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Biowaste and Algae Knowledge for the Production of 2nd Generation Biofuels

Final Report Summary - BIOWALK4BIOFUELS (Biowaste and Algae Knowledge for the Production of 2nd Generation Biofuels)

Executive Summary:
The BioWALK4Biofuels Project (B4B) aimed to develop an alternative and innovative system for the treatment of biowaste and to use GHG emissions to produce biofuels, using macroalgae as a catalyser, in a multidisciplinary approach. The project, seeking for a synergy between biowaste and macroalgae for a clean biogas production, allows to by-pass the high nitrogen content problem of biowaste: in fact macroalgae, needing nitrogen and phosphate to grow, represent an optimal interface, capable to transform the negative eutrophication potential of such a biowaste in a benefit, in terms of increasing algae growth-rate.
In the Project demonstration area (Augusta Bay, Sicily, Italy) an algae cultivation system was realized together with a biogas production plant including the following sections:
- pre-treatment plant;
- anaerobic digestion process (Hydrolysis, Acidogenesis, Acetogenesis, Methanogenesis);
- co-generator (CHP)
The plant was designed to have a capacity of 40 Nm3 of biogas/h providing 90 kWth of thermal power and about 45 kWel of electric power. This could be obtained by an anaerobic co-digestion process of different substrates, such as seaweeds, manure, oil mill waste water and rumen, from areas close to the plant.
The management of the B4B process was possible thanks to a well-structured management plan, supported by an automatic control system developed on a Labview software platform - National instrument. After all the necessary steps in order to pass the blank tests of the process, the system was ready to load biomass and start to produce biogas. Initially the system was fed with an activation mix made of rumen and corn, then, after a few days, it was loaded with rumen, chicken manure, OMWW and finally algae. The amount of algae was 90 kg wet per day.
The amount of biogas produced (6 Nm3/h) has been limited due to the relatively little time available to manage the plant during the final period of the project, as well as to the numerous problems of pipeline blockage. Also the amount of biogas has been limited during the final period of research; this has allowed turning on the torch and the co-generator for the time strictly necessary (although sufficient), to demonstrate the effectiveness of the process.
The single system processes, like pre-treatment, hydrolysis, methanation, biogas cleaning and digestate oxidation were tested properly.
Biogas, within the end of the project, was produced by a mix of algae and biowaste.
Power energy and thermal energy were produced in order to demonstrate the B4B energy process sustainability.

