In a 300 m2 greenhouse, lettuce (Lactuca sativa L., var. Romana) were transplanted into 2 L and 16 cm diameters pots, containing the A and B soils; growing density was 16 pots m2. The experiment was performed from April to June, 2009, at temperatures range of 15-28°C.

Treatments consisted in a factorial combination of two increasing N doses (200 and 400 kgN*ha-1), applied as solid fraction of digested swine livestock manure, not-digested solid fraction of swine livestock manure and granular urea [CO(NH2)2], taken as conventional mineral fertilization; not-fertilized soils were also considered as control treatments. Even if the N recommended dose for lettuce growth is about 90-100 kgN*ha-1, the choice of such high N supply was made on the basis of the need to overcome the limit of 170 KgN ha-1 by substituting these organic biomasses rich in N to the mineral fertilization, without incurring in undesired effects on plant and environment: the dose of 400 kgN*ha-1 was just applied for evaluating its potential phyto-toxicity on lettuce in relation to the different fertilization treatments. The corresponding fertilizers’doses per pot were: 128.6 g and 257.2 g for ND, 153.2 g and 306.3 g for D and 1.4 g and 2.6 g for urea.

Treatments were arranged in a randomized complete-block design with six replicates. Drip irrigation was managed in relation to plant water-demand, as reported in Figure 2.

Lettuce plants were harvested 70 days from sown; biomass dry weight (g), dry matter (%), leaf area (cm2) and leaf number were determined for each plants. In order to evaluate the effect of alternative fertilization treatments on micro and micronutrient uptake by lettuce, N, P, K, Mg, Cu, B, Fe, Mn leaf contents, plant material was incinerated at 400°C for 24 hours; ashes were then redissolved in HCl 0.1N and the supernatant filtrated to obtain a limpid solution; the nutrient content was determined by simultaneous plasma emission spectrophotometer (ICP-OES) on obtained solution and calculated in relation to dry matter.

In any biomass to biofuels conversion process, above mentioned three reaction pathways can be considered to be quite significant. Depending on the process conditions such as temperature, pressure, the resident time and the type of catalyst, the degree to which these reactions would take place may vary. Basically the major processes that are exploited during deoxygenation pathways are secondary cracking, fast catalytic cracking and hydrodeoxygenation.

3.1. Secondary pyrolysis

This is the simplest process with no catalyst involved for the deoxygenation of biomass oxygenates and occurs with fairly low efficiencies. The concept behind this process is simply to route the pyrolysis vapor through a second reactor which is maintained at a high temperature. The thermal cracking reaction initiated in the secondary reactor would remove oxygen as H2O, CO2 and CO. Due to the thermodynamic nature of this reaction, increased resident times would favor formation of thermodynamically more stable species such as CO2 and CO. The significant drawback of this method is the higher tendency of losing carbon in the forms of CO2 and CO.

Millable stalk population at harvest was significantly higher due to application of filter mud and boiler ash + 100% recommended dose of fertilizers (RDF) than all other treatment combinations. However, there was no significant effect on stalk length, number of internodes and stalk weight due to different treatments. Cane and sugar yields were significantly higher by 30.2 tc/ha and 3.8 ts/ha respectively due to application of filter mud and boiler ash + 100% RDF. The data are presented in Table 3.

Cane-mill by-products (filter mud and boiler ash) do contain valuable amounts of N, P, K, Ca, and several micronutrients "in [15]". These in addition to the inorganic fertilizers applied considerably increased cane yields as compared to treatments which received only inorganic fertilizers. In [11] it is also showed that organic wastes-including filter mud and bolier ash could be used as an alternative source of nutrients in cane cultivation.

Over the years, many species have been or being evaluated for potential of biofuel crops in the USA and Europe, in which the perennial grasses are dominant (Tables 1 and 2) [12]. In the USA, switchgrass was determined as a model species. In Europe, miscanthus, reed canarygrass, giant reed and switchgrass were chosen for more extensive research programs [12]. In addition, legume species and mixture of multi-species also been evaluated as bioenergy crops [5,13].

