nitrites and nitrates by chemolithotrophs: Nitrosomonas, Nitrosococcus and Nitrosospira in first phase (hydroxylamine is the intermediate product) and Nitrobacter, Nitrospira, Nitrococcus in the second phase, what can be described in form [35]:

NH+ + 1,5O2 ^ NO — + H2O + 2H+ — 278kJ / mol

NO — + 0,5O2 ^NO- — 73kJ / mol

Nitrifying bacteria, as autotrophs, take the energy from carbon dioxide and carbonates. The utilisation rate for Nitrosomonas is equal to 0.10 gdmo/gN-NH4, and for Nitrobacter — 0.06 gdmo/gN — no2 [1]. The important process parameters are: oxygen supply (4.6 g O2/1gN-NH4), temperature (5 — 300C), sludge/biomass age (more than 6 days is recommended), biomass organic compounds loading (over 0.2 g BZT5/gdm is recommended) and BOD/N ratio: when the value is more than five — organic compounds removal dominates, when is lower than three — the nitrification is a prevailing process. The decrease in alcanity is the result of nitrification (theoretically: 7.14 g CaCO3/1 g N-NH4) and it causes decrease in pH from 7.5 — 8.5 to 6.5.

Nitrates are transformed in the dissimilation reduction process (Pseudomonas, Achromobacter, Bacillus and others) to the nitrogen oxides and gaseous nitrogen correspondingly to the path:

2NO3 ~4e > 2NO3 ~2e+2 H > 2 NO ~2 e > N2O ~2 e > N2

In the terms of dissolved oxygen deficiency (anaerobic or anoxic conditions) those organisms use nitrates as H+ protons acceptors.

Denitrifying bacteria, as the heterotrophs, need organic carbon for their existence. The source of organic carbon can be: organic compounds in wastewater (internal source), easy assimilated external source of organic carbon, e. g. methanol/ethanol, or intracellular compounds as an energetic source. This ratio of organic carbon should be in range of 5 — 10 g COD/g N-NO3 [35].

In the figure 3 the elementary processes of nitrification and denitrification in nitrogen removal are shown [11]. It is easy to recognise that denitrification is partly the reverse process to the nitrification. Due to the fact that some nitrifying bacteria can live without oxygen and some denitrifying bacteria can survive in oxygen conditions there is the possibility to carry out the simultaneous nitrification and denitrification (SND) in one reactor. As the SND reactor both continuous and sequencing batch reactors can be used. In
the SBR-SND reactor high removal efficiency for organic and nitrogen compounds can be achieved — 79% and 96% respectively [36]. The biomass growth rate can be in range of 0.3 — 0.75 gSDM/gsub rem [37]. Similarly high nitrogen removal efficiency in SND process in continuous reactor (about 90%) can be achieved at certain pH and N-NH4 concentration [38]. The nitrite concentration rise indicates that the nitrification process has stopped after the first phase. The process can run at low C/N ratio.

The short version of SND process needs preservation of certain technological conditions. Important conditions are aerobic and anoxic conditions, what in one reactor system can be achieved by intermittent aeration and non aeration. It causes the characteristic variability of parameters such: pH, redox potential or nitrogen compounds concentration [39]. Examples of changes of some variables in bioreactor with intermittent aeration are presented in figure 4 [40].

The blockage or limitation of second phase of nitrification can be achieved by limitation of oxygen availability up to approximately 0.7 mg O2/dm3 [41] or by free ammonia inhibition, which concentration is impacted by the temperature and pH of wastewater. Anthonisen et al. [42] identified the limiting values of partial and full inhibition of second phase of nitrification: 0.1 g N-NH4/m3 and 1.0 g N-NH4/m3 respectively. They proposed the description of free ammonia concentration in the reactor in form [42]:

S _ 17 sn-nh4 *10P

NH3 14 exp (6344/T) + 10pH

where: Sn-nh4 — ammonium nitrogen concentration, mg/dm3,

T — temperature, K.

The free ammonia concentration has an impact on the ammonium nitrogen removal velocity, what is described by substrate inhibition model presented by Haldane [43]:

where: vnh — ammonia nitrogen and ammonium removal velocity, mgN/mgsmh,

rNHmax — ammonia nitrogen and ammonium removal maximum velocity, mgN/mgsm, KsNH3 — saturation constant, mgN-NH3/dm3,

KiNH3 — inhibition constant, mgN-NH3/dm3.

For the mathematical description of organic and nitrogen compounds removal many existing models are used with certain modifications, e. g.: substrate inhibition in Brigs — Haldane model [44] or ASM1 model [45], intermittent aeration or oxygen limitation inhibition in ASM1 [46-48], or two stages process of nitrification and denitrification in ASM3 model [49].

The BFT has been successfully applied for grow-out, but little is known about biofloc benefits on breeding. For example, in the shrimp industry with the global spread of viruses, the use of closed-life cycle broodstock appeared as a priority to guarantee biosecurity, avoiding vertical transmissions. Moreover, such industry places a considerable interest on penaeid breeding program, often performed in closed facilities, controlling the production plan through successive generations. These programs were frequently associated with large animals, disease resistance as well as the enhancement of reproductive performance. However, nutritional problems remain unresolved [61] and alternatives should be evaluated.

As an alternative for continuous in situ nutrition during the whole life-cycle, breeders raised in BFT limited or zero water exchange system are nutritional benefited by the natural productivity (biofloc) available 24 hours per day. Biofloc in a form of rich-lipid-protein source could be utilized for first stages of broodstock’s gonads formation and ovary development. Furthermore, production of broodstock in BFT could be located in small areas close to hatchery facilities, preventing spread of diseases caused by shrimp transportation.

In conventional systems breeders used to be produced in large ponds at low density. However, risks associate with accumulation of organic matter, cyanobacteria blooms and fluctuations of some water quality parameters (such as temperature, DO, pH and N — compounds) remains high and could affect the shrimp health in outdoor facilities. Once the system is stable (sufficient particulate microbiota biomass measured in Imhoff cones), BFT provides stabilized parameters of water quality when performed in indoor facilities such as greenhouses, guaranteeing shrimp health.

