In addition to microorganism safety, toxic and hazardous compounds may be produced in actual operation of O3-BAC process. Those toxic and hazardous compounds can be divided into two parts in general, namely ozonation byproducts and biodegradable byproducts respectively.

4.1.1. Ozonation byproducts

Ozonation byproducts can be divided into two kinds according to its origin, which are produced under the condition of humic substances and bromide existing in water. The former is caused by the reaction of O3 and hydroxyl radicals, and the latter is caused by hypobromous acid. Composition of byproducts would become more complicated when ammonia nitrogen and amino acid exist at the same time[66].

4.1.1.1. Byproducts caused by humic substances

HA is the main composition of NOM in water, the molecular weight of material after ozonation is lower than that of NOM, and contains more oxygen during the reaction. The main byproducts are shown in Table 7. Among these byproducts, carbonyl compounds especially aldehydes should be paid most attention. Animal experiments indicated that formaldehyde, acetaldehyde, glyoxal and methylglyoxal have acute toxicity as well as and chronic toxicity, and furthermore, tube test also implied that these substances have genotoxicity, carcinogenicity, mutagenicity in different levels.

Biomass has been intimately connected with the everyday life of people since prehistoric times. With the advent of fire by rubbing splinters, humans began to exploit the chemical energy of biomass to produce heat. Ever since, various methods have been developed to convert chemical energy present in biomass to useful heat energy. These conversion methods can be summarized as shown in figure (1).

Figure 1. Different categories of biomass conversion

Physical conversion of biomass typically involves pressing the plant (or animal) matter to produce triglyceride oils. Triglycerides cannot be used directly as transport fuels and needs to be processed further. Triglycerides can be converted into a renewable fuel widely known as biodiesel using the transesterification process. This process converts triglyceride in the presence of alcohol to fatty acid alkyl esters.

Biochemical conversion primarily involves using microorganisms or enzymes to breakdown complex chemicals present in biomass into simpler sugars or alcohols. Biomass conversion to alcohols such as ethanol has attracted wide interest in the recent past. The corn to ethanol technology is mature and is commercial in the US. Nevertheless, this technology has created some debate in the context that corn is still a primary food to many around the world. To circumvent this issue, significant strides have been made in the use of lingo-cellulosic biomass as a source for ethanol. In this method enzymes are used to breakdown cellulose to its monomers and subsequently subjected to fermentation under anaerobic conditions using microorganisms.

Thermochemical conversion is another key process that uses heat to induce chemical transformations in the biomass constituents to produce energetically useful intermediate and/or end products. Conversion techniques available under this umbrella can be categorized into four main processes as represented in figure (1). Energy generation by combustion of biomass can be considered as the most archaic [7]. However, increasing demand for transport fuels has led to the development of other processes that involve converting biomass into liquid and gaseous products [8] such as gasification, pyrolysis and liquefaction. Gasification is the conversion of biomass to a mixture of gases called synthesis gas (or syngas) that primarily consists of hydrogen, carbon monoxide, carbon dioxide and methane. During gasification, biomass is heated under an oxygen-lean environment [9]. Synthesis gas can be directly used in an internal combustion engine or can be converted to liquid fuels using a method known as Fisher-Tropsch (FT) synthesis [7, 10]. Fisher-Tropsch process is considered to be quite energy intensive and therefore, is not yet believed to be economical to compete with petroleum fuels. Nevertheless, active research is still in progress to improve the process [11, 12]. Fermentation of synthesis gas to alcohols (primarily ethanol) using microorganisms is also an active area of research [13].

Pyrolysis and liquefaction are two closely related routes targeted towards producing liquids — called bio-oil or bio-crude [14, 15]. Although not universally accepted, the term bio-oil generally refers to the highly oxygenated liquid product that directly exits a pyroloysis reactor. The term bio-crude represents a more deoxygenated liquid product.

