The metabolism of carbon during fermentation process towards hydrogen is based on transformation of pyruvate in presence of majority of microorganisms active in this reaction. The first step of dark fermentation is based on glycolysis occurying in cytosol of cell, also known as Embden-Mayerhof-Parnas (EMP) pathway (Stryer, 1999). This pathway is
initiated by one molecule of glucose, catalyzed by different enzymes and further transformed into 2 molecules of pyruvate. The energy liberated during oxidation of 3- phosphoglycerol aldehyde is sufficient for phosphorylation of generated acid towards 1,3- bisphosphoglycerol and reduction of NAD+ to NADH. This reaction is catalyzed by 3- phosphoglycerol dehydrogenase. Transformation of glucose to pyruvate is during glycolysis is accompanied by formation two molecules of ATP and two molecules of NADH.

Glucose is not the only substrate in glycolysis. Simple sugars such as fructose or galactose as well as complex sucrose — saccharose, lactose, maltose, cellobiose or cellulose can be used as the initial substrate for glycolysis. However, the incorporation of these complex sugars into glycolysis pathway require initial hydrolysis to the simple carbohydrates.

Glycerol can be considred as a good substarate for glycolysis. A part of glycerol is oxidized into dihydroxyacetone by glycerol dehydrogenase. Next, dihydroxyacetone is phosphorylated into phosphodihydroxyacetone in the presence of dihydroxyacetone kinase. Thanks to triozophosphate isomerase phosphodihydroxoacetone is transformed into 3- phosphoglycerol aldehyde and further participate in EMP pathway.

There are known also other anaerobic pathways transforming glucose into pyruvate as e. g. Entner-Daudoroff or phosphate pentose pathway (Schlegel, 2003, Dabrock, 1992, Vardar — Schara, 2008, Chin, 2003).

Entner-Doudoroff pathway goes from glucose to pyruvate and is known also as 2-keto-3-detoxy-6-phosphogluconate. Here, glucose-6-phosphate is transformed with phosphogluconate dehydrogense into 6-phosphogluconate. In the next step, the removal of water from 6-phosphogluconate leads to formation of 2-keto-3-deoxy-6-phosphogluconate. This process is followed by formation of pyruvate and 3-phosphoglycol phosphate. These transformations are analogous to glycolitic pathway already described. One molecule of glucose is transformed into molecules of pyruvate with simultaneous formation of one NADPH (reduced dinucleotide nicotinoamine adenine phosphate and one molecule of ATP (Schlegel, 2003).

Pentophosphate pathway is based on initial phosphorylation of glucose to glucose-6- phosphate with help of hexokinase. Further steps are more complicated. The glucose-6- phosphate dehydrogenaze transfer hydrogen to NAD simultaneously forming of gluconolactone. The phosphate gluconolactone dehydrogenase helps to generate 6- phosphogluconate acid. The last phase is based on decarboxylation of the acid into ribuloso-6- phosphate. The transfer of this compound into riboso-5-phosphate and xylulose-5-phosphate starts a non-oxidative phase. At this stage of reaction the reversible reaction between these compounds occurs with formation of sedoheptulose-7-phosphate and 3-phosphoglycerate aldehyde. Subsequent reactions can generate fructose-6-phosphate an erythrose-4-phosphate. In further reactions erythrose-4-phosphate is transformed into 3-phosphoglycerate aldehyde and fructose-6-phosphate. Thus, one cycle of pentophosphate pathway generates 2 molecules of fructose-6-phosphate, one molecule of 3-phosphateglycerol aldehyde, 3 molecules of CO2 and 6 molecules of NADPH. The pentophosphate pathway with glycolysis leads finally to the pyruvate formation (Schlegel, 2003).

