The enormous global daily consumption of liquid fuels is of the order of 80 million barrels/day (e equivalent of 12.7 million m3/day). The sugar cane area required to produce the same volume of ethanol is about 700 million hectares, assuming a yield of 6.5 m3/ha/ year of ethanol. This area is equivalent to 100 times the sugar cane cultivated area in Brazil, the second largest bio-ethanol producer in the world. Biofuels definitely face an issue of scale. In 2010, fuel ethanol and biodiesel combined displaced a mere 3% of oil in the world.

Figure 1[4] below illustrates the scale issue by showing how much land it would take for the USA to grow its own fuel.

It appears that algae require the least area to meet the large scale demand of liquid fuels in the USA, whereas the area required by soybeans is larger than the USA’s 48 continental states. The area required by corn is substantial. This suggests that the current biofuels production base of the USA would not be able to meet demand, and imports would be required to meet the colossal American energy appetite.

Fig. 1. How much land would it take for the USA to grow its own fuel?

The scale challenge posed to biofuels relates to the labour, management, land, water and sunshine required to produce the biomass and the processing that originates them. These are scarce resources that are also needed to grow food, feed and fibre to ultimately meet various human demands. These are resources that have an opportunity cost from competing markets. To develop biofuels in the scale of commercial liquid fuels require massive financing, a resource that may have alternative uses as well. The mobilization of private capital under a perception of market and other uncertainties is another issue that biofuels have to resolve in order to thrive.

The production of biofuels is accompanied by local environmental issues that need addressing. For instance, in the case of sugar cane ethanol, stillage the liquid residue of distillation, has a high chemical and biological oxygen demand and requires appropriate processing before final disposal. From a global climate change perspective, designed and managed properly, a biofuels production system would add minimally to greenhouse gas emissions. But, in practice, many biofuel production systems in the world are contributing net GHG emissions.

A bone of contention in the development of the biofuels industry is the present competition for feedstocks between the food and fuel industries. In the case of biodiesel, all commercial
vegetable oils that are used in preparing food are also convertible to biodiesel. A similar situation exists with respect to fuel ethanol, especially for the starch-based feedstocks (corn and wheat). However, the hike in food prices that happened globally in 2007/8 and is happening in 2010/1 derive mostly from other causes such as droughts and other climate related phenomena, higher oil prices and market speculation.

Since the cost of biofuels is dominated by feedstocks cost, access to feedstocks in the required amounts, timing and at adequate prices is key to the success of the biofuels economy. The combination of the food versus fuel conundrum with the need to have reliable and economic access to feedstocks is shifting the industry towards non-food feedstocks and to the market penetration of second generation technologies to convert cellulosic biomass into liquid biofuels.

Concern in important consuming markets about the sustainability of biofuels producing systems is putting pressure on suppliers to abide by sustainability protocols subject to certification. The sustainability of biofuels is actually linked to freer international trade, which would tend to phase out unsustainably produced biofuels in favour of regions of the world that can meet sustainable production requirements. A valuable discussion on this matter was hosted by the Rockefeller Foundation in 2008 at its Bellagio Centre and produced a sustainable biofuels consensus. The objective was to understand the many drivers for sustainable trade, consumption and production of biofuels, and the comparative advantage of supplying regions combined with demand and technology from consuming regions [5].

However, much remains to be done to achieve free international trade of biofuels. The World Trade Organization Doha rounds have reached an impasse. Currently, biodiesel is considered an industrial product, whereas fuel ethanol is categorized as an agricultural product, which allows more protectionism. What is needed is a unified treatment of biofuels, where they are classified under environmental goods and services. But, irrespective of these drawbacks, a sign pointing to a larger role for biofuels in the future are the new biofuels technology initiatives by large oil companies, such as BP, Chevron, Exxon and Shell. The development of the international trade in biofuels is likely to distribute more evenly the production and consumption of biofuels in the world. For the time being, biofuels production is overwhelmingly concentrated in the USA, Brazil and the European Union, as shown in Fig. 2 below[6].

DDGS from most modern U. S. fuel ethanol plants typically contains about 30% protein, 10% fat, at least 40% neutral detergent fiber, and up to 12% starch (Rosentrater and Muthukumarappan, 2006). Composition, however, can vary between plants and even within a single plant over time, due to a number of factors. For example, Table 1 summarizes composition of DDGS samples collected from five ethanol plants in South Dakota. On a dry basis, crude protein levels ranged from 28.3 to 31.8%; crude lipid varied between 9.4 and 11.0%; ash ranged from 4.1 to 13.3%. In terms of within-plant variability, the crude protein, crude lipid, and starch content all exhibited relatively low variation, whereas neutral detergent fiber (NDF), acid detergent fiber (ADF), and ash all had substantially higher variability.

Furthermore, DDGS from 49 plants from 12 states were analyzed for proximate composition (Table 2) and amino acid profiles (Table 3) (UMN, 2011). Dry matter content varied from 86.2% to 92.4%, while protein varied from 27.3% to 33%. Crude fat content displayed even higher variability, and ranged from 3.5% to 13.5%; crude fiber ranged from 5.37% to 10.58%; and ash content varied from 2.97% to 9.84%. On average, geographic trends were not readily apparent for any of the nutrient components. In terms of amino acids, lysine ranged from 0.61% to 1.19%, but again, no geographic trends were apparent.