According to studies performed with the blue shrimp L. stylirostris [18] and the pink shrimp F. duorarum broodstock [62], BFT could enhance spawning performance as compared to the conventional pond and tank-reared system, respectively (i. e. high number of eggs per spawn and high spawning activity; Fig 4). Such superior performance might be caused by better control of water quality parameters and continuous availability of food (biofloc) in a form of fatty acids protected against oxidation, vitamins, phospholipids and highly diverse "native protein", rather than conventional systems which "young" breeders are often limited to pelletized feed. These nutrients are required to early gonad formation in young breeders and subsequent ovary development. The continuous availability of nutrients could promote high nutrient storage in hepatopancreas, transferred to hemolymph and directed to ovary, resulting in a better sexual tissue formation and reproduction activity [18].

Regarding to shrimp broodstock management, one of the most important management procedures is related to control of solids and stocking density. High levels of solids negatively affect shrimp health, particularly with shrimp weight higher than 15g [47]. Settling solids or "biofloc volume" should be managed below than 15mL/L (measured in Imhoff cones) [38, 47]. Excess of particulate organic matter covered breeder’s gills and could limit oxygen exchange, might resulting in mortalities.

Stocking density has to be carefully managed, mainly in sub-adult/adult phase (i. e. >15g). High density or high biomass will lead to an increase in organic matter, TSS levels and N — compounds in tanks or in ponds [63]. Moreover, physical body damages are prevented at low density, improving breeder’s health. For review, a suggested stocking density is well described in [64].

For fish, no literature is available regarding BFT and application in breeders. The same trend observed in penaeid shrimp might be observed in fish. The continuous consumption of diverse microbiota (biofloc) should improve nutrients transfer, gonad formation and reproduction performance in fish. Lipid is a well-known nutrient that plays a key role in reproduction of aquatic species. In tilapia, breeders fed with crude palm oil based-feed (n-6 fatty acid rich source) presented high concentration of acid arachidonic or "ARA" (C20:4 n — 6) in gonads, eggs and larvae of tilapia as compared to fish oil or linseed oil-based feeds [65]. As a result, better reproductive performance was observed in terms of higher total number of eggs per fish, larger gonad sizes, shorter latency period, inter spawning interval and higher spawning frequency. ARA is an essential fatty acid crucial in reproduction, acting as hormone precursor [66]. In the study [33] was found high ARA content in biofloc harvested in tilapia culture freshwater tanks. Bioflocs in freshwater bioreactors contained high ARA content using glucose and glycerol as a carbon source [67]. These findings suggested that biofloc (according its nutritional profile, for review see section 5.0) might positively influence the reproductive performance in fish, supplying nutrients for gonad development, possibly also enhancing larval quality a posteriori. BFT in tilapia broodstock could be an effective method to increase tilapia fry production and further research is need in this field.

Several biological methods for lignocellulose recycling based on the enzymology of cellulose, hemicelluloses, and lignin degradation have been developed. To date, processes that use lignocellulolytic enzymes or microorganisms could lead to promising, environmentally friendly technologies. The relationship between cellulose and hemicellulose in the cell walls of higher plants is much more intimate than was previously thought. It is possible that molecules at the cellulose-hemicellulose boundaries, and those within the crystalline cellulose, require different enzymes for efficient hydrolysis.

Cellulase: Cellulases responsible for the hydrolysis of cellulose are composed of a complex mixture of enzymes with different specificities to hydrolyze the p-1,4-glycosidic linkages (Fig. 2A). Cellulases can be divided into three major enzyme activity classes (Goyal et al., 1991; Rabinovich et al., 2002). These are endoglucanases or endo-1-4-p-glucanase (EC 3.2.1.4), exoglucanase or cellobiohydrolase (EC 3.2.1.91), and p-glucosidase (EC 3.2.1.21). Endoglucanases, are thought to initiate attack randomly at multiple internal sites in the amorphous regions of the cellulose fiber, which opens-up sites for subsequent attack by the cellobiohydrolases. Cellobiohydrolases remove cellobiose from the ends of both sides of the glucan chain. Moreover, cellobiohydrolase can hydrolyze highly crystalline cellulose. p — glucosidase hydrolyzes cellobiose and in some cases short chain cellooligosaccharides to glucose.

Hemicellulase: Xylan is the main carbohydrate found in hemicelluloses. Its complete degradation requires the cooperative action of a variety of hydrolytic enzymes (Fig. 2B). Xylanases are frequently classified according to their action on distinct substrates: endo-1,4- p-xylanase (endoxylanase) (EC 3.2.1.8) generates xylooligosaccharides from the cleavage of xylan while 1,4-p-xylosidase (EC 3.2.1.37) produces xylose from xylobiose and short chain xylooligosaccharides. In addition, xylan degradation needs accessory enzymes, such as a-L- arabinofuranosidase (EC 3.2.1.55), a-4-O-methyl-D-glucuronidase (EC 3.2.1.39), acetyl xylan esterase (EC 3.1.1.72), ferulic acid esterase (EC 3.1.1.73), and p-coumaric acid esterase (EC 3.1.1.-), acting synergistically, to efficiently hydrolyze wood xylans. In the case of acetyl-4-O — methylglucuronoxylan, which is one of the most common hemicelluloses, four different enzymes are required for degradation: endo-1,4-p-xylanase, acetyl esterase (EC 3.1.1.6), a — glucuronidase, and p-xylosidase. The degradation of O-acetyl galactoglucomannan starts with the rupture of the polymer by endomannanase (EC 3.2.1.78). Acetylglucomannan esterase (EC 3.1.1.-) removes acetyl groups, and a-galactosidase (EC 3.2.1.22) eliminates galactose residues. Finally, p-mannosidase (EC 3.2.1.85) and p-glucosidase break down the endomannanase-generated oligomeric p-1,4 bonds (Thomson, 1993; Li et al., 2000; Perez et al., 2002).

The changes in soil properties were relatively small as should be expected with only one rotation. The rotational crops can contribute with organic residues, but, in general, the amounts of organic C added to the soil are usually not enough to cause significant changes in soil organic matter in the short term (Table 2).The letters in the tables represent statistical comparisons. Means followed by at least one equal letter do not differ statistically. Means followed by all the different letters differ significantly. The rotational crops also affected some soil attributes (Table 2). The organic matter content increased in the soil upper layer (0-0.2 m) with the cultivation of peanut cv. IAC-Tatu and velvet bean, and in the 0.2-0.4 m layer, with mung bean, sunflower IAC-Uruguai, and peanut cv. IAC-Tatu. The increase of soil exchangeable magnesium was also observed for peanut cv. IAC-Tatu and velvet bean, although the original Mg content was already high.