1.1.1. Switchgrass and miscanthus

Switchgrass and miscanthus are two dominant species reported in literatures for potential biofuel crops. Switchgrass, a C4 perennial grass, has been designated by the U. S. DOE as primary bioenergy crop and has been extensively studied for over two decades. Several reviews have addressed current research and development issues in switchgrass, from biology and agronomy to economics, and from production to policies [6, 14-18]. The attributes of switchgrass for biofuel production included high productivity under a wide range of environments, suitability for marginal and erosive land, relatively low water and nutrient requirements, and positive environmental benefits [17]. For biofuel purpose, switchgrass can be used to produce ethanol [2, 7, 18]. It also can be used as combustion to co-fire with coal in power plant for electricity. Currently, switchgrass production in southern Iowa is mainly used for combustion [19].

Miscanthus is another C4 tall perennial grass originated in East Asia and has been studied extensively throughout the Europe from the Mediterranean to southern Scandinavia [20]. Comparing with other C4 species (such as maize), miscanthus is more cold tolerance and winter hardy in temperate regions of Europe. It also has a low requirement of nitrogen fertilizer and pesticides. In general, miscanthus has a very high biomass yield potential when it is well established. Lewandowski et al. (2000) [20] reported that the irrigated miscanthus yield can be as high as 30 Mg/ha, and yield under rainfed conditions ranged from 10 to 12 Mg/ha. When compared biomass production in US for switchgrass and Europe for miscanthus, the average yield of miscanthus (22 Mg/ha) was twice as much as the average yield of switchgrass (10 Mg/ha), given the similar temperature, nitrogen and water regimes [21]. A side-by-side study in Illinois showed that average biomass yield in miscanthus (30 Mg/ha) can be 3 times as much as switchgrass (10 Mg/ha) [22]. Compared to switchgrass, miscanthus may require higher input costs because it must be established using rhizome cuttings, which delays full production until the second or third year [20, 21]. In Europe, the primary use of miscanthus biomass is for combustion because of the ideal chemical composition [20]. However, little information is known for the conversion of ethanol from miscanthus.

There are numerous studies developing for the optimum set of field equipment where the unit operations affect the performance of units upstream or downstream. Review of this literature is beyond the scope of this chapter. However, several simulation programs are highlighted that specifically addresses a biomass harvest and delivery system.

The Integrated Biomass Supply Analysis and Logistics Model (IBSAL) is a simulation model which simulates operations in the field [22,38,41]. ISBAL is very useful for the simulation of operations that collects feedstock from the field and examines the flow of biomass into storage. IBSAL is best used for drawing conclusions about a sequential set of events. For example, if the user has a defined number of fields, a defined set of equipment, and a target number of tons to be harvested each month (each week) then IBSAL can provide valuable guidance for optimum biomass collection. The influence of weather, equipment breakdowns, and other disturbances to the biomass system can be "played" with a series of simulations.

The BioFeed model [43] determines the overall system optimum, and integrates the important operations in the biomass chain into a single framework. It is possible that the optimal solution recommended by BioFeed may not be implemented in a real system, either due to unforeseen disturbances such as weather or due to the actions of independent stakeholders such as farmers. However, an integrated model such as Bio-Feed determines the optimal configuration, system bottlenecks, and potential improvements. Such a model can be useful in quantifying the systemic impacts of technology improvement [42].

The BioFeed model results were compared to recent studies in literature [22,31,33]. The scope of the BioFeed model was similar to the scenarios developed in [22], where the delivered cost was estimated to be about $35/Mg (d. b.) excluding biomass size reduction. The major differences between ISBAL and BioFeed were harvesting and storage costs. While Kumar and Sokhansanj [22] ignored the storage costs, they also considered a self-propelled forage harvester which had a higher throughput capacity than the mower-conditioner considered in BioFeed, thereby reducing the cost. Khanna et al. [31] reported the switchgrass delivered cost of $64.84/Mg, which was similar to the BioFeed cost estimate. The study by Duffy [33] estimated the delivered cost to be $124.30/Mg. However, the major difference in the estimates was due to a much higher establishment cost.

Since the scope of the analyses and the assumptions differed for these studies, it is impossible to make specific comparisons. However, these comparisons illustrate that the overall model predictions agree reasonably well.

Dominant microflora is selected by artificial screening and domesticating or directive breeding through biological engineering technology. Separation is the first step for getting dominant biocommunity. However, the filtered biocommunity may contain pathogenic bacteria, or with low-rated growth or high requirements for nutrition, which make it inadaptable for practical application, therefore, preliminary screening is indispensable for guaranteeing the security of selected bacterial strains. Due to the high complexity of biofacies, various species but less nutrition matrix in water, low-activated bacterial strain screened. This situation makes a relatively high disparity from the effective biological treatment. Therefore, intensification on selected bacterial strain is necessary. The process of intensification is actually the process of induction and variation of biocommunity. By changing nutritional conditions repeatedly, the bacterial strain will gradually adapt to the poor nutrition environment in fluctuation, so that the bacterial strain immobilized on activated carbon can preserve a strong ability for biodegradation[33].