In the next steps in anaerobic conditions, the oxidative decarboxylation of pyruvate occurs with acetylo-CoA and CO2 formation. This reaction is catalyzed by pyruvate oxyreductase and the reduced form of ferredoxin appears as a step in final oxidation catalyzed by hydrogenase. Here, electrons reduce protons to molecular hydrogen. The reduced ferredoxin is also formed in glycolysis as the result of NADH oxidation to NAD (Dabrock, 1992). Carbon dioxide, acetic acid, lactic acid ethanol, butanol and acetone accompany hydrogen formation:

C6H12O6 + 2H2O ^ 2CH3COOH + 2CO2 + 4H2 (4)

C6H12O6 + 2H2O ^ CH3COCH3 + 3CO2 + 4H2 (5)

C6H12O6 ^ CH3CH2CH2CH2COOH + 2CO2 + 2H2 (6)

C6H12O6 ^ CH3CH2CH2 CH2OH + 2CO2 + H2O (7)

C6H12O6 ^ 2CH3CH2OH + 2CO2 (8)

C6H12O6^2CH3CHOHCOOH (9)

These reactions indicate that theoretical yield of hydrogen should 4 moles of hydrogen per one of glucose when acetone or acetic acid are among the products (Vardar-Schara, 2008).

Cells metabolize glucose into phosphoenolpyruvate, pyruvate, and formate. Phosphoenolpyruvate is converted to succinate by fumarate reductase (FrdC), and pyruvate is converted to either lactate by lactate dehydrogenase (LdhA), to carbon dioxide (CO2) and acetate by pyruvate oxidase (PoxB), to carbon dioxide by pyruvate dehydrogenase (AceE), or to formate by pyruvate formate lyase (PFL). Hydrogen is produced from formate by the formate hydrogen lyase (FHL) system consisting of hydrogenase 3 (Hyd 3) and formate dehydrogenase-H (FDHH); the FHL is activated by FhlA that is regulated by Fnr and repressed by HycA. Evolved hydrogen is consumed through the hydrogen uptake activity of hydrogenase 1 (Hyd 1) and hydrogenase 2 (Hyd 2). Formate is exported by FocA and/or FocB and is metabolized by formate dehydrogenase-N (FDHN; FdnG), which is linked with nitrate reductase A (NarG) and formate dehydrogenase-O (FDHO; FdoG). HypABCDEF are maturation proteins for hydrogenases 1, 2, and 3 (Maeda, 2008)

Transformation of pyruvate to acetylo-CoA and formic acid occurs in the presence of puruvate-formate liase with relatively anaerobic microorganisms. Formic acid is then transformed into hydrogen and CO2 in the presence of formic-hydrogen lyase. Here, 2 molecules of hydrogen from one molecule of glucose can be generated. Similarly as in the case of completely anaerobic bacteria, pyruvate can form lactic acid (reaction 9), whereas acetylo-CoA into ethanol and acetic acid (reactions 8 and 4). These processes can lower the theoretical amounts of generated hydrogen. Additional negative effect comes from the formation of succinic acid. Namely, formate-hydrogen lyase. become active only at low values of pH what in consequence is caused by formation of acids. Thanks to the decomposition of formic acid further fermentation towards other acids can proceed (Dabrock, 1992, Hallenbeck, 2009).

The absence of photosystem II in purple non-sulphur bacteria eliminates the problem of oxygen inhibition in hydrogen generation. However, in order to decompose water molecule and generate an electron in the photobiological process, the PNS bacteria need simple organic and inorganic compounds for photosynthesis. Organic compounds are a source of carbon and electrons. The PNS bacteria can use also CO2 as a source of carbon after transformation of metabolism into photoautotrophic one. However, if the light intensity is too low to reduce CO2 then the cell can use H2 and even H2S (at low concentrations) as a source of electrons (Kars, 2010). However, CO2 absorption is the basic metabolic process in the cell developing either in autotrophic or heterotrophic systems. The removal of RuBisCO enzyme via genetic modification of PNS bacteria results in the decline of photoheterotrophic development (Akkerman, 2002). Hydrogen generation with PNS bacteria can be realized in the presence of such simple organic molecules as acetate, lactate, malate or glucose. The maximum theoretical yields of conversion of these compounds to photogenerated hydrogen are described by the following equations:

The theoretical amounts are usually much higher than those observed in experiments. The conversion of lactate and malate occurs easily with relatively high yields, but that of acetate and glucose is much more difficult and gives low yields of hydrogen (Kars, 2010). The discrepancies between theoretical values in hydrogen productivity and those obtained in experiments can be explained by different metabolic pathways of carbon in PNS bacteria (Figure 10, Koku, 2002).