Some plants are beginning to implement various fractionation processes (either pre­fermentation or post-fermentation) in order to produce multiple product streams (RFA, 2009a). These new processes can lead to additional differences in DDGS nutrient levels. For example, various techniques for dry fractionation and wet fractionation have been developed to concentrate protein, fiber, and oil components from the endosperm (which contains the starch). This allows a highly-concentrated starch substrate to be introduced to the fermentation process, and it allows the other components to be used for human food applications. Singh and Johnston (2009) have provided an extensive discussion regarding various pre-fermentation fractionation approaches. On the other hand, post-fermentation fractionation techniques have also been examined. For example, Srinivasan et al. (2005) used a combination of (air classification and sieving to separate fiber particles from DDGS. Processes have also been developed to remove corn oil from thin stillage and CDS; although the resulting corn oil fractions cannot be used as food-grade oil, they can readily be converted into biodiesel. All of these approaches, if implemented commercially, will alter the composition of the resulting DDGS.

The U. S. ethanol industry’s primary market for distillers grains has historically been as a commodity livestock feed. Most often this has been in the form of DDGS, and to a lesser degree in the form of DWG; the other coproducts are sold in much lower quantities than either DDGS or DWG and some are not always produced either). Feeding ethanol coproducts to animals is a practical method of utilizing these materials because they contain high nutrient levels, and they are digestible (to varying degrees) by most livestock. And, use of DDGS in animal feeds (instead of corn grain) helps to offset the corn which has been

DDGS use in livestock diets has continued to increase over the years. Predictions of peak potential for DDGS use in domestic U. S. beef, dairy, swine, and poultry markets have estimated that between 40 and 60 million t could be used in the U. S. each year, depending upon inclusion rates for each species (Staff, 2005; Cooper, 2006; U. S. Grains Council, 2007). Globally, the need for protein-based animal feeds continues to grow. Of the 23 million t of DDGS produced in 2008 (RFA, 2009b), 4.5 million t were exported to international markets (FAS, 2009); this accounted for nearly 20% of the U. S. DDGS production that year (Figure 6). And the potential for global exports is projected to increase for the foreseeable future (U. S. Grains Council, 2007).

О

n

n

Not only are coproducts important to the livestock industry as feed ingredients, but they are also essential to the sustainability of the fuel ethanol industry itself. In fact, the sale of distillers grains (all types — dry and wet) contributes substantially to the economic viability of each ethanol plant (sales can generally contribute between 10 and 20% of a plant’s total revenue stream (Figure 7), but at times it can be as high as 40%), depending upon the market conditions for corn, ethanol, and distillers grains. This is the reason why these process residues are referred to as "coproducts", instead of "byproducts" or "waste products"; they truly are products in their own right along with the fuel.

So the sales price of DDGS is important to ethanol manufacturers and livestock producers alike. Over the last three decades, the price for DDGS has ranged from approximately $50.71/1 up to $209.44/1 (Figure 8). DDGS and corn prices have historically paralleled each other very closely (Figure 9). This relationship has been quite strong over the last several

years. This is not surprising, as DDGS is most often used to replace corn in livestock diet formulations. DDGS has increasingly been used as a replacement for soybean meal as well, primarily as a source of protein. Even so, DDGS has historically been sold at a discounted price vis-a-vis both corn and soybean meal. This has been true on a volumetric unit basis, as well as per unit protein basis (Figure 9).

3.1.1 Biodegradability (lability) and stability of organic matter

How many labile components of organic matter are lost during anaerobic digestion in a biogas plant can be demonstrated by determination of the degree of organic matter lability. For this purpose a number of methods can be used that are mostly based on resistance to oxidation or on resistance to hydrolysis. Oxidation methods are based on oxidation with chemical oxidants, e. g. with a solution of K2Cr2O7 in sulphuric acid at various concentrations — 6 M + 9 M + 12 M (Walkley 1947, Chan et al. 2001) or with a neutral solution of KMnO4 at various concentrations (Blair et al. 1995, Tirol-Padre, Ladha 2004). The degree of organic matter lability is evaluated from the amount of oxidizable carbon in per cent of its total amount in particular variously aggressive oxidation environments or the reaction kinetics of the observed oxidation reaction is examined while its characteristic is the rate constant of the oxidation process.

In 2003 was proposed and tested the method to evaluate the kinetics of mineralisation of the degradable part of soil organic matter by the vacuum measurement of biochemical oxygen demand (BOD) of soil suspensions using an Oxi Top Control system of the WTW Merck Company, designed for the hydrochemical analysis of organically contaminated waters (Kolar et al. 2003). BOD on the particular days of incubation is obtained by these measurements whereas total limit BODt can be determined from these data, and it is possible to calculate the rate constant K of biochemical oxidation of soil organic substances per 24 hours as the rate of stability of these substances. A dilution method is the conventional technique of measuring BOD and also rate constants. It was applied to determine the stability of soil organic substances but it was a time — and labour-consuming procedure. The Oxi Top Control method was used with vacuum measurement in vessels equipped with measuring heads with infrared interface indicator communicating with OC 100 or OC 110 controller while documentation is provided by the ACHAT OC programme communicating with the PC, and previously with the TD 100 thermal printer. Measuring heads will store in their memory up to 360 data sentences that can be represented graphically by the controller while it is also possible to measure through the glass or plastic door of the vessel thermostat directly on stirring platforms. The rate of biochemical oxidation of organic substances as the first-order reaction is proportionate to the residual concentration of yet unoxidised substances:

dy/dt = K (L — y) = KLz (1)

where:

L = total BOD y = BOD at time t Lz = residual BOD k, K = rate constants

By integrating from 0 to t of the above relation the following equation is obtained:

Lz = L. erK = L. 10-kt In general it applies for BOD at time t:

y = L (1 — 10-kt) (3)

where:

y = BOD at time t L = BODtotal

k = rate constant [24 hrs-1]

Used procedure is identical with the method of measurement recommended by the manufacturer in accordance with the Proposal for German Uniform Procedures DEV 46th Bulletin 2000 — H 55, also published in the instructions for BOD (on CD-ROM) of WTW Merck Company.

The decomposition of organic matter is the first-order reaction. In these reactions the reaction rate at any instant is proportionate to the concentration of a reactant (see the basic equation dy/dt). Constant k is the specific reaction rate or rate constant and indicates the instantaneous reaction rate at the unit concentration of a reactant. The actual reaction rate is continually variable and equals the product of the rate constant and the instantaneous concentration. The relation of the reaction product expressed by BOD at time t (y) to t is the same as the relation of the reactant (L — y) at time t and therefore the equations

(L — y) = L. e-kt (4)

and

y = L (1 — e-kt)

are analogical.

If in the graph the residual concentration of carbon is plotted on the y-axis in a logarithmic scale log (L — y) and the time in days from the beginning of experiment is plotted on the x — axis, we will obtain a straight line, the slope of which corresponds to the value — k/2.303.

The quantity of the labile fraction of organic matter can also be assessed by determination of soluble carbon compounds in hot water (Korschens et al 1990, Schulz 1990) and their quality by determination of the rate constant of their biochemical oxidation (Kolar et al. 2003, 2005a, b).

Hydrolytic methods are based on resistance of the organic matter different aggressive ways of hydrolysis that is realised at different temperature, time of action and concentration of hydrolytic agent, which is usually sulphuric acid. Among many variants of these methods the hydrolytic method according to Rovira et Vallejo (2000, 2002, 2007) in Shirato et Yokozawa (2006) modification was found to be the best. This method yields three fractions: labile LP1, semi-labile LP2 and stable LP3. The per cent ratio of these three fractions, the sum of which is total carbon of the sample Ctot, provides a very reliable picture of the degree of organic matter lability.

Of course, there are a lot of methods based on the study of organic matter biodegradability in anaerobic conditions. First of all, it is the international standard ISO CD 11734: Water quality — evaluation of the "ultimate" anaerobic biodegradability of organic compounds in digested sludge — Method by measurement of the biogas production, and particularly a very important paper using the Oxi Top Control measuring system manufactured by the German company MERCK for this purpose (Sussmuth et al. 1999).

Tests of methanogenic activity (Straka et al. 2003) and tests examining the activity of a microbial system (Zabranska et al. 1985a, b, 1987) are methods that can describe the degree of organic matter lability in its ultimate effect. Our long-time work experiences in the
evaluation of a huge amount of various analyses for the study of organic matter lability have brought about this substantial knowledge:

1. The study of the ratio of organic matter labile fractions, i. e. of their quantity, is always incomplete. A more authentic picture of the situation can be obtained only if information on the quality of this labile fraction is added to quantitative data. Such a qualitative characteristic is acquired in the easiest way by the study of reaction kinetics of the oxidation process of this fraction. The process of biochemical oxidation and the calculation of its rate constant KBio are always more accurate that the calculation of its rate constant of oxidation by chemical oxidants Kchem (Kolar et al. 2009a).

2. It applies to current substrates for biogas production in biogas plants that with some scarce exceptions the degree of organic matter lability is very similar in both aerobic and anaerobic conditions. In other words: organic matter is or is not easily degradable regardless of the conditions concerned (Kolar et al. 2006).

3. A comparison of various methods for determination of organic matter lability and its degradability in the anaerobic environment of biogas plant digesters and also for determination of digestate degradability after its application to the soil showed that hydrolytic methods are the best techniques. They are relatively expeditious, cheap, sample homogenisation and weighing are easy, and the results correlate very closely with methods determining the biodegradability of organic matter directly. E. g. with the exception of difficult weighing of a very small sample and mainly its homogenisation the Oxi Top Control Merck system is absolutely perfect and highly productive — it allows to measure in a comfortable way simultaneously up to 360 experimental treatments and to assess the results continually using the measuring heads of bottles with infrared transmitters, receiving controller and special ACHAT OC programme for processing on the PC including the graph construction. But its price is high, in the CR about 4 million Kc for the complex equipment. Hydrolytic methods require only a small amount of these costs and are quite satisfactory for practical operations (Kolar et al. 2008). However, for scientific purposes we should prefer the methods that determine anaerobic degradability of organic matter, designated by DC.

The substrate production of methane VCH4S [the volume of produced methane (VCH4c) after the subtraction of endogenous production of methane (VCH4e) by the inocula] was determined by an Oxi Top Control Merck measuring system.