During IBAC treatment, the process of microorganisms immobilizing is of great complexity, which is not only related to various acting forces, but also involves microorganism growth as well as the ability for producing extracellular and external appendages[34]. DLVO theory can give a better explanation that how bacteria are attached, when bacteria are regarded as colloidal particles[35-37]. Although there is a relatively high repulsive force during the process when the bacteria approaching to the activated carbon, the bacteria will ultimately contact with the activated carbon under the bridging effect of EPSs[38]. The bacteria may take advantage of the bridging effect with surficial particles, which enables the attachment of itself with filter materials at secondary extreme point in accordance with the theoretical level diagram of DLVO theory, rather than by the certain distance or overcoming the necessary energy peak as that of non-biological particle[39], see Fig. 10. for details. Although there are various ways to immobilize microorganisms, and any method with a limitation for free — flowing of microorganisms can be used to produce and immobilize microorganisms, an ideal and universal application way for immobilizing microorganisms is still not available. There are 4 common methods of immobilization, see Table 3 [40-42] for details.

Continuous flow reactor with elevated wastewater pH was maintained at 12 hours HRT and 15/15 minutes aeration/nonaeration intervals. Values of pH were in the range of 8.0-8.5. Three rates of volume reactor filling by carriers were investigated: 60%, 40% and 20%.

The elevated pH caused the ammonia release and inhibition of the second phase of nitrification and this way the total nitrogen removal process was shorten by elimination of two elementary processes: nitratation and denitratation (figure 8).

The free ammonia concentration value, calculated accordingly to equation 6 was equal to appr. 1 mg N/dm3, what is known as a limiting value for inhibition of nitrification second phase [42]. The part of free ammonia could be released as the result of amino acids denaturization during alkalinity process, but due to relatively low concentration of organic nitrogen (mainly amino acids) in inlet wastewater (maximum 10% of total nitrogen) this factor can be neglected. In these conditions the SND process was achieved (shortened nitrogen removal process) what had been indicated by temporary nitrites accumulation. It is known and was stated by several authors [51] that as a result of ammonia inhibition of second phase of nitrification, mediate and final products are released simultaneously.

The higher removal efficiency was achieved at higher volume carriers filling of reactor (figure 9). The rise in pH value versus the rise of nitrogen compounds removal rate, what was observed by other authors [52].

The lime addition resulted in some changes in biomass, e. g. reducing organic fraction rate in biomass of 25% comparing to the process without lime addition. The lime in the liquid phase and on the carriers surface were some kind of "condensation centers" and caused the higher concentration of both biomass form. The smaller amount of carriers enabled more undisturbed carriers movement (figure 10). The lime addition caused also the less susceptibility of carriers pores for clogging by biomass.

The cost of diets in several animal cultures is predominantly due to the cost of protein component [75]. In the case of aquaculture, its massive expansion in the last decades has begun to face some important limitations like increasing prices of fishmeal, a raw material prime component of aquaculture diets. However, pressure caused in natural stocks (over­fishing) has depleted fishmeal production and, as a consequence, continuous increase in prices has been observed [76]. Moreover, growth of aquafeed industry (driven by an increase in fish/shrimp demand as the global population continues to growth), the competition with other animal cultures (such as swine and poultry) and differences in fishmeal quality also collaborated with increase in prices of fishmeal. The quality attributed to fishmeal includes high palatability, high content of digestible protein, highly unsaturated fatty acids (HUFA) and minerals.

In this context, alternatives should be evaluated opposing this non-optimism scenario. Aquaculture industry needs to investigate alternative source of proteins to replace less sustainable ones. Candidates of protein sources might have good digestibility, palatability, energy content, low ash content and present a well-balanced essential amino acids profile (EAA) [77].