Project Context and Objectives:
The world energy demand is expected to rise 60% by 2030; oil consumption has increased by 20% since 1994, while the European Union import dependency has reached almost 54% of its energy requirements in 2006. Land filling of biowaste is one of the major sources of methane emissions in Europe, contributing to 2% of GHG emissions in 2007 in the European Union (EU). In 2010 it has been reported that 93% of the greenhouse gas emissions from waste management come from the landfill sector, whilst incineration, without energy recovery and other waste treatment operations, contribute only 7% of the total emissions. Biodegradable waste that is land filled should be reduced to 35% of 1995 levels by 2016 according to the EC Landfill Directive (1999/31/EC). Direct and indirect greenhouse gas emissions (GHG) from industries are currently at about 12 GtCO2-eq.
The Council Directive 91/676/EEC and the subsequent Report from the commission to the council and the European Parliament on the implementation of this Directive, has underlined the need to control the reduction of water pollution caused by nitrates from wastes to protect human health, living resources and aquatic ecosystems. Additionally, attention is being focused on the treatment of biowaste, which requires strong support from EU legislation to address difficulties in their direct utilisation, cost-efficiency and its output product pollution.
Moreover, the need to focus on ‘non-food’ energy crops for the production of 2nd generation biofuels and develop cost-efficient solutions has been revealed and underlined in the recent report of the Food and Agriculture Organisation. In fact the production of biogas is principally carried out through anaerobic (mixed) fermentation of cereal crops and manure.
The European Council after the United Nations Climate Change Conference in Bali December 2007 with the EU directive 2009/28/CE and 2009/30/CE set two ambitious key targets: a reduction of 20% of greenhouse gases (GHG) by 2020; and a 20% share of renewable energies in EU energy consumption, of which 10% should be produced by biofuels by 2020. Hence, the need to further explore new sources of alternative renewable energy and ground-breaking solutions to reduce GHG emissions becomes of the upmost importance.
It appears fundamental to improve the know-how about a “new” biofuel production, in particular regarding 2nd generation biofuels. In order to face this new challenge, the B4B project have a multidisciplinary approach showcase for biowaste to be used as a feedstock and algae as a catalyst for producing biofuels.
The BioWalk4Biofuels project was a research and a demonstrative initiative, which aimed to develop a cost-efficient solution that could use biowaste as a feedstock for the production of 2nd generation biofuels, using macroalgae as a catalyst, while minimizing the environmental impact of biofuel production.
The main and specific objectives of the project can be pointed out as follows:
a) use of macroalgae as an interface between biowaste and energy production to allow the direct utilization of biowaste for biogas production;
b) macroalgae can be directly used in bio-digesters to produce energy without the need of mixing them with cereals, deriving from agricultural production (in such way avoiding land consumption); macroalgae can be fed to the bio-digesters together with other locally available biowastes such as chicken manure, olive mill waste water, rumen, whey.
c) drawing and construction of a a drone for algal harvesting activities. The Roboharv drone has the capacity to move the algae up to one meter depth in the cultivation system, to increase the oxygen level in the water, to push the biomass toward the pump suction from which they are sent to the preparation and homogenization facility.
d) the pre-cultivation of protoplasts in an incubator facility permits the acceleration of cell regeneration, allowing a backup of macro algal biomass and consequently a reduction in time of the macroalgae growth process.
e) different types of biowastes can be mixed and used as a feed for the Anaerobic Digester, transforming them into biogas; this aspect represents a very positive externality in the whole production process, as: 1) it avoids the need for a specific disposal of each biowaste, which would result in an expensive process, as well as still impacting from the environmental point of view; 2) biowastes become a very important resource for the purpose of energy production; 3) biowastes are collected from livestock activities and industrial assets within a few dozen kilometers from the experimental plant, in such a way producing a minimal impact in terms of transport and fully accomplishing the requirements of a short supply chain process.
f) Optimization of the two phase anaerobic digestion processes through the following activities: management of operating parameters inside the reactors; biomass loads in the preparation tank, oxidation reduction potential control, physical-chemical variables management.
g) use of a control system, designed and built for the demonstration plant, which allows a real time assessment of all process data flow, allowing at the same time a daily/periodic check of the energy balance and of the impacts of the biogas production process through the use of the Life Cycle Assessment (LCA) procedure.

Project Results:
Many activities were carried out during the project, in order to define and improve the production process (biogas and algae), the monitoring, the safety and the management of the demonstration plant; the description of these activities and of the consequent results follows:

1.3.1 Demonstration plant and biogas production
The construction of the experimental plant for biogas production from algae in Augusta was concluded on January 2014. Figure 1.3.1.A schematically shows the whole production process.
Nevertheless, during the start-up of the demonstration plant (blank tests), problems were encountered in the Anaerobic Digestion (AD) component: excessive swelling of the fermentation tank even at low pressures, failure of the seals. In figure 1.3.1.B the structural analysis carried out in order to point out the critical deformation of the methanation component is shown. In figure 1.3.1.C the licks of waster of the methanation tank together with the subsequent activities carried out in order to replace the sealing gaskets are evidenced.
Different actions were undertaken in order to solve these technical problems: all the gaskets of the digester were replaced by lifting the upper part of the component with a crane (Figure 1.3.1.C). A structural check of the AD component was carried out in order to solve the swelling problems which occurred in the fermentation process blank tests.
In the same period maintenance and repair operations were carried out on several equipments of the demonstration plant (pumps, one-way valves, process sensors) in order to allow the full operation of the production plant. After all these reparation and maintenance activities, no deformation was noticed to the volume of the digester, while the manometers have shown that the pressure inside the plant has not changed substantially. All the blank tests were, therefore, carried out successfully.
During the design phase, the operational pressure of the anaerobic digester was defined in a range from a minimum of 20 mbar to a maximum of 40 mbar. This range defined the conditions in which the biogas could be stored into the digester; nevertheless, to make operating the system at a pressure of 20 mbar, a few changes were made to the methanation process: in particular a blower (figure 1.3.1.D) was acquired and installed in the line (after the digester) in order to raise the outcome biogas pressure from 20 mbar to a maximal value of 140 mbar and overcome any eventual load losses.
Several calculation were made in order to reckon the circuit characteristic curves. In figure 1.3.1.E the characteristic curves of both the gas circuits are presented (“bypass” and “carbon filters” - the two pipelines at the output of the blower) depending of the blower rpm.
This change has also led to a substantial revision of the plant management software, of the digester control sensors and of the pipeline linking the digester and the CHP. In fact, the pipeline connections for CHP were re-designed and the automation software was modified accordingly. A biogas cleaning and drying system was installed in order to test the AD plant reliability and biogas production.
The control system of the shaft rotation speed of the blower was also studied in order to avoid aspirations, which could cause depressions to the digester: for this purpose an inverter was installed in the plant in order to vary the frequency of the blower power supply (and, therefore, the relative rotation speed of the shaft) and to adjust, in this way, the pressure inside the digester.
The biogas tests started in January 2015. Initially the system was fed with an activation mix made of rumen and corn, then, after a few days it was loaded with chicken manure, OMWW and finally algae. The amount of algae was 90 kg wet per day.
The amount of biogas produced (6 Nm3/h) has been limited due to the relatively little time available to manage the plant during the final period of the project, as well as to the numerous problems of pipeline blockage (caused by the initial underestimation of biomass flow pressure). Also the amount of biogas has been limited during the final period of research; this has allowed turning on the torch and the co-generator for the time strictly necessary (although sufficient), to demonstrate the effectiveness of the process.
The single system processes: pre-treatment, hydrolysis, methanation, biogas cleaning and digestate oxidation, were tested properly.
Biogas, within the end of the project, was produced by a mix of algae and Biowaste: seaweeds, manure, OMWW. The quality of the resulted biogas was good, as demonstrated by its characterization: the produced biogas has showed a content of CH4 and CO2 of the 55% and 43% respectively.
The cleaning system was also successfully tested: it was composed by a dehumidification system (chiller-Parker) and by an H2S removal section (carbon filters designed and relized by DICMA). The biogas optimally achieved the standards for its further use as co-generator fuel (Figure 1.3.1.F and 1.3.1.G).
Electricity was produced with a minimum load. The functioning of the electricity section of the CHP was tested, as well as its activation or deactivation under certain scheduled conditions of work like the biogas pressure value, for example. Also heat was produced by the CHP unit. The three-way valve was used to test the control system of the flows of heat from the CHP unit to hydrolysis and, alternatively, from the CHP to the cooler (Figures 1.3.1.H and 1.3.1.I).
For process optimization all input and output parameters of the anaerobic digestion plant were deeply studied and the design of the system was modified in order to maximize biogas and by-products production. Several optimization methods were studied to maintain a correct temperature inside both hydrolysis and methanation tanks. Studies on mass and heat balance were carried out on the basis of the characteristics of tanks and seawater. Heat exchangers, which provide heat transfer between the hydrolysis liquor and the seawater, have been dimensioned in order to control the temperature of the liquid in input to the methanation.
The Labview software was used to implement a control system for the whole plant. The B4B control panel allowed a real-time control of plant equipments: the parameters of the algae cultivation and harvesting, the biogas and energy production. A database collected and recorded operational data to generate detailed reports (figure 1.3.1.J).
More than 250 customized routines were developed for the B4B process and they are completely at disposal for further integration and new applications.
The design of this system was started in July 2012. The automation software was tested on 23/01/2014 in all his loads and subsequently updated in order to take into account all the technical changes made on the plant.
An automated algae harvesting system (Roboharv) was realized by the Partnership, with the technical support of the company Robotronix, in order to achieve a partially unsupervised harvesting procedure in the open ponds of the cultivation system (Figure 1.3.1.K). Many test were done in order to optimize and simplify the drone activities in open sea and in open ponds. The knowledge about the navigation control system and the ability to alignment and follow a predetermined path during the navigation was increased. Also the ability to push the harvested algae in the suction point (to transport algae in the preparation section) was improved.
The Coordinator has recently reached an agreement with the company ELMAR S.r.l. in order to develop any technological improvements to the use of the Roboharv drone both for algae collection and for its use with echo sounder instruments in lagoons and coastal areas (multisensory marine platform).
Although the installation of the cultivation system was completed on September 9th 2012 and the four floating ponds were ready and full of the right quantity of water on the 15th October 2012, the incredible amount of problems which occurred on the cultivation system in the recent years prevented the Partnership to make the four ponds operational all together. Underwater repairs were carried out for months by the Partnership. Regardless, using just one pond for cultivation purposes, the Partnership carried out four cultivation campaigns of Ulva Laetevirens and, in the last two, the Roboharv harvesting system was also used to circulate and collect the biomass.