The amount of electrons generated on absorption of organic compounds depends on the source of organic carbon. Even a slight difference in the molecular structure can lead towards completely different metabolic pathway. For example, D — and L-isomers of malate (after conversion into pyruvate) can easily join the TCA cycle. In this way the energy demand for hydrogen generation is met, whereas such a substrate as acetate is used in the other metabolic pathways: e. g. glyoxylate cycle, citramalate cycle, and ethylmalonyl-CoA pathway (Kars, 2010). The excess of electrons generated during assimilation of such substrates as glycerol or butyrate must be accepted during CO2 photoreduction. Therefore, when the only source of carbon is glycerol, it is not assimilated in significant amounts, which is changed after supplementation of glycerol with malate. Initially, malate is assimilated from the medium and evolution of CO2 occurs. In the second step of reaction, the evolved CO2 permits the use of glycerol as a substrate (Pike, 1975).

Fructose

Although the large variety of substrates can be used by photosynthetic bacteria, only a few fulfill the requirements for fast reaction rate and high yield of photogenerated H2. In general, the preferred substrates as anions of organic acids, whereas carbohydrates do not meet the above criteria (Koku, 2002).

Marianna Makadi, Attila Tomocsik and Viktoria Orosz

Research Institute of Nyiregyhaza, RISF, CAAES, University of Debrecen,

Hungary

1. Introduction

Digestate is the by-product of methane and heat production in a biogas plant, coming from organic wastes. Depending on the biogas technology, the digestate could be a solid or a liquid material.

Digestate contains a high proportion of mineral nitrogen (N) especially in the form of ammonium which is available for plants. Moreover, it contains other macro — and microelements necessary for plant growth. Therefore the digestate can be a useful source of plant nutrients, it seems to be an effective fertilizer for crop plants. On the other hand, the organic fractions of digestate can contribute to soil organic matter (SOM) turnover, influencing the biological, chemical and physical soil characteristics as a soil amendment.

Besides these favourable effects of digestate, there are new researches to use it as solid fuel or in the process of methane production.

In their experiments, Khemkhao et al. (2011) found that DGGE patterns of bacterial diversity of the three bacterial groups, hydrolytic, acidogenic and acetogenic, persisted at all operating temperatures. However, the distribution of their members among bacteria in each group did show small changes under the different operating conditions. By the end of the operating period, the UASB with chitosan addition was found to contain a lower proportion of hydrolytic bacteria and a higher proportion of acidogenic bacteria than the control. However, the diversity of acetogenic bacteria was found to be similar in the two reactors. Sulfate-reducing bacteria were detected in the control but not in the chitosan reactor.

It is known (Bitton, 1994) that hydrolytic, acidogenic and acetogenic bacteria work together to degrade complex organic matters into acetate, CO2 and H2. Hydrolytic bacteria begin the process of degradation by breaking down complex organic molecules such as proteins, cellulose, lignin and lipids into soluble monomer molecules by extracellular enzymes, i. e., proteases, cellulases and lipases. The monomer molecules produced are amino acids, glucose, fatty acids and glycerol. These monomers are then degraded by the acidogenic (acid-forming) group of bacteria which convert them into organic acids, alcohols and ketones, acetate, CO2, and H2. The organic acids produced include acetic, propionic, formic, lactic, butyric, and succinic acids. The alcohols and ketones produced are ethanol, methanol, glycerol and acetone. In the final stage, the acetogenic bacteria (acetate and H2-producing bacteria) convert the fatty acids, alcohols and ketones into acetate, CO2 and H2.