Pyrolysis, unlike gasification, takes place in an oxygen-free atmosphere. The most common technique for producing a large volume of condensable fraction is the fast pyrolysis process. Several reactor configurations used for fast pyrolysis include: ablative, entrained flow, rotating core, vacuum pyrolysis, circulating fluidized bed, and deep bubbling fluidized bed reactors [16]. During fast pyrolysis, biomass is heated at a very high heating rate (eg. 103­104 K/s)[17]. The temperature at which the thermal scission of biomass material such as cellulose and lignin take place is around 450-550 °C. The bio-oil portion is originally in the vapor-phase and obtained by quenching the volatile output. The yields of the condensable — fraction are reported to be as high as 70-80%. According to some kinetics studies conducted for different biomass, the frequency factor for pyrolysis varies between 109-10u orders of
magnitude. This is an indication of how fast the reaction would occur during a short residence time [18].

Since its chemical complexity, the biomass pyrolysis reaction has been studied by using model compounds like cellulose. A set of possible reactions paths under different heating conditions is presented in figure (2)[19, 20].

The composition of bio-oil is substantially different from crude petroleum due to the presence of high concentrations of oxygenates. Biomass to bio-oil pyrolysis stoichiometry can be represented using an empirical formula as shown in eq.(1).

Bio-oil properties are highly variable and depend heavily on the type of biomass. High fibrous biomass that contains high amounts of lignin is considered to be the most effective for the production of bio-oil for fuel applications. Further, the oil-yields and composition of bio-oils highly depend on the process (pyrolysis/ liquefaction) used. Fast pyrolysis yields higher amounts of bio-oil compared to slow pyrolysis whereas liquefaction produces low amounts of oxygenated compounds as compared to fast pyrolysis. Oxygenated compounds such as aldehydes, alcohols, ketones, and carboxylic acids can be seen in bio-oils in varying degrees. A typical product distribution of bio-oil is depicted in figure (4). It can be seen that bio-oil contains large amounts of ketones, carboxylic acids and aldehydes [22].

Bio-oil has already been tested in furnaces and gas turbines [21], as well as in space heaters and in boilers[24, 25], as represented in figure (3). Although bio-oil has potential as a crude — oil alternative, problems have been reported when bio-oil was used in such applications. These include, blocking filters by high levels of char particulates, high viscosity causing pumping issues, and corrosion from the low pH [26].

Liquefaction is another approach to producing bio-oils. The concept of oil production using biomass in hot water surfaced in the early 1920’s. However, a more robust and effective method was not available until Pittsburg Energy Research Center (PERC) in the 70’s demonstrated the use of carbon monoxide, steam and sodium carbonate catalyst at Albany Biomass Liquefaction facility in Oregon USA. Detailed information on this method can be found elsewhere [27].

The quality of bio-oil from the liquefaction process is reported to be superior to that obtained from pyrolysis. Liquefaction of biomass using super — critical methods can be considered as one of the more recent techniques under investigation.

Conversion of biomass to a product that is compatible with existing petroleum refinery infrastructure is prudent in several fronts. First, this will allow biomass to be converted into fuels and chemicals that are identical to what we use today (such as gasoline, diesel and jet fuels). So, such fuels could be used in present-day automobiles with no engine modifications. On the other hand, usage of existing fuel production and distribution of infrastructure helps long-term sustainability of biomass-to-fuels technology [28].

The challenge of converting bio-oil into a hydrocarbon fuel has been effective for the removal of functional groups that contain oxygenates (-OH, — COH, — COOH, etc.). The oxygen content of biomass-derived bio-oil is estimated to be 35-40% with a heating value between 16 and 19 MJ/kg [8]. In the recent years, there has been an unprecedented growth of research and developmental efforts related to conversion technologies and the information is scattered. Accordingly, the overall objective of this review is to assimilate this information and compare the status of key deoxygenation technologies.

The overall suitability assessment involved the use of the partial suitability maps of temperature, rainfall and soil productivity ratings (Figures 1 and 2). An overlay of the three maps gave suitability ratings for sugarcane bio-fuel feedstock.

Subtraction of gazetted areas, wetlands and water bodies produced final suitability maps and tables presented in the results. Steep areas have not been excluded since they are associated with highlands which are densely populated areas. It is hoped that soil conservation practices will be practiced where such areas are considered for production of sugarcane feedstock. Urban areas, though expanding, are negligible and have not been considered in the calculations.