1.3.2 Macroalgae cultivation parameters – species selection, growth potential and effect of waste streams on macroalgae cultivation
Macroalgae live by photosynthesis using energy from the sunlight, CO2 and nutrient to generate biomass. With focus on bioremediation of nutrient rich waste streams and production of biomass for renewable energy, macroalgae biomass potentially offers a number of benefits as compared to biomass from land plants, as their production does not require arable land and pesticides, and only minor freshwater resources. In addition, nitrogen and CO2 rich waste streams may serve as nutrient and carbon sources for optimised biomass production, thus allowing the algae production systems to provide simultaneously the dual ecosystem services of bioremediation and biomass production.
In order to optimize the efficiency of biomass production and bioremediation, macroalgae species with high growth rates, fast rates of nutrient uptake as well as robustness in cultivation, were selected as focus species for further optimization of production and bioremediation (D2.1). Growth trials were performed in laboratory and pilot scale aiming to generate knowledge to be transferred to the large-scale floating ponds in Augusta. The suitability of different available waste streams as carbon and nutrient sources for the focus species was analyzed, and growth trial using selected waste streams (raw and anaerobically digested manure, urban waste water and reject water from a wastewater treatment plant, as well as flue gas from straw/coal co-firing as well as wood pellets) were carried out in laboratory and pilot-scale to simultaneously optimize biomass production and bioremediation. All knowledge generated was summarized in a manual with recommendation for cultivation in large-scale in Augusta.

Summary of results of work
Species selection – growth and energy potential
A thorough literature survey was conducted with focus on the species relevant for cultivation in the large-scale ponds in Augusta. Selection criteria were local availability, robustness in cultivation, as well as high growth rates and energy potential. Species from the following genii were selected: Two green algae: Ulva (Figure 1.3.2.A) and Chaetomorpha (Figure 1.3.2.B) and one red algae: Gracilaria sp (Figure 1.3.2.C). An analysis of the expected advantages and disadvantages of the potential performance of the three algae in the Augusta ponds pointed to Ulva sp. as the superior candidate species, since Ulva species had demonstrated the highest rates of growth (>35 % d-1) and nutrient uptake (up to18 µmol N mg DW-1 h-1 (also with waste streams as nutrient and carbon source), high biogas yields (up to 560 ml CH4 mg VS-1) and local availability. Also there was a solid basis of experience for keeping Ulva species in larger scale cultivation, without major biofouling problems and with general adaptability to fluctuating cultivation conditions. Another major advantage was the level of progress in the protoplast isolation and regeneration from Ulva, which was promising for the success of the development of protoplast proliferation of the algae stock in this project.

Analysis of the growth and energy potential of potentially suitable algae, with focus on response to salinity, solar radiation and CO2
Following growth trials were performed in India, Jordan, Sicily and Denmark in laboratory and pilot scale to document the effect on growth and energy potential, of essential environmental parameters (salinity, as well as availability of CO2, light and nutrients) (Figure 1.3.2.D and 1.3.2.E). CO2 was delivered “on demand” from the algae, as it was automatically added to keep pH in the range optimal for growth – between 7 and 8. pH values out of this range are suboptimal or even detrimental.
The most significant results in this task ware that addition of CO2 tended to increase Ulva growth rates with up to 20% in lab. When tested in larger scale in the demonstration facility in Denmark, an even greater effect, up to 50% increase of growth rate, was documented. Another significant result was that nutrient starvation within two weeks could increase the carbon:nitrogen (C:N) ratio of the Ulva biomass from 10 to above 40, improving the quality of the biomass for biogas production, but significantly lowering the growth rates (Figure 1.3.2.F). This emphasised the inevitable trade-off between growth rate and C:N ratio, as high C:N ratios are only achieved when the algae are significantly deprived of nitrogen, pointing to the possibility of a two-phased cultivation system, phase one with focus on biomass production and nutrient removal, phase two with focus on nitrogen starvation and the resulting carbon accumulation in the biomass produced in phase one.
The floating ponds in Augusta were, during spring 2013, inoculated with local Ulva sp. Approximately one metric ton of Ulva was produced in the ponds and growth rates of 3-5% were estimated. Direct quantification of biomass production was not possible. The valuable experiences were summarised into a number of suggestions for future improvements in the design of pre-industrial size cultivation systems:
• pumping of CO2, which would stimulate photosynthesis and thus growth;
• a water mixing system, which could circulate to the surface the thalli of Ulva sp. lying on the bottom of the pond, allowing a constant and uniform distribution of the ambient irradiation (again, essential for photosynthesis) to all the material in culture. A water mixing system would also secure a better distribution of nutrients, essential for the growth of the biomass; the condition of "stagnant" water along with high seasonal temperatures caused a stratification of the water, which in the long run caused the death of the thalli at greater depth, and the triggering of processes of putrefaction and consequent hypoxia in the deeper layers;
• an in-situ continuous or semi-continuous monitoring system for physic-chemical parameters (N, P, pH), would allow for an optimal evaluation of the input of nutrients;
• use of sources of nutrients already experimentally tested on the growth of Ulva or, in the case continuing with the temporary use of agricultural fertilizers, of separate stock of ammonium nitrate (NH4NO3) and phosphate (P2O5), to be dosed appropriately according to the stoichiometric need of the algae.

