In order to estimate an optimal fermentation time under ultrasonic exposure in this study, parameters such as ethanol concentration, ethanol volumetric productivity, ethanol yield and lactose consumption were investigated.

The maximum values of ethanol concentration and lactose consumption were achieved when the HRT was 36 h. Under the HRT of 36 h in the ultrasound-assisted fermentation, the average ethanol concentration of 26.30 g L-1, ethanol yield of 0.532 g g-1 lactose and lactose consumption of 98,9% were obtained (Fig. 9-11). Using S. cerevisiae without ultrasound exposure gave the results as 23,60 g L-1, 0.511 g g-1, 92,4%, respectively and the differences were statistically significant (p<0.05). Shortening the HRT to 24 h allowed remaining high ethanol yield of 0.520 g g-1 with sonicated S. cerevisiae, but in the control fermentation unit it was as low as 0.487 g g-1 (p<0.05). When the HRT was 12 h the ethanol yields were 0.318 and 0.365 g g-1 depending on using ultrasounds device (Fig. 11). From the economic viewpoint, shortening the fermentation time (HRT) could reduce costs of industrial ethanol production. The study showed that there is no need to extend the HRT over 36 h or more, because most of the lactose was converted into ethanol during 24 h (95.6% in the ultrasound-assisted fermentation. Nikolic et al. (2010) stated that optimal fermentation time for free and immobilized S. cerevisiae was 38 h. Ozmihci & Kargi (2008) studied ethanol production from cheese whey powder (CWP) solution containing 50 g sugar L-1 at six different HRTs varying between 17.6 and 64.4 h by Kluyveromyces marxianus strains. Percent sugar utilization,

12 24 36

HRT (h)

9. Effects of HRT and ultrasound irradiation on the ethanol concentration

HRT (h)

effluent ethanol concentration and ethanol yield increased with increasing HRT from 17.6 to 50 h. Further increasing in HRT to 64.4 h resulted in decrease of the analyzed parameters. Moreover, the time for fermentation decreased at higher initial substrate concentration (Guimaraes et al., 2008a; Nikolic et al., 2010; Ozmihci & Kargi, 2008). According to Guimaraes et al. (2008a) the fermentations with 50-150 g lactose L-1 reached completion in about the same time of 27 h but the maximum ethanol concentration increased linearly with increasing initial lactose concentration from 6.5 g ethanol L-1 with 20 g lactose L-1 to 57 g L-1 with 200 g L-1. They also stated that increasing lactose concentration led to incomplete fermentation and impair the fermentation due to nutrient limitation.

Interestingly, the volumetric productivities of ethanol decreased at longer HRT (Table 3). Maximum productivity of ethanol of 1.060 g L-1 h-1 was observed under the HRT of 12 h when the culture has been sonicated and 0.908 g L-1 h-1 under the HRT of 24 h in the fermentation process without ultrasound irradiation (p<0.05). The volumetric ethanol productivity in the ultrasound-assisted fermentation obtained in this work was higher than that reported for batch or fed-batch fermentations with S. cerevisiae strains: 0.3 g L-1 h-1 (Rubio-Texeira et al., 1998), 0.46 g L-1 h-1 (Guimaraes et al., 2008b), 0.14 — 0.6 g L-1 h-1 (Ramakrishnan & Hartley, 1993), 1 g L-1 h-1 (Compagno et al., 1995). Ozmihci & Kargi (2007) using Kluyveromyces marxianus to ferment concentrated cheese whey powder solution obtained higher volumetric ethanol productivity over 2 g L-1 h-1, but after 120 h fermentation.

1.2.6 Effect of beating degree and rosin on wet tensile strength

strength decreased with the increase of rosin; when the beating degree was higher than 0 level, wet tensile strength increased with the increase of beating degree and rosin, the maximum value occurred when beating degree was held at 30SR°, and rosin was held at 0.4%. This is because added rosin impacted adsorption effect of fibre to wet strength agents, wet tensile strength decreased, but with the continuing increase of beating degree, the fibre sub-wire broom degree further enhanced, the adsorption effect of fibre on wet strength agent was over than the rosin.

3.4.2 Effect of grammage and wet strength agent on wet tensile strength

of the grammage and wet strength agent; the maximum occurred when wet strength agent was held at 3%, and grammage was held at 110 g/ m2, this is because the number of fibre increased and bonding effect of fibre enhanced, when wet tensile strength increased with the increase of grammage, at the same time, wet strength agent provided cationic charge, fibre strongly adsorbed wet strength agent added because of pulp fibre with anionic charge, so that the wet tensile strength of film increased.