Sunflower accumulated more above-ground dry matter of total biomass and soybean more grain yield than the other crops (Table 3). Soybean, sunn hemp, velvet bean, and sunflower extracted the greatest amounts of N and P (Table 4). Sunflower also recycled more of K, Ca, Mg, and Zn than the other rotational crops, probably as a consequence of the higher biomass yield (Tables 3 and 4).

Soybean presented the highest N content, and sunflower the lowest. No differences were observed between peanuts and velvet bean and between sunn hemp and mung bean (Table 5). Among the macronutrients, N had the highest and P the lowest accumulation in the rotational crops. On the average Fe was recycled in the highest amounts in the above­ground parts of the rotational crops and Zn in the lowest (Table 4). The same results were observed by [29]) who evaluated pigeon pea (Cajanus cajan) and stylo plants (Stylosanthes guianensis var. vulgaris cv. Mineirao).

The high AMF infection rate, which helps the uptake of micronutrients (Table 3), may explain the high amounts of Zn returned to the soil when sunflower was grown before sugarcane. There is an increasing utilization of sunflower as a crop rotation with sugarcane in Brazil, due to its use for silage, seed oil production, and to its potential as a feedstock for biodiesel [30].

The amounts of N in the above-ground parts of sunn hemp (Table 4) were relatively low compared to those of [31], who reported the extraction of up to 230 kg ha-1 of N, and to those of [12], who found 196 kg ha-1 of N. However, the amounts of N returned to the soil are directly related to the nutrient concentration in the plant, which varies with the local potential for biological nitrogen fixation (BNF) and with the growth stage of the crop at the time of cutting, and with the biomass yield, which is affected by the weather, soil, and crop growing conditions.

Table 4. Nutrient content of above ground biomass of the rotational crops, excluding the grains.

Perin [32] found substantial amounts of N derived from BNF present in the above ground parts of sunn hemp (57.0%) grown isolated and 61.1% when intercropped with millet (50% seeded with each crop). The sunn hemp+ millet treatment grown before a maize crop resulted in higher grain yield than when sunn hemp alone was the preceding rotation. This effect was not observed when N-fertilizer (90 kg N ha-1) was added. Intercropping legume and cereals is a promising biological strategy to increase and keep N into the production system under tropical conditions [32]. A large proportion of the N present in soybeans usually comes from BNF. Guimaraes [33] found that 96% of the N present in above ground parts of soybeans were derived from BNF, values which are in agreement with those obtained by [32] for sunn hemp. However, in the present study, only about 27% of the N present in the soybean residues were from BNF (Table 5), probably because of poor specific population of fixing bacteria for soybeans in the experimental site, which have been grown with sugarcane for long time. No inoculation of soybean with Bradyrhizobium was done. The contribution of BNF for the peanut varieties was significantly different: it reached 70% of the N in the cv. IAC — Caiapo but only 37.7% in the cv. IAC-Tatu (Table 5). Usually the natural population of rhyzobia is high enough to guarantee root colonization for peanuts but probably the bacteria population in the soil of the experimental site was not efficient for peanuts cv. IAC-Tatu.

The rate of natural colonization with AMF was relatively high in all crops (Table 3). Peanut cv. IAC-Caiapo and sunflower cv. IAC-Uruguai, followed by velvet bean, had at least 64% of root infection with AMF. At the same time, sunflower produced the greatest amount of above-ground biomass, followed by C. juncea and velvet bean. Soybean had the highest grain yield (Table 3) and also presented a considerable percentage of root infection with AMF: 56% (Table 3). Besides the symbiotic association with rhizobia, roots of the legumes can be colonized by fungi of the family Endogonaceae that form vesicular-arbuscular (VA) endomycorrhizas, which help enhance the uptake of phosphorus and other nutrients [34].

Results of a nursery study on the effect of a short season pre-cropping with different mycotrophic herbaceous crops on growth of arbuscular mycorrhiza-dependent mandarin orange plants at an early stage after transplantation were presented by [20]. Mandarin orange seedling plants 180 days after transplantation showed variation in shoot growth in response to single season pre-cropping with seven different crops—maize, Paspalum millet, soybean, onion, tomato, mustard, and ginger, and two non-cropped fallow treatments— non-weeded and weeded fallows. Net growth benefit to the orange plants due to the different pre-crops and the non-weeded fallow treatment over the weeded fallow treatment plants showed a highly positive correlation with mycorrhizal root mass of the orange plants as it varied with the pre-crop treatments. Increase in citrus growth varied between 0 and 50% depending upon the mycorrhizal root mass of the pre-crops and weeds, AMF spore number, and infective inoculum density of the pre-cropped soils. These pre-crop variables individually and cumulatively contributed to the highly significant positive correlation between the AMF potential of the pre-cropped soils and growth of mandarin orange plants through their effect on mycorrhizal root mass development (i. e. extent of mycorrhization) of the mandarin orange plants. The choice of a pre-crop from the available options, grown even for a short season, can substantially alter the inherent AMF potential of soils to a significant influence on the performance of the mycorrhiza-dependent orange plant. The relationship between soil mycorrhizal potential left by a pre-crop and mycorrhizal benefit drawn by the succeeding AMF responsive plant can be of advantage for the exploitation of native AMF potential of soils for growth and nutrition management of crops in low nutrient, low input-output systems of production [20].

Sugarcane yield increased more than 30%, in average, due to the rotational crops as compared with the control treatment; those benefits lasted up to the third harvest (Table 6). In the first cutting, sunflower was the rotational crop that induced the greater yield increase, followed by peanut cv. IAC-Caiapo, and soybean cv. IAC 17. [35] observed that sunn hemp residues increased the sugarcane yield; in the first harvest after the green manure, the effect of the legume crop was better than that of chemical fertilization with nitrogen. Similar results were reported later by [36], with a yield rise of 15.4 tons ha-1 of sugarcane stalks, which represented about 24% increase in relation to the control. Positive effects on stalk yields were also found by [31] when sugarcane was grown after Crotalaria spectabilis, and by [36], who cultivated sugarcane after sunn hemp and velvet bean.