Effect of biowaste and emissions on macroalgae cultivation before the transplant in open ponds
A number of relevant waste streams were tested for the first time as nutrient and carbon source for cultivation of relevant green macroalgae: manure from pigs (raw and anaerobically digested), manure from chicken, reject water of waste water sludge as well as flue gas from wood pellets and from co-combustion of coal and straw.
Addition of these waste streams in suitable concentrations increased the growth rates of Ulva lactuca, and hence proved to be suitable as nutrient or carbon source in cultivation of this species.
The most significant results from this task was that, the effect of flue gas on growth and biomass composition is equal to the effect of pure CO2, indicating no particular positive or negative effects of flue gas on biomass production, and also not on the elemental composition of the biomass (Figure 1.3.2.G). This indicated that the use of (cleaned) fluegas had no negative implications for the use of the algae biomass, but a clear positive effect on production rate, as well as on the uptake rates of nitrogen and phosphorus (Tables 1.3.2.A and 1.3.2.B). This indicates that that addition of CO2 in dual ways improves the efficiency of bioremediation of waste streams in both accelerating biomass production, as well as nutrient removal capacity of the algae tissue.
Regarding the use of different types of nutrient rich waste streams as nutrient source of the cultivation of macroalgae, it was in this project demonstrated for the first time that anaerobically digested manure as well as reject water from a sewage treatment plant are excellent sources of nutrients for cultivation of Ulva (Nielsen et al, 2012. Sode et al 2013). Both these types of waste water were anaerobically digested prior to feeding to the algae, thus reflecting the process in the Augusta plant. The results demonstrated that the maximal growth rates were achieved at nutrient concentrations of approximately 50 µM N, whereas at higher nutrient concentrations, the growth rates did not increase (Figure 1.3.2.H) whereas the algae tended to accumulate more nitrogen and phosphorus (internal concentrations increased).
The ambient nutrient concentrations should be determined by the following application of the biomass: If the biomass is to be used for biogas production, the C:N value should be kept in the rage of 20-30, and thus the nutrient concentrations should not exceed 6-12 µM N. However, if the biomass is supposed to be utilised as a protein source, high nutrient concentrations should be used with the algae. The use of waste streams as nutrient source did not elevate the content of heavy metals in the algae biomass produced, and the Ulva biomass was thus not disqualified for use as protein source in feed production (Table 1.3.2.C).

1.3.3. Protoplasts cultures and their developmental morphology of selected algae
Background
Protoplasts are intact living cells devoid of cell walls. Treatment of either thallus fragments or tissues with specific cell wall lytic enzymes results in total removal of rigid and complex cell wall polysaccharides and production of protoplasts.
The most important and promising application of such wall-less cells has been the genetic transformation aimed at trait improvement plant resource. Nevertheless, recent studies on the cultivation of green algae have utilized the protoplasts as an alternative “seed” material in place of spores for mass propagation, and emphasized the need for development of simple, efficient and cost effective method that can consistently yield reproducible results for mass production of protoplasts with ease.
In view of this, a simple enzyme based method was developed, tested its efficacy for producing consistently large yields of viable protoplasts from various other species and thereafter established its reusability for making it cost effective. Later, a customized prototype model was designed for seeding and propagation of protoplasts in the laboratory. The data generated through these experiments was ultimately used for conceptualization of a design for an outdoor facility for mass propagation of protoplasts so as to generate required volumes of germlings of Ulva needed for feeding the floating tanks.