The suitability of the land resource quality for sugarcane was based on sets of values which indicate how well each cane requirement is satisfied by each land quality say: mean annual rainfall, minimum and maximum temperatures and soil productivity. The four suitability classes (rating), assessed in terms of reduced yields, and were defined according to " in [10] ". Potential land-use conflict visualization also gives an indication of land available for the production of sugarcane bio-fuel feedstocks. Conflict visualization for food versus sugarcane was done by an overlay of suitability maps of maize with sugarcane. Land-use conflict with gazetted areas was assessed by overlaying gazetted area maps with sugarcane suitability map.

As previously mentioned, fine root turnover might be the most important pathway of C sequestration in forest ecosystems. Therefore, it is crucial to study root dynamics and turnover in the context of forest management. Our case study confirms that on average, fine root biomass (FRB) decreased with increasing stand age in HF (R = -0.28; p<0.01) but remained constant in CS. This basically reflects aboveground biomass dynamics where the CS system has a relatively balanced stand structure throughout rotational cycles. It is partly a consequence of shorter rotation cycles and therefore retains the aggradation phase [49]. Another reason lays in the continuous growing stock, since standards are kept on site during and after understorey harvest. Considering a finer resolution one may observe dynamic changes in FRB corresponding to stand development stages. FRB increased after stand reorganization, culminated at an age of 31 (CS) and 50 years (HF) and subsequently decreased as stands aged. In accordance with increasing aboveground biomass stores, coarse root C pools increased with age in HF (R= 0.87; p= 0.53), accounting for 8.0 (0.9) % of total C pool and no trend was observed in CS, where coarse root C pools accounted for 7.8 (1.0) % respectively [13]. Although on average HF has lower total belowground biomass stores (7 % less), the FRB is 32% higher as compared to CS. The root-to-shoot ratio indicates higher belowground relative to aboveground biomass accumulation rates in early successional phases. A direct comparison between HF and CS reveals two major differences:

1. In comparison with HF, there was no initial major decrease of the ratio observed in CS

2. The ratio is always lower in CS than in HF

These differences may be due to significant aboveground biomass stocks represented by standards in CS and therefore comparatively low ratios, even in the stand reorganization phase. Consequently, root/shoot ratios are in equilibrium throughout the rotation period. More favourable soil conditions in CS may lead to lower ratios throughout stand development. It was shown that drought and limited soil resources (nutrient) availability promote FRB production [50, 51]. The effect of standards harvesting was observed in our case study as a slightly higher ratio in the 15-year old stand compared with the one-year old stand in CS. On average, the root C pool represented 28.0(3.0)% of total phytomass C stores when excluding the youngest HF stand where the root C pool was 1.6 times as high as aboveground phytomass stores [13].

The advantage of round bales is that the rounded top sheds water and bales can be stored in ambient storage without the expense of covered storage. A second important advantage of round bales is that round balers are conventional technology throughout the United States. They are widely used to harvest forage. Compared to a large rectangular baler, a round baler has lower capital cost.

A significant advantage is realized by the farmers if their round balers can be used for both their existing livestock enterprise and a bioenergy enterprise. Warm-season grasses are harvested during the winter for the bioenergy market. Thus, the biomass harvest does not conflict with the hay harvest. The advantage gained by the biorefinery is that no need for their feedstock producers to invest in new equipment. Requiring capitalization of new equipment will make the contacting with feedstock producers more difficult.

The disadvantage of round bales is that they are hard to stack and thus the handling and transport costs are higher. System performance was analyzed and field capacities were determined based on field measurements [16]. Individual handling of bales (either round or rectangular) is not cost effective. The high cost is caused by long loading and unloading times. Multi-bale handling units have been designed, and these units are discussed in Section 9 of this chapter.

2.1.2. Mechanism and characteristics of Оз-BAC process

In practice, there are still some problems when BAC technology is used alone, for example, some difficult biodegradable materials can not be removed effectively and the working life of BAC would be reduced. Meanwhile, in order to ensure the safety of water distribution system, disinfection is indispensable after biological treatment. When chlorine treating potable water is used, large amounts of halogenated organic byproducts will be produced during the reaction between the organics and the chlorine. Among those byproducts, THMs, HAAs, etc., are carcinogenic. Therefore, before BAC treatment, pre­ozonation, the O3-BAC Process, is widely used, which concludes 3 procedures: ozonation, adsorption effect of activated carbon and biodegradation[19]. When O3-BAC Process is used, organic will be firstly oxidized into small degradable molecules by strong oxidation of ozone, then the small degradable molecules will be adsorbed onto the activated carbon and degraded by microorganism, simultaneously the oxygen discomposed from ozone will enhance the level of DO, which makes DO in raw water be saturated or approximately saturated, which in turn, provides necessary condition for biodegradation[20-25]. Fig. 4. shows a simplified model of mutual effects among the main factors during O3-BAC Process [26].