Sunflower was the best rotational treatment, causing an yield increased of around 46% in the first harvest after the rotational crops (Table 6). Meanwhile, in the average of three cuttings, peanut showed an yield increase of around 22% whereas sunflower presented a 10% yield increase; these results are in agreement with those of [31, 36].

The sugar content of sugarcane stalks is important because the raw material remuneration takes into account this parameter. Some crops that preceded sugarcane had a high effect on sugar yield (Table 7); this was observed mainly in the first harvest in areas where sunflower, peanuts and C. juncea were previously cultivated (Table 2). The 3-year average data showed a sugar yield increase, in the best treatment, of 3 t ha-1 in relation to the control. These results were already observed by [35, 31] who found an average increase of 2.98 ton-1 ha due to green manure crops grown before sugarcane.

Studying crop rotation with legume plants in comparison with a control with and without a mineral N addition, [35] observed that, after a crop rotation, the sugarcane yield was higher after C. juncea and velvet bean, with 3.0 and 3.2 stalk tons ha-1 increase, respectively. The treatments with an addition of N fertilizer but no-rotation with green manure resulted in only 1.1 tons ha-1 of a sugar yield increase, in the average of three years, suggesting that the beneficial influence of leguminous plants is not restricted to the N left by the leguminous plants after harvest.

Farmers must combine the resources of land, labor, management, and capital in order to derive the most profit. Since resources are usually scarce, maximizing returns on each one is important. Crop rotations provide income diversification. If profitability of one crop is reduced because of price variation or some unpredicted reason, income is not as likely to be adversely affected as if the whole farm was planted to this crop, provided that a profit potential exists for each crop in a rotation. This is especially important to the farmer with limited capital.

Some of the general purposes of rotations are to improve or maintain soil fertility, reduce the erosion, reduce the build-up of pests and diseases, best distribute the work load, reduce the risk of weather damage, reduce the reliance on agricultural chemicals, and increase the net profits. Crop rotations have fallen somewhat into disfavor because they require additional planning and management skills, increasing the complexity of farming operations.

Crop rotation can positively affect yield and increase profit (Table 8). Except for peanuts, all other rotational crops contributed to raise the net income. This was true both for the green manures (crotalaria juncea and velvet bean), as for the grain crops (soybean, sunflower and mung bean). Peanuts caused an increase in the sugarcane stalk yields relative to the control, especially in the first harvest (Table 8), but the high cost of production of this grain somewhat cancelled out the benefit of this rotation. However, in many sugarcane regions in Sao Paulo State peanuts are extensively grown in rotation with sugarcane, probably because in those sites yields are higher and the cost of production, lower. Mung beans are a niche crop. Although it provided a relatively high net return in the present study (Table 8), the risks may be high due to the market restrictions and price fluctuations.

Table 8. Economic balance1 of sugarcane production including revenues and costs of crop rotation.

2. To evaluated the recovery of nitrogen by sugarcane when applied green manure crop and mineral N

To evaluate the utilization of nitrogen by sugarcane (Saccharum spp.) fertilized with sunn hemp (SH) (Crotalaria juncea L.) and ammonium sulfate (AS):

The presence of a green manure crop and mineral N applied together caused some soil alterations that could be detected in samples collected in the sugarcane planting and harvesting seasons (Table 9). There was an increase in calcium and magnesium availability, and consequently in base saturation and pH, in relation to the AS-15N treatment, at planting. Similar results were obtained by [38], who worked with four velvet bean cultivars, velvet bean, Georgia velvet bean, cow itch, and cratylia. The presence of green manure caused a significant sum of bases increase, due to increases in calcium and magnesium; consequently, treatments involving velvet bean showed higher CEC values. The presence of organic acids in the plant mass could be the reason for this change.

During sugarcane harvest, increases in Mg concentration, pH, and base saturation (V%) were observed in the treatments containing SH-15N+ AS in relation to the treatment containing AS-15N alone. Also, a significant reduction in potential acidity was observed in treatments containing SH-15N+ AS in relation to the treatment containing AS-15N alone (Table 9).

Table 9. Chemical characterization of the soil (0.0-0.2 m depth) in the sugarcane planting and harvesting seasons.

The presence of organic acids in decomposing plant residues can help Mg movement in the soil [39]. Crops with high C:N ratio may release N more slowly and cause an increase in N uptake by succeeding crop In addition, rotational plants that were grown before sugarcane could recycle nutrients that would otherwise be leached contribute with N derived from BNF and keep some elements in plant available forms, which could be transformed into more recalcitrant forms if the soil lie fallow for some time.

There was no variation in nutrient contents for macronutrients N and P, and for micronutrients B and Zn in sugarcane stalks at harvest time (Table 10). However, there were differences in Ca and K contents; the latter showed higher values in treatments involving fertilizer application, either mineral or organic, while Ca showed a higher value in the treatment with green manure and mineral N, indicating better nutrition with this element in the treatment containing higher nitrogen supply.

Nitrogen and potassium absorption is greatly influenced by moisture; this relation has been known for a long time [40], and the fact that treatments involving green manure crops maintained environments with higher moisture due to soil mulching with plant mass could have favored better potassium nutrition. With regard to calcium, nitrogen seems to favor absorption [41].

When sugarcane was cultivated for five years and was harvested three times. 15N recovery was evaluated in the two first harvests. In the sum of the three harvests, the highest stalk yields were obtained with a combination of green manure and inorganic N fertilizer; however, in the second cutting the yields were higher where sunn hemp (SH) was used than in plots with ammonium sulfate (AS) (Table 11).

Millable stalk yields of the first cycle (plant cane, harvested 18 months after planting) were higher than those of the second and the third cycle (Table 11). The yield decline with time is common, especially in the cases such as the present experiment when only the first cycle crop was fertilized in order to evaluate the residual effect of N application in the mineral or green manure forms. In the first year the stalk yield was numerically higher in plots fertilized with a combination of green manure and AS; however, in the second year the plots that received SH produced more cane than those fertilized only with AS or the control treatment, indicating that the green manure applied before planting still affected plant growth and yield after 34 months. In the third cycle, there were no differences among the treatments, showing that the residual effect of both N sources had disappeared (Table 11). In the sum of three cuttings, the combination of AS and green manure resulted in highest yields.