The calculation is based on this equation of state:

n = p x V/RT (6)

where:

n = number of gas moles V = volume [ml]

P = pressure [hPa]

T = temperature [°K]

R = gas constant 8.134 J/mol °K

and the number of CO2 and CH4 moles in the gaseous phase of fermentation vessels is calculated:

where: p0 = initial pressure

Fermentation at 35° C and continuous agitation of vessels in a thermostat lasts for 60 days, the pressure range of measuring heads is 500 — 1 350 kPa and the time interval of measuring pressure changes is 4.5 min. Anaerobic fermentation is terminated by the injection of 1 ml of 19% HCl with a syringe through the rubber closure of the vessel to the substrate. As a result of acidification CO2 is displaced from the liquid phase of the fermentation vessel. The process is terminated after 4 hours. The number of CO2 moles is calculated from the liquid phase:

nCO2 l = {[p2 (Vg — VHCl) — pi x Vg]/RT} x 10-4 (9)

The injection of 1 ml of 30% KOH into the rubber container in the second tube of the fermentation vessel follows. The sorption of CO2 from the gaseous phase of the vessel is terminated after 24 hours and the total number of CO2 moles in gaseous and liquid phases is calculated from a drop in the pressure in the vessel:

ncO2 l, CO2 g = {[рз (Vg — VhCI — Vkoh) — P2 (Vg — VHCl)J’RT} x 10-4 (10)

where:

Ap = difference in pressures [hPa]

Vg = the volume of the gas space of the fermentation vessel [ml] p1 = gas pressure before HCl application [hPa] p2 = gas pressure before KOH application [hPa] p3 = gas pressure after KOH application [hPa]

R = gas constant = 8.134 J/mol °K T = absolute temperature = 273.15 + X °C VHCl = the volume of added HCl [ml]

VKOH = the volume of added KOH [ml]

Based on the results, it is easy to calculate the number of CO2 moles in the gaseous phase and by the subtraction from nCO2 g CH4 the number of moles of produced methane:

nCH4 = (nCO2 g CH4 + nCO2 l) — nCO2 l CO2 g (11)

The total number of moles of the gases of transported carbon:

nCO2 g CH4 + nCO2 l = ntotal (12)

Baumann’s solution A + B in deionised water of pH = 7.0 is used as a liquid medium (Sussmuth et al. 1999).

The standard addition of the inoculum corresponds roughly to an amount of 0.3% by volume (aqueous sludge from the anaerobic tank of the digester). Instead of Baumann’s solution it is possible to use a ready-made nutrient salt of the MERCK Company for this system.

The operation of the Oxi Top Control measuring system was described in detail by Sussmuth et al. (1999).

Methane yield was calculated from the substrate production of methane VcH4S by division by the initial quantity of the added substrate:

where:

Verne = methane yield of C-source

Verne = methane yield of the added inoculum

S = substrate quantity at the beginning [g]

Lord’s test and other methods suitable for few-element sets and based on the R range of parallel determinations were used for the mathematical and statistical evaluation of analytical results including the computation of the interval of reliability.

Anaerobic degradability is given by the equation:

e

Dc =-£- .100
e

where:

es = total C content in the sample

eg = C content in methane released during the measurement of anaerobic degradability The value of Cg is computed from the substrate production of methane VeH4S:

e = ^2 p vch4S
g RT

(because 1 mol CH4 contains 12 g C) where:

K = temperature (°K)

R = gas constant

P = pressure

VeH4S = the volume of produced methane after the subtraction of endogenous production by the inoculum from total production

This method, which determines organic matter lability in anaerobic conditions, is so exact that it allows to investigate e. g. the digestive tract of ruminants as an enzymatic bioreactor and to acquire information on its activity, on feed utilisation or digestibility and on the influence of various external factors on the digestion of these animals (Kolar et al 2010a) or to determine the share of particular animal species in the production of greenhouse gasses (Kolar et al 2009b).

At the end of this subchapter dealing with the degree of organic matter lability and its changes after fermentation in a biogas plant these experimental data are presented:

A mixture of pig slurry and primary (raw) sludge from the sedimentation stage of a municipal waste water treatment plant at a 1 : 1 volume ratio was treated in an experimental unit of anaerobic digestion operating as a simple periodically filled BATCH-system with mechanical agitation, heating tubes with circulating heated medium at a mesophilic temperature (40°C) and low organic load of the digester (2.2 kg org. dry matter/m3) and 28- day fermentation.

Acid hydrolysis of sludge, slurry and their mixture was done before and after anaerobic fermentation. The hydrolysis of samples was performed with the dry matter of examined sludge and its mixture with pig slurry including the liquid fraction after screening the material through a 250-pm mesh sieve. The method of hydrolysis according to Rovira and Vallejo (2000, 2002) as modified by Shirato and Yokozawa (2006): 300 mg of homogenised sample is hydrolysed with 20 ml of 2.5 M H2SO4 for 30 min at 105°C in a pyrex tube. The
hydrolysate is centrifuged and decanted, the residues are washed with 25 ml water and the wash water is added to the hydrolysate. This hydrolysate is used to determine Labile Pool I

(LP I).