There is a little research related to the interactions between activated sludge flocs and biofilm e. g. migration of the organisms. These interactions are complex, and both relations: between flocs and biofilm, and between heterotrophs and autotrophs in the biofilm should be considered in modeling [15] and operation. Albizuri et al. [15] assumed that these interactions could act with mediation of colloidal components. It is well know fact that there are many grazing species (e. g. Ciliata) which creep on the flocs/biofilm surface or swim near the flocs and biofilm surface. on the other hand there is some number of species existing in deeper layers of biofilm, which probably can not migrate. It is worth to note that the structure of activated sludge flocs is heterogeneous and deep layers of biomass in flocs are anaerobic, what results in different species composition (anaerobic bacteria).

The biological composition of flocs in hybrid bioreactors is similar to typical biological content of activated sludge flocs. Similarly the size of hybrid bioreactor flocs in hybrid reactors is close to typical activated sludge flocs — diameter in the range of 150-500 pm [16].

In hybrid bioreactors various nitrogen removal processes pathways are possible, including autotrophic processes, e. g. anammox [17]. A sufficiently thick layer of biofilm or flocs is needed for complex nitrogen process transformations including denitrification. on the other hand a relatively thin biofilm (due to shearing stress) results in high activity of biomass [18]. Some authors [19,18] found that in sequencing batch biofilm reactors (SBBR) the biofilm is fully penetrated by substrates and electrons acceptors can be released.

Similarly as in case of other attached biomass systems, e. g. trickling filters in MBBR design procedure, surface area loading rate should be the design parameter [5,11]. This approach is based on some typical range of biofilm thickness (in this case surface area can be the indicator of the biomass concentration). From this point of view the size and shape of carriers seem to be less important. The substrate to biomass loading rate is base but not sole design criterion. Important but poorly recognised factors are: access to total biofilm surface area and access to aerobic biofilm surface area. Some authors indicated that carriers of high total surface area thanks to micropores should have some amount of macropores, enabling fluid reach in oxygen contact with deeper inner spaces of carriers, e. g foamed cellulose carriers [20]. The macropores are important for nitrifying biomass, which needs contact with dissolved oxygen. Micropores are often filled completely with biofilm preventing the oxygen penetration. Oxygen access factor is crucial for biofilm thickness, porosity and surface roughness.

The ratio of the suspended to the attached biomass can vary accordingly to many factors and conditions. The amount of attached biomass can reach over 90%. Plattes et al. [21] indicated 93% of biomass in form of biofilm attached to the carrier elements and only 7% of biomass — as suspended in the bulk liquid. Detachment (or sloughing) of biomass is variable in time [5]; probably this phenomenon is similar to sloughing of excess biomass from biofilm growing in others attached biomass systems, e. g. trickling filters. Some authors [22] suggested that in such systems detachment process occurs periodically.

Due to mechanical contact with others carriers and shear stress, the biomass grows mainly on the internal area of carriers, what was reported by several authors [16, 23], excepting carriers having outgrowths on the outside walls surface.

The common forms in typical activated sludge system are aggregated flocs and planktonic free-swimming cells, and bacterial communities are dominated by: Betaproteobacteria, Alphaproteobacteria, Gammaproteobacteia and more less frequent: Bacteroidetes and Firmicutes [24]. Some authors [24] observed in biofilms in MBBR limited bacterial diversity and Firmicutes domination. The research of Biswas and Turner [24] indicated that MBBR communities differ from communities existing in conventional activated sludge reactors. The characteristic feature of MBBR bacteria community was a presence of two distinct communities: suspended biomass with fast-growing aerobic bacteria and biofilm biomass, which was dominated by anaerobic bacteria [24]. In biofilms of WWTP which were studied by these authors the prevailing forms were Clostridia (38% of clones) and sulfate-reducing bacteria (Deltaproteobacteria members). The another forms were less abundant: Desulfobacterales (11-19%), Syntrophobacterales (8-10%), Desulfovibrionales (0.5-1.5%). The other groups were also observed: Bacteroidetes, Synergistes, Planctomycetes, Verrucomicrobia and Acidobacteria.