[36] showed evidence of the positive effect of green manure fertilization with sunn hemp in sugarcane, with greater sugarcane yield increase than with the application of 40 kg ha-1 mineral N to the soil. [43], studying lupine in maize, and [44], studying velvet bean and sunn hemp in rice, could not find response to mineral N applied after green manure, and no N fertilizer was needed when vetch (Vicia spp.) was grown after wheat, and when cotton followed faba beans [42].

The effect of fertilizer source on sugar concentration was less evident. In the average of three cuttings, the value of pol in plots, treated with both AS + SH was higher than in that observed in plots that received no N (Table 11). Pol in cane juice was higher in the third cutting than in the two previous ones. Variations in pol measurements among cropping seasons are usually more affected by environmental conditions (temperature and drought) that determine cane maturation than by nutrition. However, high N tends to decrease sugar content and delay maturation [45]; therefore, after two years with no N fertilization, sugar content in cane plants was more likely to be high.

The recovery of N by the first two consecutive harvests accounted for 19 to 21% of the N applied as leguminous green manure and 46 to 49% of the N applied as AS.

Nitrogen derived from AS and SH in the leaves and top parts of the sugarcane plant, excluding the stalks, varied from 6.9 to 12.3 % of the total N at the end of the first cycle (plant cane) and was not affected by N source (Table 12). But the amounts of N from both sources accumulated in the leaves and tops were in the range of only 4.5 to 6.0 kg ha-1, which represent a recovery of 6.4 to 8.1% of the N applied as AS and 2.7 and 3.1% of the N from the green manure (Table 12). The recovery of 15N in the second cycle decreased when the N source was the inorganic fertilizer. In the second year the percentage of N derived from sunn hemp was greater than that from the AS, indicating a slightly higher residual effect of the green manure (Table 12).

Sampling Treatments2 Mean ± SEM3

dates AS15N SH-15N + AS SH15N AS15N+ SH

————————— Ndff,%—————————

Table 12. Percentage (Ndff) and quantity (QNdff) of nitrogen in leaves derived from the labeled fertilizer source and nitrogen recovery (R) in samples taken in the first and second harvests1.

The percentage of N derived from the AS or SH accumulated in the stalks harvested in the first cycle were similar and ranged from 7.0 to 10.5% of the total N content. In the plant cane cycle the amounts of N in the stalks that had been applied as inorganic or organic fertilizers were higher than those measured in the leaves and tops and varied from 27.3 to 24.1 kg N ha-1 (Table 13). The recovery of N derived from inorganic fertilizer — 30.1 to 34.4% — was higher than that of the sunn hemp — 8.8 to 9.8%. However, in the second harvest the N the green manure supplied more N to the cane stalk than AS (Table 13). The difference in the amounts of N in the sugarcane plants derived from green manure and mineral fertilizer in the ratoon crop was around 1to 2 kg ha-1 N in leaves and tops (Table 12) and 4 to 7 kg N ha-1 in the stalks (Table 13), which were relatively small compared to the amounts of N accumulated in the ratoon plants (179 kg N ha-1 in plants supplied with AS and 243 kg ha-1 N in the SH treatments, or a 64 kg N ha-1difference — Table 13). These results suggest that the effect of green manure on the yield of the second ratoon crop (Table 11) may not be only due to the extra N supply, but rather to other beneficial role of green manure on soil physical — chemical or biological activity properties.

Adding up the amounts of N taken up by the sugarcane plant and contained in the above­ground parts of the plant (leaves, tops and stalks), AS supplied 32.4 to 34.2 kg N ha-1or about 46 to 49% of N recovery; the N taken up by sugarcane from sunn hemp varied from 37.4 to 40.0 kg ha-1, which represented 19.1 to 20.8% N recovery (Tables 12 and 13).

The recovery of N from fertilizers by sugarcane is usually lower than that of grain crops: the latter varies from 50 to 70% [46] whereas for sugarcane the figures vary from 20 to 40% [47­49, 25]. Results of several studies show that the utilization of N from green manure by subsequent crops rarely exceeds 20% [43, 50, 12, 51] and most of the N remains in the soil, incorporated in the organic matter fraction. In the present study the application of AS along with SH increased N utilization by sugarcane plants. This result is in line with that of [44] who used an organic source isolated or combined with an inorganic fertilizer in rice crops and concluded that the green manures improved the mineral N utilization, resulting in N use efficiency of up to 79%.

In a study in pots [51] observed that maize plants took up more N from sunn hemp incorporated to a sandy soil (Paleudalf) than to a clayey soils (Eutrudox) and that the N derived from the roots was more recalcitrant than that of the shoots. Between 50 and 68% of the 15N of the sunn hemp shoots remained in the soil whereas the figures for roots varied from 65 to 80%. Unaccounted for 15N, probably lost in gaseous forms, varied from 5 to 15% of the sunn hemp N [51].

In a detailed account of the first year of the present experiment, [12] showed that 8 months after planting, the recovery by sugarcane plants (above ground parts) of the N derived from AS or from sunn hemp was similar: 3 to 6% of the added N. However, 12 and 15-month-old sugarcane plants recovered between 20 and 35% of the AS but only 6 to 8% of the sunn hemp-derived N.

The percentage of recovery of the inorganic fertilizer N contained in the stalk when the sugarcane plants were harvested after 18 months of planting varied from 30 to 34%; the corresponding figures for the N derived from sunn hemp were significantly lower: around 9

to 10% (Table 13). The residual effect of the N from both sources in the second harvest of the sugarcane plant was similar: between 4 and 6% of the N supplied at planting as AS or SH was recovered in the stalks of the sugarcane plant 31 months after planting (Table 12).

9.0 ± 1.2

2.6 ± 0.1

20.4 ± 2.78

6.4 ± 0.8

20.8 ± 1.9

4.8 ± 1.9

222.6 ± 9.2 A

217.6 ± 9.2 A
accumulation during the last three months, as also observed by [49]. The nitrogen contents found in the above-ground part of sugarcane, Table 10, are in agreement with results of [47].

As the amounts of N applied as AS or SH to sugarcane in the first cycle were different (70 kg N ha-1as AS and 196 kg N ha-1 as SH), the quantities of N derived from the green manure in the second harvest were larger than those from the inorganic fertilizer, although the percentage of N recovery was similar (Table 12).