The washed residue is dried at 60°C and hydrolysed with 2 ml of 13 M H2SC>4 overnight at room temperature and continuous shaking. Such an amount of water is added that the concentration of the acid will be 1 M, and the sample is hydrolysed for 3 hours at 105°C at intermittent shaking. The hydrolysate is isolated by centrifugation and decantation, the residue is washed again with 25 ml of water and the wash water is added to the hydrolysate. This hydrolysate is used for the determination of Labile Pool II (LP II). The residue from this hydrolysis is dried at 60°C and Recalcitrant Pool (RP) is determined from this fraction.

Ctot is determined in all three fractions.

Degradability of organic matter of the test materials was studied by modified methods of Leblanc et al. (2006) used to examine the decomposition of green mulch from Inga samanensis and Inga edulis leaves. These authors conducted their study in outdoor conditions (average annual temperature 25.1°C) and we had to modify their method in the cold climate of this country. At first, the liquid phase of sludge, slurry and mixture was separated by centrifugation; the solid phase was washed with hot water several times and separated from the solid phase again. By this procedure we tried to separate the solid phase from the liquid one, which contains water-soluble organic substances and mineral nutrients. Solid phases of tested organic materials were mixed with sandy-loamy Cambisol at a 3:1 weight ratio to provide for inoculation with soil microorganisms and volume ventilation of samples with air. After wetting to 50% of water retention capacity the mixtures at an amount of 50 g were put onto flat PE dishes 25 x 25 cm in size. The material was spread across the surface of the dish. Cultivation was run in a wet thermostat at 25°C, and in the period of 2 — 20 weeks dishes were sampled in 14-day intervals as subsamples from each of the four experimental treatments. The agrochemical analysis of the used topsoil proved that the content of available nutrients P, K, Ca and Mg according to MEHLICH III is in the category "high" and pKKCl = 6.3. After drying at 60°C for 72 hours the content of lipids, crude protein, hemicelluloses, cellulose, lignin, total nitrogen and hot-water-insoluble dry matter was determined in the dish contents.

After twenty weeks of incubation organic substances were determined in the dish contents by fractionation into 4 degrees of lability according to Chan et al. (2001).

The content of hemicelluloses was calculated from a difference between the values of neutral detergent fibre (NDF) and acid detergent fibre (ADF), lignin was calculated from ADF by subtracting the result after lignin oxidation with KMnO4. Because ADF contains lignin, cellulose and mineral fraction, it was possible to determine the cellulose content by ashing the residue in a muffle furnace and by determination of mineral fraction. These methods were described by Van Soest (1963), modifications used by Columbian authors (Leblanc et al. 2006) were reported by Lopez et al. (1992).

Ion exchange capacity [mmol. chem. eq./kg] was determined in dry matter of the examined materials according to Gillman (1979), buffering capacity was determined in samples induced into the H+-cycle with HCl diluted with water at 1 : 1 and washed with water until the reaction to Cl — disappears. In the medium of 0.2 M KCl the samples were titrated to pH = 7 with 0.1 M NaCH and buffering capacity was calculated from its consumption.

Tab. 1 shows the analyses of a mixture of pig slurry and primary sludge used in the experiment. Cbviously, compared to the values reported in literature our experimental materials had a somewhat lower content of organic substances in dry matter, and perhaps this is the reason why anaerobic fermentation reduced the content of organic substances by 39% only although the usual reduction by 45 — 65% for primary sludge was expected as reported in literature (Pitter 1981) and by 40 — 50% for pig slurry (StehHk 1988). As a result of the organic dry matter reduction the content of nutrients in sludge after anaerobic fermentation is higher, nitrogen content is lower by about 20%. In this process organic nitrogen is converted to (NH^)2CO3, which partly decomposes into NH3 + H2O + CO2 and partly passes into the sludge liquor. Roschke (2003) reported that up to 70% of total nitrogen might pass to the ammonium form at 54% degradation of organic substances of dry matter. Even though concentrations of the other nutrients in dry matter of the aerobically stabilised sludge increased as a result of the organic dry matter reduction, their content in the sludge liquor also increased (Tab. 2).

Taking into account that the amount of water-soluble nutrients in the sludge liquor and organic forms of N and P dispersed in the sludge liquor in the form of colloid sol (but it is a very low amount) is related not only to the composition of the substrate but also to technological conditions of anaerobic digestion, digester load and operating temperature, it is evident that the liquid fraction of anaerobically stabilised sludge contains a certain amount of mineral nutrients, approximately 1 kg N/m3, besides the others, although differences in the concentration of P and K in the liquid fraction before and after fermentation are generally negligible. It is a very low amount, and there arises a question whether the influence of the liquid fraction on vegetation is given by the effect of nutrients or water itself, particularly in drier conditions.

After anaerobic digestion the solid phase of sludge still contains a high amount of proteins and other sources of organic nitrogen that could be a potential pool of mineral nitrogen if the degradation of sludge after fermentation in soil is satisfactory.

The results of hydrolysis in Tab. 3 prove that pig slurry has 59% of its total carbon in LP I, which indicates great lability, corresponding to the hydrolysability of cereals and grasses according to Shirato and Yokozawa (2006). Primary sewage sludge is still better from this aspect, having almost 70% C in LP I. The degree of lability of the sludge and slurry mixture is relatively high and corresponds to the component ratio. After methanisation carbon content in LP I of the sludge and slurry mixture decreases to less than a third of the original amount and carbon of non-hydrolysable matters increases even almost four times in the RP fraction. The sum of LP I and LP II, i. e. the labile, degradable fraction of carbon compounds of the sludge and pig slurry mixture, was reduced by anaerobic digestion from 83% to 34%, that means approximately by 50%. These are enormous differences and they prove that mainly very labile organic substances are heavily destroyed by the anaerobic process even though a reduction in the content of organic substances during anaerobic fermentation is lower (by 39% in our experiment).