In the past years, BFT has been emerged not only as promising alternative to grow-out system, but also as a method to obtain protein for compounds diets originated from its diverse microbiota. Collected in tanks/ponds [46, 62] or produced in bioreactors [17, 39, 67] biofloc (Fig 8) is a raw material to produce "biofloc meal". In bioreactors, biofloc production can clean up effluent waters from aquaculture facilities, converting dissolved nutrients into single-cell protein [78]. Usually, two types of bioreactors have been employed: sequencing batch reactors (SBRs) and membrane batch reactors (MBRs), both controlling ammonia, nitrite and suspended solids with great efficacy (for review of bioreactors and its employ, see Kuhn et al 2012). Moreover, excess of solids removed from culture tanks or ponds and/or concentrated into solid removal devices [28] could also be a recyclable source of biofloc for biofloc meal production. This sustainable approach of protein source is getting more attention in the aquaculture industry. The microbial particles can provide important nutrients such as protein [33, 46], lipids [10, 37], aminoacids [80] and fatty acids [33, 67, 81].

Biofloc meal (also called "single-celled" protein), added to compounded feed is currently focus of intensive research in nutrition fields [17, 78]. However, to produce this protein ingredient some processes are required such as drying, milling and storage. In this context, nutritional characteristics could be affected (by i. e. temperature during drying), which the "native" properties could be altered.

Nutritional composition of biofloc differs according to environmental condition, carbon source applied, TSS level, salinity, stocking density, light intensity, phytoplankton and bacteria communities and ratio, etc. Regarding to age of bioflocs, in "young" biofloc heterotrophic bacteria is mainly presented as compared to "old" biofloc dominated by fungi [79]. In biofloc particles, protein, lipid and ash content could vary substantially (12 to 49, 0.5 to 12.5 and 13 to 46%, respectively; Table 2). The same trend occurs with fatty acids (FA) profile. Essential FA such as linoleic acid (C18:2 n-6 or LA), linolenic acid (C18:3 n-3 or ALA), arachidonic acid (C20:4 n-6 or ARA), eicosapentanoic acid (C20:5 n-3 or EPA) and docosahexaenoic acid (C22:6 n-3 or DHA), as well as sum of n-3 and sum of n-6 differ considerably between 1.5 to 28.2, 0.04 to 3.3, 0.06 to 3.55, 0.05 to 0.5, 0.05 to 0.77, 0.4 to 4.4 and 2.0 to 27.0% of total FA. Type of carbon source, freshwater or marine water and production of biofloc biomass (in bioreactors or culture tanks) definitely influence the FA profile (Table 3 and 4). Vitamin and amino acids profile from biofloc produced in large-scale commercial bioreactors [82] in given in Table 5.

Information is still scarce about how microorganisms profile and its nutritional composition could impact animal growth. However, is already known that microorganisms in biofloc might partially replace protein content in shrimp diets, although were not always the case [10, 88]. Recent studies determined how reducing the protein content of diet would affect growth performance of shrimp reared in biofloc conditions. In the study [15] was found that at least 10% of protein content in pelletized feed can be reduced when F. paulensis postlarvae are raised in BFT conditions. In [89] was observed that shrimp fed with less than 25% crude protein under biofloc conditions performed similarly to shrimp raised under regular clear­water intensive culture with a 37%-protein diet. The biofloc system also delivered more consistent survival rates, especially at higher density. A low-protein biofloc meal-based pellet (25% CP) was evaluated as a replacement of conventional high-protein fishmeal diet (40% CP) for L. vannamei in a relatively low temperature (25oC) under biofloc conditions

[35] . The results showed that is possible to replace 1/3 part of a conventional diet by alternative low-protein biofloc meal pellet without interfering survival and shrimp performance.

Also, recent studies have been demonstrated that fishmeal in shrimp diets can be partially replaced by other protein sources under biofloc conditions or by biofloc meal. In [90] was evaluated two fishmeal replacement levels (40 and 100% of replacement) by other ingredients (soyabean meal and viscera meals) in diets for Litopenaeus vannamei reared in a biofloc system. The authors observed that fishmeal can be replaced in a level of 40.0% without interfering on growth performance and water quality. On the other hand, incorporating treated solids (microbial flocs) generated from tilapia effluent into shrimp feed, [91] demonstrated that shrimp performance was significantly increased as compared to untreated solids (settling basins of tilapia culture units). In [92] a trial performed in clear­water conditions detected that fishmeal can be completely replaced with soy protein concentrate and biofloc meal (obtained from super-intensive shrimp farm effluent) in 38% CP diets without adverse effects on L. vannamei performance. Moreover, [17] observed that biofloc produced in SBRs bioreactors using tilapia effluent and sugar as a growth media could offer an alternative protein source to shrimp feeds. Microbial floc-based diets significantly outperformed control fishmeal-based diets in terms of weight gain per week with no differences in survival.