In a multiple response question, respondents were asked to mention the constraining factors towards biogas use adoption. The main factors mentioned were that the installation cost was too high (95.8%) and lack of credit facility (95%). Other reasons were lack of expertise (91.7%) and inadequate water (60%) to run the plants. Only a small proportion of 3.3% out of the 120 respondents said they do not need the facility. This may suggest that if the access to biogas is facilitated either through subsidy or access to credits many households in the district could adopt the technology. A comparison across the categories suggest that

respondents from the poor and less poor were more in demand for credits and also mentioned the facility to be too costly (Table 6), suggesting a different kind of approach to induce them adopt the technology.

The term liquid gas (Liquefied Petroleum Gas[22] it refers to C3 and C4 hydrocarbons or mixtures thereof. It is generated as a by-product in petroleum refining and as an associated gas from the extraction of oil and natural gas. LPG is gaseous at room temperature under atmospheric conditions, but can be liquefied at low pressures. In liquid form, its specific volume is about 260 times smaller than in the gaseous state. Therefore, large amounts of energy can be transported and stored in relatively small containers.

The transportation of LPG is carried out worldwide by tanker ships, barges, pipelines, by rail tank cars, road tankers or in liquefied gas cylinders. LPG is stored in stationary tank facilities or in gas cylinders. Up to a tank size of 2.9 t capacity, the above-ground installation does not require a permit. From a tank capacity of 2.9 tonnes, the federal emission regulations need to be considered when granting a permit. The technical conditions for setting up tank installations are defined in TRB 801 No.25 "LPG storage tank facilities".

Commercial LPG consists of at least 95 percent by mass of propane and propene, whereby the propane content must predominate. The remainder may consist of ethane (C2H6), ethene (C2H4), butane (C4H10) and butene (C4H8) isomers. The classification for commercial propene, butane and butene is equivalent. Note also the degree of purity according to DIN 51 622 [DIN 1985]: Data on sulphur or sulphur compounds are listed here.

In DIN 51624 "automotive fuels — natural gas requirements and test methods" [8-15]upper limits for the propane/butane mole fractions in natural gas of 6% / 2% in the total mixture and a methane number > 70 are required. EASEE-gas CBP (EASEE, 2005) specifies a hydrocarbon dew point of -2 ° C at 1-70 bar. For the calculations shown below, a typical LPG composition of propane / butane, 95 / 5 is used.

The political barriers that exist are mainly in the area of sovereignty rights and the will to initiate national biogas technology programmes. Another problem is the high number of armed conflicts and political instability in the continent which together with the region’s debt burden have reduced the region’s credibility. Hence, providing capital even for modest investments will prove difficult. African governments need to commit themselves to renewable energy programmes. Government constant commitments to the development and promotion of renewable energy sources have been instrumental in promoting an ambitious alcohol fuel in Brazil, biogas programmes in Europe, China and India. It could be helpful to learn from the experiences gained in the developed world but adapted to the needs and situation in developing countries. However, in some African countries, the hostile social climate and political instability prevent opportunities of international collaboration and support.

The anaerobic reactor was initially charged with 300 mL of distilled water, 500 mL of the inoculum and 200 mL of a nutrient-trace element solution. The composition of this nutrient — trace element solution is given in detail elsewhere (Borja et al., 2001).

The start-up of the reactor involved stepped increases in COD loading using an influent substrate concentration of 17.2 g COD/L. During this period the organic loading rate (OLR) was gradually increased from 0.25 to 0.50 g COD/ (L d) between 1 and 15 d, 0.75 g COD/ (L d) between 16 and 30 d, 1.00 g COD/(L d) between 31 and 45 d and finally 1.25 g COD/(L d) between 46 and 60 d.

After the preliminary step, the reactor was fed in series of semicontinuous experiments using OLRs of 0.9, 1.2, 1.4, 1.7, 2.1, 2.8, 3.5, 4.1 L COD/(L d) for the OWSW1, which correspond to hydraulic retention times (HRTs) of 40.0, 28.6, 25.0, 20.0, 16.6, 12.5, 10.0 and 8.3 d, respectively. After these experiments with OMSW 1 five different OLRs were assessed for the OMSW 2, 3.0, 6.0, 9.05, 12.0 and 15.0 g COD/ (L d), these OLRs corresponded to HRTs of 50.0, 25.0, 16.6, 12.5 and 10.0 d, respectively.