Tab. 4 shows the analysis of raw materials (sludge and pig slurry) and their mixture before and after anaerobic fermentation while Tab. 5 shows the analysis of their liquid fraction. The same results (Tab. 4) are provided by the incubation of the solid phase of sludge, pig slurry and their mixture before and after anaerobic fermentation when incubated with soil at 25°C and by the contents of lipids, crude protein, hemicelluloses, cellulose, lignin, total nitrogen and hot-water-insoluble dry matter; the same explicit conclusion can be drawn from the results of the fractionation of organic matter lability of the experimental treatments after 20- week incubation with soil according to Chan et al. (2001) shown in Tab. 5. A comparison of the results in Tab. 3 and 5 indicates that as a result of the activity of microorganisms of the added soil in incubation hardly hydrolysable organic matter was also degraded — differences between the most stable fractions F 3 and F 4 in Tab. 5 are larger by about 73% after anaerobic fermentation while in the course of acid chemical hydrolysis the content of non-hydrolysable fraction was worsened by anaerobic fermentation because it increased by about 290%. But it is a matter of fact that the soil microorganisms are not able to stimulate the anaerobically fermented sludge to degradation as proved by more than % of total carbon in fraction 4.

The table results document that 20-week incubation decreased more or less the per cent content of examined organic substances except lignin (total N 5 — 8%, cellulose 17 — 25%, hemicellulose 13 — 22%, proteins 9 — 12%, lipids 4 — 7%, and the content of hot-water — insoluble dry matter by 10 — 15%) factually in all experimental treatments except the treatment of the anaerobically fermented mixture of primary sludge and pig slurry where a reduction in these matters is low or nil. Hence, primary sludge, pig slurry and their mixture can be considered as organic fertilisers but only before anaerobic fermentation. We recorded a substantially lower degree of degradation of selected organic substances in sludge, pig slurry and their mixture during incubation with 25% of sandy-loamy soil (5 — 25%) than did Leblanc et al. (2006) with phytomass of Inga samanensis and Inga edulis leaves, who reported about 50% degradation of total mass, hemicelluloses and nitrogen in mass. We are convinced that it is caused by a very different content of hemicelluloses in our materials compared to the materials used by the above-mentioned authors. No easily degradable hemicelluloses are present in sewage sludge or in pig slurry any longer, and obviously, only more stable forms pass through the digestive tracts of animals and humans. It is also interesting that after anaerobic fermentation and after 20-week aerobic cultivation at 25°C only the compounds (lipids + proteins + hemicelluloses in mixture II D account roughly for 11%) that could be considered as labile remained in the mixture of slurry and sludge. These are apparently their more stable forms as confirmed by the results in Tab. 5 which illustrate that to approximately 11% of organic carbon compounds it is necessary to add the % proportions of the first and second fraction on the basis of oxidisability according to Chan et al. (2001). Literary sources report that the sum of lipids, proteins and hemicelluloses in the anaerobically stabilised sludge from municipal waste water treatment plants amounts to 13% — 39.6% of dry matter, so it is quite a general phenomenon.

The ion exchange capacity of sludge, pig slurry and their mixture before fermentation, before incubation and after incubation is very low and does not reach the values that are typical of sandy soil. It is increased by anaerobic fermentation along with incubation markedly but practically little significantly to the level typical of medium-textured soils. The same relations were observed for buffering capacity, which is not surprising. The results document that degradability of the organic part of anaerobically stabilised sludge worsened substantially and that it cannot be improved very markedly by the use of soil microorganisms and soil.

We have to draw a surprising conclusion that sludge as a waste from the processes of anaerobic digestion is a mineral rather than organic fertiliser and that from the aspect of its use as organic fertiliser it is a material of much lower quality than the original materials. We cannot speak about any improvement of the organic material by anaerobic digestion at all. Their liquid phase, rather than the solid one, can be considered as a fertiliser. If it is taken as a fertiliser in general terms, we do not protest because besides the slightly higher content of mineral nutrients available to plants (mostly nitrogen) it has the higher ion exchange capacity and higher buffering capacity than the material before anaerobic fermentation, but this increase is practically little significant.

The use of membranes for gas cleaning is a well established technology in chemical industries. The membrane is a porous material that let some gases permeate through its structure. Employing an adequate material, it is possible to have selectivity between the gases of the mixture to be separated. For this particular application, two different streams are obtained: a permeate gas (mainly CO2, water and ammonia) and the retentate (concentrated CH4). The most commonly employed materials are hollow fibres made of different polymers. Several companies provide this technology, being Air Liquide the largest company in this area (Air Liquide, 2011). In their process, the biogas is compressed to 16 bars and then routed to a two-stage membrane process where methane with purity higher than 90% can be obtained. To upgrade CH4 to a higher purity, a PSA process can be used in series.