Because less N derived from the green manure was recovered by the sugarcane plant in the first cycle it would be expected that a higher proportion of that N would be taken up in the second cycle (first ratoon), but this did not happen. It seems that the residual N that is incorporated to the soil organic matter has a somewhat long turnover. Other authors have reported low recovery (about 3.5% of the N) by the second crop after sunn hemp cover crop

[52] or hairy vetch (Vicia villosa Roth) plowed into the soil [53]. Low recovery of residual N has also been observed for inorganic fertilizer sources: less than 3% of the N derived from fertilizers was taken up by soybeans (Glicine max (L) Merril) [54], maize (Zea Mays L.) [52],

[53] or sugarcane (Saccharum spp) [55], results similar to those obtained in the present study (Table 12 and 13).

The amounts of inorganic N, derived from both N sources, present in the 0-0.4 m layer of soil in the first season after N application and were below 1 kg ha-1.

The average concentration of inorganic nitrogen in the 0-40 cm layer of soil was relatively low in most samples taken after 8, 12, 15, and 18 months of planting (Table 14). Samples taken in February, in the middle of the rainy and hot season, presented somewhat higher values of (NH4+ + NO3-)-N probably reflecting higher mineralization of soil organic N (Figure 2). Later in the growing season (samples of May and Aug 2002) soil inorganic N content decreased again. This coincides with beginning of the dry season with mild temperatures, when the sugarcane plant reached maturity and probably had already depleted the soil for most of the available N.

Table 14. Soil mineral N (NH4+ + NO3-) determined in four sampling dates during the plant cane cycle. Data are average of samplings of the 0-0.2 and 0.2-0.4 m soil layers.

The percentage of the inorganic N derived from AS or SH present in the soil from the 8th to the 18th month after sugarcane planting represented only 1 to 9% of total inorganic N (Table 15). The proportion of N that was originated from AS decreased with time whereas that from the green manure increased, indicating that the mineralization of this organic source could supply more N at the end of the season (Table 15). Indeed, [12] showed that sugarcane stalks sampled in 15-month old plants had significantly higher percentage of N derived from AS than from SH; in the 18th month that difference had disappeared. Nonetheless, throughout the season, the amounts of inorganic N in the soil derived from either AS or SH were of very little significance for the nutrition of the sugarcane plant — less than 1 kg ha-1 of inorganic N in a 40 cm soil layer (Table 15), indicating that little residual N is expected in soils grown with this crop. Although the rate of N applied as SH was almost 200 kg N ha-1, little nitrate leaching losses are expected under the conditions of this experiment.

Table 15. Percent (Ndff) and amount (QNdff) of soil mineral N (NH4+ + NO3-) derived from the labeled fertilizer source (Ndff). Data are average of samplings of the 0-0.2 and 0.2-0.4 m soil layers.

Soil N is often the most limiting element for plant growth and quality. Therefore, green manure may be useful for increasing soil fertility and crop production. With regard to fertilization, organic matter such as a green manure can be potentially important sources of N for crop production [56].

Sugarcane is a fast growing plant that produces high amounts of dry matter. Therefore, it tends to rapidly deplete the soil of inorganic N, especially in soils fertilized with small rates of soluble N as in the case of this study. Cantarella [25] reviewed several Brazilian studies showing little nitrate leaching losses in sugarcane. More recently, [57] showed that only 0.2 kg ha-1 NO3—N derived from 120 kg ha-1 of N as urea enriched to 5.04 15N At% applied to the planting furrow leached below 0.9 m in a sugarcane field, although the total N loss reached 18 kg ha-1 N, mostly derived from soil organic matter mineralization or residual N already present in the soil. As in the present study, the data of [57] refer to N applied at the end of the rainy season when excess water percolating through the soil profile is limited (Figure 2).

5. To evaluate the effect of biomass on the occurrence of nematodes (Pratylenchus spp.) and sugarcane yield after five cuts

The legume most productive was sunn hemp crotalaria juncea IAC-1 with 10,264 kg ha-1, followed by velvet-bean with 4,391 kg ha-1 and peanuts IAC-Caiapo and IAC-Tatu with 3,177 kg ha-1 and 1,965 kg ha-1, respectively.

There was an increase of Stalk yield of sugarcane in the average of the five cuts, compared to control treatment (Table 16). It can be seen that the effect of planting green manure in the fields of sugarcane promoted reform of benefits in terms of increased productivity of sugarcane, and this is lasting reaching in this case until the fifth cut, and the only treatment that stood out was the rotation of the witness with sunn hemp. Notably, the sunn hemp had higher dry matter production, and this may be a positive influence on growth of sugarcane. After five harvests, sunn hemp crotalaria was the leguminous crop that induced the greatest sugarcane yield, with 30% increase in cane yield and 35% in sugar yield.

Crop rotation with non-host species of nematodes, when well planned, can be an efficient method for integrated control of nematodes. It is common in some areas, the practice of cultivation of Fabaceae in the period between the destruction of ratoon sugarcane field and planting the new. There are several plants used in systems of crop rotation with sugarcane, the most common are crotalarias, velvet beans, soybeans and peanuts. However, depending on nematode species occurring in the area, some of these cultures may significantly increase the population of these parasites. Thus, the sugarcane crop, can be greatly impaired by increasing the inoculum potential of the nematodes [58, 59].

The peanut IAC-Caiapo and velvet bean were the leguminous crops that resulted in the greater percentage of AM fungus. The lowest population of Pratylenchus spp. was found in the treatments with peanut IAC-Tatu and IAC-Caiapo (Figure 3).

Thet peanut IAC-Caiapo showed a minimum of 10 nematodes per 10 g of roots and a maximum of 470, while on the control this variation was from 80 to 2,510, indicating the smaller presence of the nematode in treatments with peanut IAC-Caiapo (Figure 3).

Peanut cv. Peanut cv. Control Sunn hemp Velvet bean ІАС-Tatu IAC-Caiapo IAC1

Treatments

Figure 3. Number of nematodes of the genus Pratylenchus spp. by 10 grams of roots of sugar sugarcane cultivation influenced by the previous legume species. Adapted from [16].