Once steady-state conditions were achieved at each feed flow-rate, the daily volume of methane produced, and total and soluble COD, pH, total volatile fatty acids (TVFA) and volatile solids (VS) of the different effluents were determined. The samples were collected and analysed for at least 5 consecutive days. The steady-state value of a given parameter was taken as the average of these consecutive measurements for that parameter when the deviations between the observed values were less than 3% in all cases. Each experiment had a duration of 2-3 times the corresponding HRT.

The organic loadings applied in this work were increased in a stepwise fashion in order to minimise the transient impact on the reactor that might be induced by a sudden increase in loadings.

1.3 Gas parameters, gas quality figures (G 260, G 685)

The gas used in the networks for the final customer has to fulfill quality and composition requirements. According to the standard defined by DVGW G 260 working sheet two main types of natural gas (gas families) are distinguished which stem from different sources and production locations:

• H-Gas, high calorific value (Russian source, typically)

• L-Gas, low calorific value (North Sea source, mainly)

The calorific value is generally used for the billing, as the final consumer/customer must receive his bill with the energy value included, meaning in the unit of kWh in a period (i. e. a year, a month). The energy value yields from multiplication of accumulated flow and calorific value, e. g. 3000 m3/a * 10.1 kWh/m3 equal to 30300 kWh/a.

Nora Niemetz1, Karl-Heinz Kettl1, Manfred Szerencsits2 and Michael Narodoslawsky1

1Graz University of Technology, 2Okocluster, Austria

1. Introduction

Biogas production is discussed controversially, because biogas plants with substantial production capacity and considerable demand for feedstock were built in recent years. As a consequence, in most cases corn becomes the dominating crop in the surrounding and the competition on arable land is intensified. Therefore biogas production is blamed to raise environmental risks (e. g. erosion, nitrate leaching, etc.). Furthermore it is still discussed, that a significant increase of biogas production could threaten the security of food supply. The way out of this dilemma is simply straight forward but also challenging: to use preferably biogenous feedstock for biogas production which is not in competition with food or feed production (e. g. intercrops, manure, feedstock from unused grassland, agro-wastes, etc.). However, the use of intercrops for biogas production is not that attractive since current biogas technology from harvest up to the digestion is optimized for corn. Additionally current reimbursement schemes do neither take the physiological advantages and higher competitiveness of corn into account nor compensate lower yield potentials of intercrops which are growing in late summer or early spring. Higher feed-in tariffs for biogas from intercrop feedstock, as they are provided for the use of manure in smaller biogas systems, would not only be justified, as shown below, but also stimulating. Beyond that, the plant species used as intercrops as well as the agronomic measures and machinery used for their growing seem to provide lots of opportunities for optimization to increase achievable yields. Moreover, adaptations of biogas production systems, as discussed in this chapter, facilitate biogas production from intercrops.

Further advantages of intercrops growing are that they contribute to a better soil quality as well as humus content and reduce the risk of nitrous oxide emissions. Simultaneously intercrops allow a decrease of the amount of chemical fertilizer input, because the risk of nitrate leaching is reduced and if leguminosae are integrated in intercrop-mixtures, atmospheric nitrogen is fixed. This is important, because conventional agriculture for food and feed production utilizes considerable amounts of mineral fertilizers. Due to the fact that the production of mineral nitrogen fertilizers is based on fossil resources, it makes economically and ecologically sense to reduce the fertilizers demand.

In the case study, a spa town in Upper Austria, the set-up of the supply chain is seen as key parameter. An important issue in this case are more decentralized networks for biogas production. This can be achieved e. g. with several separated decentralized biogas fermenters which are linked by biogas pipelines to a centralized combined heat and power plant.

Genetic engineering is one of the methods for improvement of activity in hydrogen generation by microorganisms. Although the yields of generated hydrogen can be performed by optimization of the reaction conditions, genetic modifications seems to be the appropriate solution at the moment. The main idea of modification rely on implantation of other genes into the bacteria strains containing hydrogenase.