Two different L gas target properties have been described. Because of their basic constitutions, one bio-methane mixture is conditioned with air and the other is conditioned with a combination of air and LPG.

Table 14 shows a summary of the admixtures with which a target calorific value-oriented mixture for the low calorific base gas property can be achieved.

In the case of simple air addition, particular attention should be paid to compliance with the maximum O2 volume fraction. This should not exceed 3 % vol. in dry networks according to DVGW worksheet G 260. This quantity is reached when adding pure air to the processed biogas, at an admixture of 15 vol -% of air. In the low caloric L gases (e. g. Weser Ems gas), this limit is never reached.

Furthermore, a minimum air addition may also be necessary, in order to achieve the required Wobbe Index according to DVGW worksheet G 260.

Table 15 shows the minimum air addition for the individual processing grades of methane to achieve an L gas compliant Wobbe Index of under 13.0 kWh/m3 (NTP).

For the high-caloric L gas mixtures (target properties according to Holland II L gas) the processed biogas is conditioned with air and LPG. Table 16 shows the correlating LPG-air additions, to reach the calorific value range (+ / -2%).

The gray-shaded areas show where a compliant combination of air and LPG additions is impossible. With increasing LPG additions, the necessary addition of air is limited by the maximum O2 volume fraction of 3 %. If too little LPG is added, only the lower calorific value range can be covered. The broadest coverage of the calorific value range lies in between and is marked by the wider bandwidth of air additions.

Ghana has a population of 24.8 million of which 48.5% live in the rural areas (United Nations, 2007). Netherlands Development Organization (2007) estimates that Ghana has a potential to realise 280 000 domestic biogas plants, that is capable of producing 6000 m3 of liquid fertiliser, which would increase yield by 25%. However, low perception of biogas has modern energy has made Ghana not to realise the full potential of biogas utilisation (Bensah and Brew-Hammond, 2008). Bensah and Brew-Hammond (2010) highlights the status of the biogas development in Ghana, in which only about 200 units have been installed, thus lagging behind in comparison with other African countries such as Rwanda, Kenya and Tanzania. Some initiatives such as Biogas Technology West Africa Ltd[6] , funded by UNIDO has implemented a number of biogas digesters in Ghana for schools, hospitals and colleges. These are mainly underground masonry dome systems in the range of 60 m3 to 160 m3 volume. One example of these projects is Keta secondary school plant for 1200 users, and has a capacity of 80 m3. The plant is built in sandy, water logged area and it makes use of human waste. The gas is used for cooking. The future development of biogas in Ghana will however not be left to private investors and initiatives if the benefit to the rural communities is to be realised. Bensah and Brew-Hammond (2010) argues that, for successful future development of biogas in Ghana, there is a need for establishing a government body that solely focuses on promoting biogas.

1.1.3 Mozambique

Mozambique has a population of 22.6 million people, in which 61.6% reside in rural area in 2010 (United Nations, 2007). Similar to Ghana, the Mozambique government does not have an agency solely supporting the development of biogas. Some initiative such as Biogas Technology West Africa Ltd[7] is however, also undertaking a biogas power project in the country. The project is an electric power system powered by a biogas-fired internal combustion engine generator at Mpunsa Village, Chicualacuala District, Gaza Province in Mozambique.

As far as the anaerobic digestion process is concerned, it is more appropriate to discuss alkalinity and pH together because these parameters are related to each other and very promising to ensure a suitable environment for successful methanogenesis process. Alkalinity is produced in the wastewaters as results of the hydroxides and carbonates of calcium, magnesium, sodium, potassium or ammonia and may also include borates, silicates and phosphates (Tchobanoglous and Burton, 1991). The alkalinity plays an important pH controlling role in the anaerobic treatment process by buffering the acidity derived from the acidogenesis process (Gerardi, 2003; Fannin, 1987).

Methane producing methanogens are known to be strongly affected by pH (Poh and Chong, 2009) and could only survive on a very narrow range of pH (Table 2) (Gerardi, 2003).