The suspended biomass observed in two MBBR reactors by Biswas and Turner [24] was consisted mainly of aerobic microorganisms: Alphaproteobacteria (Rhizobiales, Rhodobacterales), Gammaproteobacteria (Pseudomonadales, Aeromonadales), Betaproteobacteria (Burkholderiales, Rhodocyclales). Majority of Firmicutes was represented by Clostridia and one MBBR reactor suspended biomass was reach in Campylobacteraceae (54% of clones).

The differences in microbial composition can appear not only between biofilm and activated sludge in MBBR reactor but also between MBBR bioreactors themselves. Biswas and Turner [24] observed the biomass, both black with sulfurous odour in one MBBR reactor and grayish-brown without obvious odour — in other MBBR reactor. Some authors indicated that in continuous-flow MBBR in which SND process was established, the microbial community structures of biofilm are related to C/N ratios [25]. In MBBRs the volume concentration ratio

of biofilm to the activated sludge flocs cab be even higher: 5-13 [26] than for separated attached biomass and suspended biomass systems. Some important differences between biofilm and flocs features in MBBRs were found by Xiao end Garnczarczyk [26]. They observed 3 — 5 times higher geometric porosity in biofilm than in activated sludge flocs. Biofilm boundary fractal dimension was higher than activated flocs one. These authors observed also some similarities: two different space populations both in biofilm and in flocs were indicated and both attached and suspended biomass shifted some of their structural properties to larger values (thickness, density) with the increased hydraulic loading.

The particulate organic matter and other organisms in the microbial food web have been proposed as potential food sources for aquatic animals [25]. In BFT, microorganisms present a key role in nutrition of cultured animals. The macroaggregates (biofloc) is a rich protein- lipid natural source available "in situ" 24 hours per day [14]. In the water column occurs a complex interaction between organic matter, physical substrate and large range of microorganisms such as phytoplankton, free and attached bacteria, aggregates of particulate organic matter and grazers, such as rotifers, ciliates and flagellates protozoa and copepods

[26] (Fig 3). This natural productivity play an important role recycling nutrients and maintaining the water quality [27,28].

The consumption of biofloc by shrimp or fish has demonstrated innumerous benefits such as improvement of growth rate [10], decrease of FCR and associated costs in feed [9]. Growth enhancement has been attributed to both bacterial and algae nutritional components, which up to 30% of conventional feeding ration can be lowered due to biofloc consumption in shrimp [29]. In reference [9] was reported that more than 29% of daily food consumed for L. vannamei could be biofloc. In tilapia, in [30] was estimated that feed utilization is higher in BFT at a rate of 20% less than conventional water-exchange systems.

Also, consumption of macroaggregates can increase nitrogen retention from added feed by 7-13% [31, 32]. In this context, BFT has driven opportunities to use alternative diets. Low protein feeds and feeds with alternative protein sources different than marine-based products (i. e. fishmeal, squid meal, etc) have been successfully applied in BFT [28, 33-35], leading "green" market opportunities.

Regarding to maintenance of water quality, control of bacterial community over autotrophic microorganisms is achieved using a high carbon to nitrogen ratio (C:N) [30], which nitrogenous by-products can be easily taken up by heterotrophic bacteria [36]. High carbon to nitrogen ratio is required to guarantee optimum heterotrophic bacteria growth [14, 37], using this energy for maintenance (respiration, feeding, movement, digestion, etc), but also for growth and to produce new cells. High carbon concentration in water could supersede the carbon assimilatory capacity of algae, contributing to bacteria growth. Aerobic microorganisms are efficient in converting feed to new cell material (40-60% of conversion efficiency), rather than higher organisms that spend about 10-15% to rise in weight [16]. Bacteria and other microorganisms act as very efficient "biochemical systems" to degrade and metabolize organic residues [36]. In other words, they recycle very efficiently nutrients in a form of organic and inorganic matter (un-consumed and non-digested feed, metabolic residues and carbon sources applied as fertilizers) into new microbial cells.