decomposition of formic acid in presence of formate-hydrogen liaze (FHL) representing the set of enzymes localized in the inner cell membrane. Hydrogenase 3 coded as hycA and formate dehydrogenase known as fdhF are the main components of the FHL. The presence of hycA gene limits the synthesis of fhlA, responsible for better activity of FHL towards hydrogen. Therefore the removal of hycA increases the fhlA gene expression and in consequence hydrogen production by 5-10%. (Hallenback, 2009). The research of the FHL genes expression were performed by Bisaillon et al. and other authors (Bisaillon, 2006, Turcot, 2008, Penfold, 2003) and they found almost two times higher rate of hydrogen generation for modified strain of E. coli HD701. Genes responsible for nickel-iron hydrogenases (hydrogenase I and II) coded by hya and hyb operons were found in the E. coli genom as well. It was found that elimination of these enzymes by genetic modification can result with almost 35% higher production of hydrogen (Hallenback, 2009, Bisaillon, 2006, Turcot, 2008). Other profits originating from genetic engineering are related to deactivation of enzymes responsible for transformations of glucose into lactic, succinic and fumaric acids. The removal of ldhA (lactic acid) and frdBC (succinic and fumaric acids) genes results in increase of hydrogen formation. The 1.4 fold higher amount of hydrogen were found by Yoshida et al. (Yoshida, 2006) in this situation. The new mutant strain of SR 15 can produce 1.82 mol H2/ mol glucose what is close to the theoretical value (2 mol H2/mol glucose). Studies performed by Maeda et al. (Maeda, 2007) showed that bacteria BW2513 with seven modified genes (hyaB, hybC, hycA, fdoG, frdC, ldhA and aceE) generate 4.6 fold more hydrogen than wild-type strain.

The nitrogenase and uptake hydrogenase play an important role in the photofermentation process of hydrogen generation by PNS bacteria. The engineering of the mutants free of uptake hydrogenase is the basic task of gene modifications. Genes coding hydrogenase (hup) can be modified by resistance gene insertion into the hup genes or by deletion of hup genes (Kars, 2009, Kars, 2008, Kim, 2006). Appropriately modified Rhodobacter spheroids can generate hydrogen also in the absence of light (Kim, 2008).

Production of polyhydroxybutyrate (PHB) accompany hydrogen generation by PNS bacteria what applies the excess of reducing equivalents in other metabolic pathway. The PHB is the storage material stored in cytoplasm. This compound is formed in the environment rich in carbon compounds but lean in nitrogen (Kemavongse, 2007). The PHB is unwanted competition product accompanying hydrogen generation. The removal of genes responsible for formation of PHB syntase effectively stops generation of the polymer (Kim, 2006). Low activity in PHB formation not always results in an increase of hydrogen yield. Whereas in presence of lactate, malate or malate the amount of photogenerated hydrogen is not influenced by PHB (Hustede, 1993) the presence of acetate can increase photofermentation towards hydrogen. However, the importance of PHB as biodegradable polymer significantly increased in recent years. Therefore, simultaneous photogeneration of hydrogen and PHB gained economic dimension (Yigit, 1999).

There are genetic modifications influencing changes in the amount of LHC (light harvesting complexes). The reduction of pigment present in LHC diminish the self-shadow effect and therefore better access of light into deeper located cells. The decrease of amount of LH1 (Vasilyeva, 1999) complexes with maximum of absorption at 875 nm or those with absorption maximum at 800 and 850 nm (LH2) (Kim, 2006) can increase the amount of photo generated hydrogen. Genetic manipulations cannot lead to total elimination of the pigments (Kim, 2006).

The negative influence of ammonium ions on nitrogenase is well recognized. Therefore, genetic modifications of nonsensitive to NH+ ions should be the subject for considerations. Among many methods reducing the role of ammonium ions in photofermentation is blockage of Calvin cycle via mutation of genes coding the RuBisCO enzyme. This way the excess electron stream is directed to nitrogenase even in the presence of NH4+ ions. Another modification can be achieved by disruption of proteins transporting NH4+ ions through cytoplasmic membrane. Strains of this type ( e. g. Rhodobacter capsulatus) loose their ability to regulate nitrogenase in presence of ammonium ions. (Qian, 1996). Such modifications allow to perform photofermentation even in the presence of molecular nitrogen. Although the amount of generated hydrogen is lower than in nitrogen free atmosphere but economically much more favorable (Yakunin, 2002).

Genetic modifications can be very effective but also troublesome and very expensive. Therefore other methods of process improvement are under investigations. Optimum value of pH equals 7. Photofermentation with Rhodobacter sphaeroides starting at pH=6.8 and ending at pH=7.5 results in significant drop of activity ( 7 times) but PHB concentration is tripled (Jamil, 2009).

Photofermentative bacteria belongs to mesophilic microorganisms and operate between 30 and 35 oC. Therefore, any critical temperatures act against high yield of hydrogen. For example Rhodobacter capsulatus operating at temperatures varying from 15-40 oC produce 50% less hydrogen than the same bacteria kept at constant temperature of 30 oC (Ozgura, 2010).