Bioethanol is produced from simple sugar-rich raw materials or from starchy feedstock, from which simple sugars can be easily processed and released, which are fermented to produce ethanol. Bioethanol production comprises three steps. Firstly, the complex sugars are hydrolysed to release glucose. Subsequently, the glucose is subjected to a second fermentation step carried out by yeasts such as Saccharomyces cerevisiae; for example, yielding ethanol and carbon dioxide. The third step consists of a thermochemical process and is based on the distillation of the diluted ethanol to obtain highly concentrated ethanol. When using lignocellulosic raw materials such as agricultural residues (corn stover, straw, sugar cane bagasse), forestry waste, wastepaper and other cellulosic residues, a chemical or enzymatic hydrolysis pretreatment to degrade the lignin is needed. This additional step reduces the efficiency of the process. Some improvements have been achieved by the engineering of cellulases from the Trichoderma genus fungi (Fukuda et al., 2006) and the utilization of microorganisms able to simultaneously express the cellulase and enzymes needed for the ethanol fermentation pathway, such as piruvate descarboxilases and alcohol dehydrogenases (Lu et al., 2006; van Zyl et al., 2007; Jegannathan et al., 2009; Rahman et al., 2009; van Dam et al., 2009). However, these improvements have still not generated an efficient and economically affordable process.

With regard to biodiesel, it consists of a mixture of fatty acid alkyl esters (FAAE) obtained by the transesterification of fatty acids and straight chain alcohols (generally ethanol or methanol), mainly from vegetable oils. When methanol is the alcohol of choice, the term used to refer to the biodiesel is fatty acid methyl esters (FAME), while the ethanol-derived biodiesel is known as fatty acid ethyl esters (FAEE). The properties of the biodiesel obtained from ethanol or methanol are very similar, but methanol is the preferred alcohol in spite of its toxicity and fossil fuel origin because of its low cost and wide availability (Ranganathan et al., 2008; Fjerbaek et al., 2009).

The commercially delivered biodiesel is mainly obtained by the chemical transesterification of the triglycerides contained in sunflower, rapeseed or palm oil. This process can be carried out by acid and alkaline liquid catalysts (Kawahara & Ono, 1979; Jeromin et al., 1987; Aksoy et al., 1988; Fukuda et al., 2001), or heterogeneous solid catalysts such as supported metals, basic oxides or zeolites (Cao et al., 2008). The preferred catalysts are the liquid ones, particularly the alkaline ones, because these catalysts are cheap, very versatile and yield less corrosive fuel than the acid catalysts. Also, liquid catalysts are preferred because the reusable solid catalysts are still withdrawn with mass transfer and reactant diffusion problems. However, the alkaline catalysis has several limitations, especially the futile consumption of the catalyst, problems of viscosity, mass transfer and recovery of biodiesel and by-products owing to the saponification of the catalyst and free fatty acids in the presence of water (Freedman et al., 1984; Zhang et al., 2003; Jaruwat et al., 2010). These problems are bypassed by high temperature reaction conditions, addition of organic solvents to manage the water presence or enhance the interface formation, or increase of the alcohol:catalysts ratio (Kawahara & Ono, 1979; Fukuda et al., 2001). Thus, the process requires high energy inputs to maintain high temperatures conducive to viable

transesterification rates, and to separate methanol. Besides, the process generates alkaline waste water that requires treatment prior to its disposal (Jaruwat et al., 2010). Jointly, all these negative factors raise doubts about the sustainability and environmental benefits of the biodiesel industry.

Saccharomyces cerevisiae (baker’s yeast) has been used for industrial ethanol production from hexoses (C6 sugars) for a thousand years. However, a significant amount of pentoses (C5 sugars) derived from the hemicellulose portion of the lignocellulosic biomass is present in the hydrolysate from the pretreatment process. Modern biotechnologies enable fermenting microorganisms to use both C5 and C6 sugars available from the hydrolysate. This further increases the economic competitiveness of ethanol production and other bio-products from cellulosic biomass.

Recently, microorganisms for cellulosic ethanol production, such as Saccharomyces cerevisiae, Zymomonas mobilis and Escherichia coli, have been genetically engineered using metabolic engineering approaches. Lau et al. (2010) compared the fermentation performance of Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST) and Zymomonas mobilis AX101 for cellulosic ethanol production. Three microorganisms resulted in a metabolic yield, final concentration and rate greater than 0.42 g/ g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. They concluded that Saccharomyces cerevisiae 424A(LNH-ST) is the most promising strain for industrial production because of its ability to ferment both glucose and xylose.

Vasan et al (2011) introduced an Enterobacter cloacae cellulase gene into Zymomonas mobilis strain and 0.134 filter paper activity unit (FPU)/ml units of cellulase activity was observed with the recombinant bacterium. When using carboxymethyl cellulose and 4% NaOH pretreated bagasse as substrates, the recombinant strain produced 5.5% and 4% (V/V) ethanol respectively, which was three times higher than the amount obtained with the original E. cloacae isolate.

In 2010, Purde University scientists improved a strain of yeast that can produce more biofuel from cellulosic plant material by fermenting all five types of the plant’s sugars: galactose, manose, glucose, xylose and arabinose. Arabinose makes up about 10 percent of the sugars contained in cellulosic biomass (Casey et al., 2010).