Table 2. The optimum pH range for selected methanogens (Gerardi, 2003; Steinhaus et al.2007, Tabatabaei et al., 2011)

As such, the methanogenic activity will be severely affected once the optimum pH range is not met. Steinhaus and coworker studied the optimum growth conditions of Methanosaeta concilii using a portable anaerobic microtank (Steinhaus et al., 2007). They reported an optimum pH level of 7.6 revealing that even little variations on both sides of the optimum pH suppressed the growth of the methanogens. Several studies have also reported reactor failure or underperformance simply due to pH reduction caused by accumulation of high volatile fatty acids in the anaerobic treatment system (Fabian and Gordon, 1999; Poh and Chong, 2009; Tabatabaei et al., 2011).

In a study using synthetic wastewater in the thermophilic temperature, was found that at the pH of above 8.0, the methanogenesis was strongly inhibited and the value recorded for acetotrophic methanogenic test was zero (Visser et al., 1993). When investigating the role of pH in anaerobic degradation test; Fabian and Gordon (1999), found out that the acidification led to the low performance of the anaerobic degradation, however the biodegradation was significantly increased once the wastewater when the pH was adjusted to above 6.5.

Intermeshing is a basic concept in pipeline/ network planning: it provides intrinsic redundancy for gas delivery in case of trouble/break at single points of pipeline. It supports continuous operation and pressure of the system; as it is most important to keep the whole pipeline system under pressure all the time (if the pressure would drop to zero, oxygen could enter the pipeline system exposing some areas or the system to the risk of explosion). Exaggerated use of intermeshing lead to higher investment cost in pipes and decrease a cost- efficient network structure in principle (besides, an item of passionate discussions among planners). Intermeshed networks need, because of their complexity, simulation support to get detailed insight into physical state and variables of the pipeline system.

2.5 Online — and offline-simulation

An advanced feature of simulation or operating mode is online-simulation. Here the simulation is coupled with a SCADA system and is executed corresponding to the cycle of the data acquisition (minutes to hours). It is a good tool to watch and control the network by additional detailed information almost in real time. This type of simulation is very demanding as it requires correct and complete data all the time what must be carefully prepared. Offline-simulation is the normal application it can be executed when needed — at arbitrary time.

Table 9 overviews the results of the three optimizations described before.

It turned out that the profitability of a fermenter on location 2 is lower than on the other locations. It was never preferred in any optimum structure. The other locations have one advantage — the shared usage of biogas pipelines whereas low additional costs for location 1 have to be born. There are never heating pipelines from the different locations to the center considered in the optimum technology networks. Just the biogas is transported; heat is produced centrally and distributed within a district heating network, although additional biomass furnaces are required. In scenario 1 the missing corn silage availability was compensated by a higher amount of intercrops, referring to the CH4 content, and it shows the best revenue, because of higher plant utilization and higher revenue for electricity and heat production. Although in the optimal scenario the amount of corn relating to the total feedstock was not even 17 % of the total (dry matter) the compensation for corn with intercrops results in higher revenue. For more corn that intercrops compensate in the input the impact would be even higher. Therefore it is obvious that intercrops can be a profitable feedstock to run a biogas plant. For the case study the availability of intercrops would have to be raised as described before which would lead to the best technology network for the region.

The system has two limiting factors; on the one hand the distances between the fermenter locations and the feedstock providers accompanying different transport costs and on the other hand the limited resource availability. It could be shown that it is not lucrative to run a central CHP with higher capacity (500 kWel) as feed-in tariffs are lower and less revenue can be gained. Nevertheless, from the point of view of sustainability, it would be preferable to substitute two smaller CHPs with a bigger one. An adaptation of reimbursement schemes to the solutions presented is recommended.

1.1.2.1 Research status of biodegradable film materials

Biodegradable materials research began in the 1960s. The initial study was mainly to add natural polymers with biodegradable properties (such as starch, etc.) to generic plastic, then get the so-called biodegradable materials. St. Lawrence starch-company developed a starch — polyethylene or polypropylene blends in Canada (Qiu Weiyang, 2002). With human understanding of biodegradability of macromolecule, the research focus began to turn to biodegradable materials (Qiao Haijun, 2007), which can be classified as microorganism synthetic polymer, chemical synthetic polymer and natural polymer.

biodegradable film, it includes structural degradation film, biodegradable film containing inorganic salts and adding starch. (3) photo — biodegradable film; (4) plant fibre film.