The carbon sources applied in BFT are often by-products derived from human and/or animal food industry, preferentially local available. Cheap sources of carbohydrates such as molasses, glycerol and plant meals (i. e. wheat, corn, rice, tapioca, etc) will be applied before fry/post-larvae stocking and during grow-out phase, aiming to maintain a high C:N ratio (~15-20:1) and to control N compounds peaks. Also, a mix of plant meals can be pelletized ("green-pellet") and applied into ponds [38]; or low protein diets containing high C:N ratio can also be carried out [16,33]. The carbon source serves as a substrate for operating BFT systems and production of microbial protein cells [36]. There are many considerations for its selection such as costs, local availability, biodegradability and efficiency of bacteria assimilation. In Table 1 is summarized some studies with different species and carbon source applied in BFT system.

Not all species are candidates to BFT. Some characteristics seems to be necessary to achieve a better growth performance such as resistance to high density, tolerance to intermediate levels of dissolved oxygen (~3-6 mg/L), settling solids in water (~10 with a maximum of 15 mL/L of "biofloc volume", measured in Imhoff cones) [38] and N-compounds, presence of filtering apparatus (i. e. tilapia), omnivorous habits and/or digestive system adaptable to better assimilate the microbial particles.

Khanok Ratanakhanokchai, Rattiya Waeonukul, Patthra Pason, Chakrit Tachaapaikoon, Khin Lay Kyu, Kazuo Sakka, Akihiko Kosugi and Yutaka Mori

Additional information is available at the end of the chapter http://dx. doi. org/10.5772/51820

1. Introduction

To develop a bio-based economy for sustainable economic growth, it is necessary to produce chemicals and fuels from renewable resources, such as plant biomass. Plant biomass contains a complex mixture of polysaccharides, mainly cellulose and hemicellulose (mainly xylan), and other polysaccharides (Aspinall, 1980). The hemicelluloses, as well as the aromatic polymer lignin, interact with the cellulose fibrils, creating a rigid structure strengthening the plant cell wall. Therefore, complete and rapid hydrolysis of these polysaccharides requires not only cellulolytic enzymes but also the cooperation of xylanolytic enzymes (Thomson, 1993). Many microorganisms that produce enzymes capable of degrading cellulose and hemicellulose have been reported and characterized. Two enzyme systems are known for their degradation of lignocellulose by microorganisms. In many aerobic fungi and bacteria, endoglucanase, exoglucanase, and ancillary enzymes are secreted individually and can act synergistically on lignocellulose. The most thoroughly studied enzymes are the glycosyl hydrolases of Trichoderma reesei (Dashtban et al., 2009). On the other hand, several anaerobic cellulolytic microorganisms such as Clostridium thermocellum (Lamed & Bayer, 1988), C. cellulovorans (Doi et al., 2003), C. josui (Kakiuchi et al., 1998) and C. cellulolyticum (Gal et al., 1997) are known to produce a cell-associated, large extracellular polysaccharolytic multicomponent complex called the cellulosome, in which several cellulolytic and xylanolytic enzymes are tightly bound to a scaffolding protein (core protein). Thus, the cellulosome provides for a large variety of enzymes and attractive enzymatic properties for the degradation of recalcitrant plant biomass. So far, anaerobic microorganisms have been identified as producing the multienzyme complex, cellulosome

© 2013 Ratanakhanokchai et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons. org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(Doi & Kosugi, 2004; Demain et al., 2005). However, when compared with aerobic enzymes, production of those enzymes by anaerobic culture presents a high cost because of the high price of medium, slow rate of growth and low yield of enzyme, while only a little information has been reported on cellulosome-like multienzyme complex produced by aerobic bacteria (Kim & Kim, 1993; Jiang et al,, 2004; van Dyk et al., 2009). Therefore, the multienzyme complexes, cellulosomes, produced by aerobic bacteria show great potential for improving plant biomass degradation. A facultatively anaerobic bacterium, P. curdlanolyticus strain B-6, is unique in that it produces extracellular xylanolytic-cellulolytic multienzyme complex under aerobic conditions (Pason et al., 2006a, 2006b; Waeonukul et al., 2009b). In the following years, the characteristics, function, genetics and mechanism of the xylanolytic-cellulolytic enzymes system of this bacterium has been the subject of considerable research. In light of new findings in this field, this review will describe the state of knowledge about the multienzyme complex of strain B-6 and its potential biotechnological exploitations.