6. Conclusions

Crop rotation can positively affect yield and increase profit, contributed to raise the net income. This was true both for the green manures (sunn hemp and velvet bean), as for the grain crops (soybean, sunflower and mung bean). Peanuts caused an increase in the sugarcane stalk yields relative to the control, especially in the first harvest, but the high cost of production of this grain somewhat cancelled out the benefit of this rotation.

However, in many sugarcane regions in Sao Paulo State (Brazil) peanuts are extensively grown in rotation with sugarcane, probably because in those sites yields are higher and the cost of production, lower. Mung beans are a niche crop. Although it provided a relatively high net return in the present study, the risks may be high due to the market restrictions and price fluctuations.

The biomass of green manure induced a complete N substitution in sugarcane and can cause positively affect yield and increase Ca and Mg contents, sum of bases, pH, and base saturation, and decreasing potential acidity and increase profit.

The combination of inorganic fertilizer and green manure resulted in higher sugarcane yields than either N source separately. The recovery of N from ammonium sulfate was higher in the first year whereas in the green manure presented a longer residual effect and resulted in higher yields of cane in the second cycle. The recovery of 15N — labeled fertilizers by two successive sugarcane crops summed up 19 to 21% of the N applied as sunn hemp and 46 to 49% of the N applied as ammonium sulfate. Very little inorganic N was present in the 0-40 cm soil layer with both N sources.

The sugar content of sugarcane stalks is important because the raw material remuneration takes into account this parameter in Brazil. Some crops that preceded sugarcane had a high effect on sugar yield; this was observed mainly in the first harvest in areas where sunflower, peanuts, velvet beans and sunn hemp were previously cultivated. The 3-year average data showed a sugar yield increase, in the best treatment, of 3 t ha-1 in relation to the control.

The peanut IAC-Caiapo, sunflower and velvet bean were the leguminous crops that resulted in the greater percentage of AM fungus. The lowest population of Pratylenchus spp. was found in the treatments with peanut IAC-Tatu and IAC-Caiapo.

After five harvests, sunn hemp crotalaria was the leguminous crop that induced the greatest sugarcane yield, with 30% increase in cane yield and 35% in sugar yield.

Like any other crops, optimizing biomass yield and maintaining quality stands require fertilizer inputs for biofuel crops. Currently, nitrogen (N) remains the primary fertilizer used in biofuel crops; therefore, most studies just consider the N application. Although some perennial species such as swithgrass and miscanthus are tolerant to low soil fertility conditions, studies showed that biomass yield responded to N application [16, 69]. Lemus et al. (2008) used 4 N rates (0, 56, 112 and 224 kg/ha) in switchgrass southern Iowa. They found that N application generally improved the biomass yield but the yield response declined as N level increased [19].

The amount of N fertilizer required for any biofuel crop is a function of several factors including yield potential of the site, cultivar, management practices, soil types, and so on. Therefore, the optimum N rate can vary from place to place. For example, a study in Texas using lowland switchgrass cultivar ‘Alamo’ determined that the optimum N rate was 168 kg/ha [70]. In another study in CRP land of NGP, however, the N rate of 56 kg/ha was optimum for upland switchgrass cultivars [30]. Gunderson et al. (2008) [71] summarized the response of biomass yield of upland switchgrass cultivars to N fertilizer rate. They showed that switchgrass yield even decreased as N rate was over 100 kg/ha (Figure 1). Among the management practices, perennial grass rotating with legume crops or mixture of grass and legumes may reduce N fertilizer inputs and improve their energy balance [71].

Some perennial grasses (e. g., switchgrass and miscanthus) can recycle N from the aboveground shoots to the crown, rhizome, and root in the fall for use in over-wintering and regrowth in the following spring [72]. This mechanism makes an efficient use and reuse of N by plant. However, there is still little information on when and how much of N recycles among plant organs, and how much the N cycling can contribute to over N balance in biofuel crops [7].

Another factor affecting crop N balance is fertilizer use efficiency and N use efficiency (NUE). Take switchgrass as an example, biomass yield varied considerably (up to 5 fold) at the same N application level (Figure 1). Certainly, N was not used efficiently at low yield level. Therefore, improving both fertilization use efficiency and NUE is very important for increasing biomass yield in biofuel crops. In addition, increased efficiency will ultimately reduce the N inputs.

The producer grows the sugarcane. The sugar mill takes ownership of the crop in the field and harvests and transports it for continuous operation during the processing season. Sugarcane must be processed within 24 hours of harvest so storage is not an option within the sugarcane system.

The advantage of the Texas model is that the producer does not own any harvesting or hauling equipment. The disadvantage of the system is the producer does not have control over the time of harvest. Sugar content peaks (point of maximum compensation) in the middle of the season and a producer who has their cane harvested early, or late, sells less sugar. Fortunately, the issue is addressed in their contract.

2.2.1. System construction and research method

The adopted system device is shown in Fig. 23., ozone is dosed via titanium microporous aerator from the bottom column of contact reaction to realize the contact of gas and water in a reversal way. Generally the dosage of ozone is 1.5mg/mgTOC, and the retention time is 11 min. After ozonation, the outflow will equivalently enter the bio-ceramsite filter column (the diameter of ceramic particles is 2-3mm) and bio-activated carbon filter column through the high level cistern and the control valve respectively. Two filter columns are both made of organic glass with 70mm inner diameter and 1650mm height, and the effective height of each filer is 1050mm. For each column, there are sample outlets at height of 100mm, 300mm, 500mm and 700mm along the top of each filter. The empty bed contact time for each column is 15min, and the cycle period of backwash is 3d. Under the same condition, by analyzing the removal effect of BAC and BCF towards DOC and BDOC to conduct quantitative analysis of organics removed by BAC adsorption and biodegradation, the theory on removal of pollutants in water by BAC will be discussed and the removal induced by the synergy effect of BAC adsorption and biodegradation[56,57] will be determined accordingly in this part.

Figure 23. Pilot test arrangement

Vermicomposting is defined as a bio-oxidative process in which detritivore earthworms interact intensively with microorganisms and other fauna within the decomposer community, accelerating the stabilization of organic matter and greatly modifying its physical and biochemical properties [75]. Epigeic earthworms with their natural ability to colonize organic wastes; high rates of consumption, digestion and assimilation of organic matter; tolerance to a wide range of environmental factors; short life cycles, high reproductive rates, and endurance and resistance to handling show good potential for vermicomposting [76]. The earthworm species Eisenia andrei, Eisenia fetida, Perionyx excavatus and Eudrilus eugeniae display all these characteristics and they have been extensively used in vermicomposting facilities.