In China, the major research was additive photo-degradation film and synthetic photo­degradation film. The research focused on using light stabilizers to control degradation period. Since 1997, 944-polymeric efficient light stabilizer, BW-6911 new light stabilizer were developed, which replaced the severe irritation and sensitization GW-504/2002 anti-aging system. American Dupont CO. , Ltd produced copolymers of ethylene and CO, American OCC and DOW CO. , Ltd had used this technology to produce film and develop industrial production (Xiong Hanguo, 2004). The disadvantages of photo-degradation film were susceptible to external environment, which was difficult to control the degradation period, and covering field, the part into soil can not be degraded, so its application was limited (Xu Xiangchun, 2006).

The degradation of biodegradable film was caused by microbes in natural environmental condition. It was divided into additive biodegradable film and completely biodegradable film according to degradation mechanism and damage style.

At present, additive biodegradable film was composed of plastic, starch, compatibility agents, self-oxidants, processing additives. Typical varieties were polyethylene starch biodegradable film (Liu Ming et al., 2008). There were institutes of physics and chemistry, Beijing University of Technology, Guangdong biodegradable plastic CO., Ltd more than 20 research institutes. The research focused adding starch or modified starch into PE.

The main varieties of completely biodegradable film were PLA, PCL, and PHB and so on. United States used PCL to produce synthetic polymer biodegradable film (KAM Abd I J — kader, 2002). Warner-Lambert developed a new type of resin, which was made of 70% amylopectin starch and 30% amylose starch (He Aijun, 2002). It had good biodegradability, was considered a significant development in material science.

Photo-degradation film was made of additive photo-sensitizer, auto-oxidants, and anti­oxidants as microbial culture medium in general polymer.

Plant fibre film has good ventilation, wet and dry strength and good biodegradability. Chinese academy of agricultural sciences successfully developed the environmentally — friendly hemp film (Fu Dengqiang, 2008). In addition, paper films composed of different materials were produced. South China Technology University used sugar cane and starch as materials to manufacture a kind of fully degradable film (Tan Chengrong, 2002). Japan manufactured biodegradable film with 1%-10% chitosan cloth softwood mechanical pulp original paper in 1990. The demand of environmental film increased in Washington State University, France, Germany, Italy, Canada, Netherlands and South Korea and other countries, leading to the environmentally-friendly film industry rapid development (Han Yongjun, 2008).

Crop yield is very important economical parameter of plant production but nowadays the quality of foods is becoming more and more important. Digestate treatment seems to be very effective to increase the protein content of plants. Banik and Nandi (2004) investigated biogas residual slurry manures (solid digestate) used as supplement with rice straw for preparation of mushroom beds. The application of biomanure increased the protein content of mushroom 38.3-57.0%, while the carbohydrate concentrations were decreased. Results can be seen in Table 10.

respectively. Changes in amino acid composition of test plants were also very favourable, because almost every essential and non-essential amino acid quantity was increased significantly after digestate treatment. In line with these results the oil content of the treated plants decreased significantly.

Qi et al (2005) examined the effect of fermented waste as organic manure in cucumber and tomato production in North China. Before the vegetables transplantation, the diluted fermented residual dreg was applied 20-30 cm below the soil surface at a rate of 37,500 kg ha-1, while liquid digestate was sprinkled to the soil surface in three vegetables growing stages and on the vegetable leaves once time. They found increasing yield (18.4% and 17.8%) and vitamin C content (16.6% and 21.5%) of treated cucumber and tomato, respectively.

As the results show, the digestate application in solid or liquid form could result significant improvement in the quality of foods without damaging the environment, which is very important for the sustainable environment and healthy life.