The utilization of nitrogen by sugarcane (Saccharum spp.) fertilized with sunn hemp (SH)(Crotalaria juncea L.) and ammonium sulfate (AS) was evaluated using the 15N tracer technique in the experiment 2, that consisted of four treatments with four replications in a randomized block design as fallow: a) control with no N fertilizer or green manure; b) ammonium sulfate (AS) at a rate of 70 kg ha-1 N; c) sunn hemp (SH) green manure; d) and sunn hemp plus ammonium sulfate (SH + AS). Microplots consisting of three rows of sugarcane 2-m long were set up in plots c and d with the 15N-labeled sunn hemp.

N was added at the rate of 196 and 70 kg ha-1 as 15N labeled sunn hemp green manure (SH) and as ammonium sulfate (AS), respectively. Treatments were: (i) Control; (ii) AS15N; (iii) SH15N + AS; (iv) SH15N; and (v) AS15N + SH. Sugarcane was cultivated for five years and was harvested three times. 15N recovery was evaluated in the two first harvests.

Sunn hemp (Crotalaria juncea L, cv IAC-1) was sown at the rate of 25 seeds per meter on the 4 Dec 2000 and emerged in nine days. Microplots, consisting of 6 rows, 2- m long and spaced by 0.5 m within the sunn hemp plots were used for 15N enrichment as described by Ambrosano [24]. After 79 days the sunn hemp was cut, and the fresh material was laid down on the soil surface. Total dry mass of sunn hemp was equivalent to 9.15 Mg ha-1, containing 21.4 g kg-1 N, corresponding to 195.8 kg ha-1 N with an 15N enrichment of 2.412 atoms % excess.

Microplots with AS-labeled fertilizer (3.01 ± 0.01 atoms % 15N), with two contiguous rows 1­m long, were set up in plots b and also in plots d; therefore, these plots had microplots for both sunn hemp and AS-labeled materials.

Ammonium sulfate was sidedressed to sugarcane 90 days after planting in both main plots and microplots. N rate (70 kg ha-1) is within the range (30 to 90 kg ha-1 N) recommended for the plant cane cycle in Brazil [25]. A basal fertilization containing 100 kg ha-1 P2O5 as triple superphosphate and 100 kg ha-1 K2O as potassium chloride was applied to all treatments to ensure a full sugarcane development. Cane yield was determined outside the microplots by weighing the stalks of three rows of sugarcane, 2-m long.

Stalks yields were measured after 18 months (plant-cane cycle, on 24 Aug 2002), 31 months (1st ratoon crop, on 8 Oct 2003), and 43 months after planting (2nd ratoon crop, on 20 Sep 2004). Samples consisting of ten stalks were used for the determination of apparent sucrose content (Pol) in the cane juice, according to [23]. The expressed cane juice was analyzed for Pol (apparent sucrose) by a saccharimeter. Just before harvesting of the plant cane (24 Aug 2002) and of the first ratoon (8 Oct 2003) whole plants were collected from 1-m row of plants in the center of the microplots. Leaves and stalks were analyzed separately for determination of 15N abundance and N content in a mass spectrometer coupled to an N analyzer, following the methods described in [26].

The fraction and amount of nitrogen in the plant derived from the labeled source (Ndff) and the fraction of N recovery of the labeled source (R%) were calculated based on the isotopic results (atoms %), according to Trivelin [26], Equations 1 to 3:

where: Ndff (%) is the fraction of nitrogen in the plant derived from the labeled source, a and b are 15N abundance values (atoms % excess) in the plant and in the labeled source (AS or SH), respectively; QNdff (kg ha-1) is the amount of nitrogen in the plant derived from the labeled source, TN (kg ha-1) is total cumulative nitrogen in the sugarcane plant (kg ha-1 ); R% is the fraction of N recovery of the labeled Sugarcane cultivar IAC — 87-3396 was planted on Mar or April on plots with ten sugarcane rows, 10-m long and spaced at 1.4 m.

The biological nitrogen fixation (BNF) by leguminous plants was determined by natural abundance of 15N technique (S15N) [27], and sunflower was the non-N fixing specie. The chemical analysis of plants to determine macro and micronutrient contents were performed according to the methods proposed by [28].