Vermicomposting systems sustain a complex food web that results in the recycling of organic matter and release of nutrients [77]. Biotic interactions between decomposers (i. e., bacteria and fungi) and earthworms include competition, mutualism, predation and facilitation, and the rapid changes that occur in both functional diversity and in substrate quality are the main properties of these systems [77]. The biochemical decomposition of organic matter is primarily accomplished by the microbes, but earthworms are crucial drivers of the process as they may affect microbial decomposer activity by grazing directly on microorganisms [78-79], and by increasing the surface area available for microbial attack after the comminution of organic matter [8]. Furthermore, earthworms are known to excrete large amounts of casts, which are difficult to separate from the ingested substrate [8]. The contact between worm-worked and unworked material may thus affect the decomposition rates [80], due to the presence of microbial communities in earthworm casts different from those contained in the material prior to ingestion [81]. In addition, the nutrient content of the egested materials differ from that in the ingested material [82], which may enable better exploitation of resources, because of the presence of a pool of readily assimilable compounds in the earthworm casts. Therefore, the decaying organic matter in vermicomposting systems is a spatially and temporally heterogeneous matrix of organic resources with contrasting qualities that result from the different rates of degradation that occur during decomposition [83].

The impact of earthworms on the decomposition of organic waste during the vermicomposting process is initially due to gut — associated processes (GAPs), i. e., via the effects of ingestion, digestion and assimilation of the organic matter and microorganisms in the gut, and then casting [79] (Figure 1). Specific microbial groups respond differently to the gut environment [84] and selective effects on the presence and abundance of microorganisms during the passage of organic material through the gut of these earthworm species have been observed. For instance, some bacteria are activated during the passage through the gut, whereas others remain unaffected and others are digested in the intestinal tract and thus decrease in number [78]. These findings are in accordance with a recent work that provides strong evidence for a bottleneck effect caused by worm digestion (E. andrei) on microbial populations of the original material consumed [79]. This points to the earthworm gut as a major shaper of microbial communities, acting as a selective filter for microorganisms contained in the substrate, thereby favouring the existence of a microbial community specialised in metabolising compounds produced or released by the earthworms, in the egested materials. Such selective effects on microbial communities as a result of gut transit may alter the decomposition pathways during vermicomposting, probably by modifying the composition of the microbial communities involved in decomposition, as microbes from the gut are then released in faecal material where they continue to decompose egested organic matter. Indeed, as mentioned before, earthworm casts contain different microbial populations to those in the parent material, and as such it is expected that the inoculum of those communities in fresh organic matter promotes modifications similar to those found when earthworms are present, altering microbial community levels of activity and modifying the functional diversity of microbial populations in vermicomposting systems [80]. Previous studies have already shown that a higher microbial diversity exists in vermicompost relative to the initial substrate [19,85]. Upon completion of GAPs, the resultant earthworm casts undergo cast — associated processes (CAPs), which are more closely related to ageing processes, the presence of unworked material and to physical modification of the egested material (weeks to months; Figure 1). During these processes the effects of earthworms are mainly indirect and derived from the GAPs [17].

Overall, vermicomposting includes two different phases regarding earthworm activity: (i) an active phase during which earthworms process the organic substrate, thereby modifying its physical state and microbial composition [86], and (ii) a maturation phase marked by the displacement of the earthworms towards fresher layers of undigested substrate, during which the microorganisms take over the decomposition of the earthworm-processed substrate [17-18]. The length of the maturation phase is not fixed, and depends on the efficiency with which the active phase of the process takes place, which in turn is determined by the species and density of earthworms, and the rate at which the residue is applied [8]. During this aging, vermicompost is expected to reach an optimum in terms of its nutrient content and pathogenic load, thereby promoting plant growth and suppressing plant diseases [8]. However, unlike composting, vermicomposting is a mesophilic process (<35 °C), and as such substrates do not undergo thermal stabilisation that eliminates pathogens. Nevertheless, it has been shown that vermicomposting may reduce the levels of different pathogens such as Escherichia coli, Salmonella enteritidis, total and faecal coliforms, helminth ova and human viruses in different types of waste [75]. In a recent work [78], a reduction by 98% in the number of faecal coliforms of pig slurry was detected after two weeks of processing in the presence of E. fetida, which indicates that the own earthworm digestive abilities play a key role in the reduction of the pathogenic load of the parent material. In a previous study, these authors found that the decrease in pathogenic bacteria (i. e. total coliforms) as a result of gut transit differed among four vermicomposting earthworm species (Eisenia fetida, Eisenia andrei, Lumbricus rubellus and Eudrilus eugeniae) [87]. This was consistent with the fact that specific microbial groups respond differently to the gut environment, depending on the earthworm species. The pathogen considered is another important factor controlling the reduction in the pathogenic load during the process. Parthasarathi et al. [88] observed that earthworms did not reduce the numbers of Klebsiella pneumoniae and Morganella morganii, whereas other pathogens such as Enterobacter aerogenes and Enterobacter cloacae were completely eliminated. In a recent study [89] a decrease in the abundance of faecal enterococci, faecal coliforms and Escherichia coli was recorded across the layers of an industrial-scale vermireactor fed with cow manure; whereas no changes were reported for total coliforms, Enterobacteria or Clostridium. These findings are of great importance for the optimisation of the vermicomposting process because despite the pioneering studies of Riggle [90] and Eastman et al. [91], little is known about this process in industrial-scale systems, that is, vermicomposting systems designed to deal with large amounts of wastes. This selective effect on pathogens indicates that earthworms not only modify the abundance of such pathogenic bacteria but also alter their specific composition. According to [89], the unaffected pathogens could benefit as a result of the overall decrease in bacterial and fungal biomass across the layers of the reactor, thereby diminishing possible competition for resources.

Collectively, the aforementioned studies highlight the importance of monitoring the changes in microbial communities during vermicomposting, 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 rate of organic matter, thereby influencing the vermicompost properties, which is critical to guarantee a safe use of this end-product as an organic amendment and thus benefit both agriculture and the environment.