Regarding to biofloc meal production, one bottleneck seems to be the large amount of wet biofloc biomass required to produce 1kg of dry biofloc meal. Estimative indicates that biofloc plug in 1L settling cones contained only 1.4% of dry matter [14]. The reference [17] indicated that 1 kg of microbial floc could be produced per 1.49 kg of sucrose in bioreactors. Certainly more research is needed on this field. On the other hand, other applications of

biofloc meal in animal industry should be evaluated, mainly considering its nutritional profile and relatively low costs as compared to other protein sources (i. e. fishmeal) [17]. In aquaculture, biofloc meal could be included into broodstock pelletized feed, prior or after eyestalk ablation. Further research is encouraged in this field.

Among several Bacillus strains, isolated from various sources and cultivated under aerobic conditions, P. curdlanolyticus strain B-6 shows important evidences for multienzyme complex producing bacterium (Pason et al., 2006a) as follows: high production of cellulase and xylanase, presence of CBMs that have ability to bind to insoluble substances, adhesion of bacterial cells to insoluble substances, and production of multiple cellulases and xylanases in the form of a high molecular weight complex. Thus, strain B-6 exhibits great promise bacterium in the production of multienzyme complex under aerobic conditions. Some properties of bacterial cells and cellulase and xylanase from strain B-6 compared with other Bacillus spp. are shown in Table 2.

P. curdlanolyticus strain B-6 was a facultative, spore-forming, Gram-positive, motile, rod-shaped organism and produced catalase. Thus, this bacterium was identified as a member of the genus Bacillus according to Bergey’s Manual of Systematic Bacteriology (Sneath, 1986). The bacterium was also identified by 16S rRNA gene sequence analysis. The use of a specific PCR primer designed for differentiating the genus Paenibacillus from other members of the Bacillaceae showed that this strain had the same amplified 16S rRNA gene fragment as a member of the genus Paenibacillus. Based on these observations, it is reckoned that this strain was transferred to the genus Paenibacillus (Shida et al., 1997). The 16S rDNA sequence of this strain had 1,424 base pairs and 97% similarity with Paenibacillus curdlanolyticus (Innis & Gelfand, 1990).

The biopolyester was recovered and purified by dissolving the non-PHA cell mass (28% w/w) in sequential treatments consisting of acid pretreatment, base treatment, hypochlorite whitening, washing and drying [22].

Acid pretreatment: The cell mass in acidic solution was heated to boil and maintained for one hour under ambient conditions. The pretreated cellular solids were cooled to room temperature and separated from acid solution with centrifugation at 5,000 g for 10 min. The dissolved microbial biomass in the supernatant solution is referred to acid hydrolysates. The insoluble wet pellets were subjected to next base treatment.

Base treatment: The insoluble solids from the acid pretreatment were re-suspended in an equivalent volume of water to form slurry of about 200 g DM/L. The solution pH was raised to 10 to 11 with 5 M NaOH solution and stirred under ambient conditions for 30 to 60 min.

A small amount of surfactants such as sodium dodecylsulfate (SDS, CH3(CH2)ttOSO3Na) might be added to a concentration of 5 to 10 g/L. The slurry was then heated to boiling and maintained for 10 min under ambient conditions. After centrifugation, the dissolved biomass in the supernatant solution is referred to base hydrolysates. The sequential acid and base treatments above could also be performed without separation of the acid hydrolysates.

In this case, sodium hydroxide was directly added into the acidic cell slurry and the pH was raised to 10-11 for base treatment. After centrifugation, the supernatant solution contained the hydrolysates generated from both acid and base treatments and is refereed to acid-base hydrolysates.

Whitening and washing: The insoluble wet pellets from base treatment were re-suspended in a commercially available bleaching solution containing 6% w/w of hypochlorite. The slurry was stirred for 1 to 2 hours under ambient conditions. The white PHB pellets were recovered with centrifugation. A small amount of biomass was dissolved and mineralized in the bleaching solution because of chemical oxidation, which is not considered for reuse in this work. The wet PHA pellets were washed two times with water and dried in oven. The final PHB product is a white powder.