The access of photobacteria to the light with appropriate length and intensity play a crucial role m hydrogen photogeneration. Better access of light induce better phosphorylation and in consequence more effective synthesis of ATP and better yield of photofermentation (Kars, 2010).

Although the PNS bacteria absorb light in wide spectrum 400-950 nm the range of 750-950 nm is the most important (Eroglu, 2009, Ko, 2002). The light intensity is as well important as their wavelength. For Rhodobacter sphaeroides the amount of generated hydrogen grows linearly from 270 W/m2 (4klx) to 600 W/m2 (~ 10 klx). Below 270 W/m2 no activity of bacteria is observed (Miyake, 1999, Uyar, 2007).

Application of illumination with wavelength longer than 900 nm results in overheating of the system. This require additional cooling systems because of decrease the amount of generated hydrogen. An application of appropriate filters cutting the unwanted range of spectrum seems to be the only solution in this situation (Ko, 2002). Considering natural irradiation one should remember about day-night periodicity. It was found, however, that amount of generated hydrogen is even higher under periodic irradiation than under the continuous one (Eroglu, 2010, Koku, 2003). The day-night illumination induces better activity of nitrogenase what results from better adjustment of PNS bacteria to live in natural conditions (Meyer, 1978).

The presence of organic compounds, also those containing nitrogen (except NH4+ ions) is the key issue for the photofermentation. However, presence of macro and microelements at appropriate concentration can influence the hydrogen productivity. Iron belongs to the most important ones. This element exists mainly as the cofactor of proteins engaged in metabolism. Process of photofermentation, strictly related to the transport of electrons.

There are many electrons carriers such as cytochromes (proteins containing Fe) or ferredoxin. Moreover, the main enzyme in photofermentation — nitrogenase contains 24 atoms of iron in each molecule. The presence of iron ions in medium containing PNS bacteria is one of the very important factors influencing hydrogen productivity. At concentrations of Fe2+ ions lower than 2.4 mg/l there is no hydrogen in products. At concentrations higher than 3.2 mg/l the gradual decrease of evolved hydrogen is observed. It was assumed that non physiological coagulation of the cells can occurs (Zhu, 2007). Molybdenum is the second microelement playing an important role in photofermentaive hydrogeneration. The optimal concentration of molybdenum is 16.5 pmol/l (Kars, 2006).

The substrate yield in hydrogen production can be significantly improved by adding other strains of bacteria into the liquid medium. Improvement in photofermentation was achieved by adding halofilic archeons of Halobacterium salinarum type. The integral membrane protein — bacteriorhodopsin as the pump for the light excited electrons. The H+ ions are pumped out from cytoplasm outside the cell. The proton gradient is then engaged in ATP synthesis by Rhodobacter sphaeroides and this way increasing hydrogen generation. In this case, it is advised to use strains of PNS bacteria tolerating high concentrations of salts (Zabut, 2006) because of the high activity of bacteriorodopsyne in aqueous solution with high ionic strength.

For protection of the environment, the recycling of organic materials has essential role. The anaerobic digestion (AD) is an important method to decrease the quantity of organic wastes by utilization them for energy and heat production. The by-product of this process is the digestate.

In an AD process, different organic materials could be used alone or in mixture of animal slurries and stable wastes, offal from slaughterhouse, energy crops, cover crops and other field residues, organic fraction of municipal solid wastes (OFMSW), sewage sludge. The quality of digestate as a fertilizer or amendment depends not only on the ingestates but also on the retention time. The longer retention time results in less organic material content of the digestate because of the more effective methanogenesis (Szucs et al., 2006).

Biogas technology is known to destroy pathogens. The thermophilic AD increases the rate of elimination of pathogenic bacteria, therefore the amounts of fecal coliforms and enterococcus fulfilled the requirements of EU for hygienic indicators (Paavola & Rintala, 2008). Mesophilic digestion alone may not be adequate for correct hygienization, it needs a separate treatment (70 oC, 60 min., particle size<12 mm) before or after digestion, especially in the case of animal by-products (Bendixen, 1999; Sahlstrom, 2003).

Two types of digestate are the liquid and the solid ones which are distinguished on the bases of their dry matter (DM) content. The liquid digestate contains less than 15% DM content, while the solid digestate contains more than 15% DM. Solid digestate can be used similar to the composts or could be composted with other organic residues and can be more economically transported over grater distances than the liquid material (Moller et al., 2000).