The term biofuel refers to liquid, gas and solid fuels that are predominantly produced from biomass. The production of biofuels may ignite concerns about security, the environment, trading, and socioeconomic issues related to the rural sector. Biofuels include bioethanol, biomethanol, vegetable oils, biodiesel, biogas, bio-synthetic gas (bio-syngas), bio-oil, bio­char (charcoal created by biomass pyrolysis), Fischer-Tropsch liquids and biohydrogen. Biogas and bio-oil are primary products, the preliminary processing of which is almost reduced to collecting the raw material. Most traditional biofuels (such as ethanol from corn, wheat, or sugar beets and biodiesel from oil seeds) are produced from classic agricultural food crops that require high-quality agricultural land for growth. The biofuel economy will grow rapidly during the 21st century (Demirbas, 2008a).

The processing of the rapeseed to obtain SVO to be used as engine fuel is made through three mechanical steps: cleaning of seed, pressing and purification (see Fig. 2). The first step consists of cleaning the seeds from stones, metal pieces and straw. In this process it is very important to reduce the risk of damaging the press.

The second step is a cold pressing of the oil seed with the screw press to obtain oil. This step must be done carefully to reduce the incorporation of undesirable materials from the solid by-product (rapeseed cake) The pressing process influences the content of phosphorus, calcium and magnesium as well as the content and dimension of the particles. The variability of those elements depends on the speed and the pressing temperature. A low speed (low throughput) increases the oil yield and the content of particles. A high speed

(high throughput), produces the opposite effect, decreasing the oil yield and also the particles. It is possible to find an optimal compromise according to the necessities of production and capacity of filtering. The oil yield should be between 32-36% of rapeseed mass, due to the amount of undesirable particles obtained in the oil if the pressure is too high or if a second pressing is done (Ferchau, 2000).

As a final step, purification of raw oil obtained from the press is needed. It is recommended to use a press filter and to perform a security filtration after a decantation. A general filtration procedure must be done after decantation in order to remove the suspended particles from the oil. Usually a pressure filter is used, either a chamber filter or a vertical one. As a final step, a security filtration of a defined pore size (between 1 and 5 pm) is recommended to remove the finest particles that still remain in the oil. In this step is very important to pass the quality control exposed in section 4.5. After this final step and after complying with the quality control, the oil is prepared for combustion in a modified diesel engine.

The cake meal and the filter cake obtained in the process to obtain SVO both have a high content of protein and are suitable for being incorporated as part of animal fodder There is a variation of this process to extract more oil from the seed using a solvent. The abovementioned process is the first step. About 70% of oil from the seed is extracted, leaving 30% in cake meal. The next stage is a process of extraction using hexane as solvent. It reaches up to 95% extraction of the seed oil. In this stage, a solvent (hexane) is mixed with rapeseed cake. The solvent dissolves the oil remaining in the rapeseed cake. After its evaporation, the solvent is recovered for its use. The outline of the process is shown in Figure 3. In case of hexane extraction, the cake meal obtained has less protein than when just pressing the seed. Even though, there is no problem to use it as animal food.

Another promising lipids source, still not implemented but currently being studied worldwide, is represented by microalgae. Microalgae have a high potential as biodiesel precursors because many of them are very rich in oils, sometimes with oil contents over 80% of their dry weight, although not all species are suitable as biodiesel production oils (Chisti, 2008; Manzanera, 2011). Besides, these microorganisms are able to double their biomass in less than 24 hours, achieving a reduction between 49 and 132 fold in the medium culture time required by a rapeseed or soybean field. Furthermore, microalgae cultures require low maintenance and can grow in wastewaters, non-potable water or water unsuitable for agriculture, as well as in seawater (Mata et al., 2010). The production of microalgae biodiesel could be combined with the CO2 removal from power generation facilities (Benemann, 1997), the treatment of waste water from which microalgae would remove NH4+, NO3- and PO43- (Aslan & Kapdan, 2006), or the synthesis of several valuable products, from bioethanol or biohydrogen to organic chemicals and food supplements (Banerjee et al., 2002; Chisti, 2007; Rupprecht, 2009; Harun et al., 2010). However, microalgae biomass-based biofuels have several problems ranging from the optimization of high density and large surface units of production to the location of the microalgae production unit. Anyway, the main decisions to take are the adoption of open or closed systems, and the election of batch or continuous operation mode. As will be discussed below, depending on the system and mode of operation choice, there will be different advantages and drawbacks.

The term »reforming» was originally used to describe the thermal conversion of petroleum fractions to more volatile products with higher octane numbers, and represented the total effect of many simultaneous reactions such as cracking, dehydrogenation and isomerisation (Yaman, 2004). Reforming also refers to the conversion of hydrocarbon gases and vaporized organic compounds to hydrogen containing gases such as synthesis gas, which is a mixture of carbon monoxide and hydrogen. Synthesis gas can be produced from natural gas, for example, by such processes as reforming in the presence of steam (steam reforming) (Klass, 1998).

Fast pyrolysis of biomass followed by catalytic steam reforming and shift conversion of specific fractions to obtain H2 from bio-oil was presented as an effective way to upgrade biomass pyrolysis oils. Production of hydrogen from reforming bio-oil was investigated by NREL extensively, including the reactions in a fixed bed and a fluidized bed (Wang et al., 1997,1998; Czernik et al., 2007). Commercial nickel catalysts showed good activity in processing biomass derived liquids (Ekaterini & Lemonidou, 2008).