Summary of results of work
The seaweed samples investigated in this study for protoplast studies were collected from intertidal region during low tides from different coastal areas along the Saurashtra coast of Gujarat, India and Messina, Italy (Table 1.3.3.A). Selected healthy thallus was chopped, cleaned from debris and finally incubated in 5 ml of enzyme mixtures (Table 1.3.3.B) on rotary shaker (50 rpm) for 2-3 h in dark at 20º ± 1º C.
During the incubation period, periodic microscopic observations were made to ascertain the protoplast release. Protoplast yield in the present study varied with individual species investigated in this study. Table 1.3.3.C summarizes protoplast yield and regeneration studied for different species of Ulva and M. oxyspermum. Among the investigated species, U. lactuca registered the highest protoplast yield of 4.0 ± 0.4 × 108 cells/g of fresh weight while U. ovata showed the lowest of 1.2 ± 1.2 × 105 cells/g of fresh weight. The regeneration rate of isolated protoplasts was always more than 90% except for U. ovata (88%).
The isolated protoplasts were spherical in shape (20-32 µm) and contained a cup shape parietal chloroplast (Figure 1.3.3.A) Most of the protoplasts under optimal culture conditions (25°C under photon flux intensity of 15µm m-2s-1) regenerated cell wall after 24 h and subsequently initiated cell division. Differentiation of protoplasts from different species of Ulva showed different developmental patterns, such as sporangia, microthalli, saccate (or spherical), tubular (or spindle), irregular, and frondose. During development, sporeling phase is also observed which released the spores. The protoplasts of U. reticulata differentiated and gave rise to normal as well as variant disc type germlings.
Among the two morphotypes, the regeneration rate for normal filamentous thalli was highest with 56.84±1.35% at 20±1 °C and significantly decreased with increase in temperature, with 46.87±0.81% at 25±1 °C and 33.43±0.54% at 30±1 °C. On the contrary, the other developmental behaviour i.e. saccate like structures followed the reverse trend with highest regeneration of 66.54±0.53% at 30±1 °C and significantly decreased to 53.12±0.81% and 43.16±1.35% at 25±1°C and 20±1°C respectively. The regeneration of protoplasts into different morphotypes could be attributed to epigenetic variations.
While advocating this alternate route for seaweed proliferation and propagation, the economic viability of the entire process has also been kept in mind. As first step in this direction, the enzyme re-usability was examined by recovering and reusing the same enzyme mix for its efficacy in releasing the viable protoplast from 100 mg of algal thallus. The experiments conducted to assess the possible reusability of enzyme revealed that it can be reused at least for five times without compromising on the protoplast yield and viability (Table 1.3.3.D). The protoplast yield ranged from 2.0 × 107 to 2.0 × 105 from 1st cycle to 5th cycle, while regeneration rate remained between 91% and 81%. Although protoplast yield and viability showed a declining trend from each cycle, the variations are marginal.
Following the successful demonstration of reusability of enzyme for protoplast preparation from green seaweed Ulva lactuca, a study was undertaken to investigate the biomass yield potential of germlings raised from protoplasts as well as fragments of chopped thallus used for protoplast preparation. A 100 mg of fresh thallus gave a protoplast yield of 2.0 × 107 cells (devoid of cell wall) which showed a regeneration rate of >90% (i.e. ~1.8 × 107 seedlings) and yielded a biomass of 500 mg fresh in 15 days culture period corresponding to a daily growth rate (DGR) of 15.2% in static cultures (assuming 60 mg thallus accounted for protoplast production). These germlings were subsequently transferred into aerated flat bottom round flasks with 400 mL enriched seawater medium and cultured under optimum irradiance and temperature.
The culture medium in flasks was replenished regularly on every second day. The final biomass obtained after 20 days of culture was 53 g fresh accounting for a DGR of 16.8% (Fig. 1.3.3.C). This suggests that aerated cultures triggers growth resulting into higher DGR than static cultures. Another parameter which is of paramount importance for obtaining higher biomass production is stocking density versus volume of culture medium.
In order to understand the relationship between seeding density and volume of culture medium, different seeding densities from 50 mg to 200 mg fresh with an increment of 25 mg each were cultured in a fixed volume of 400 mL medium in aerated flat bottom round flasks for 11 days. Although DGR varied narrowly among the stocking densities investigated, maximum DGR of about 20% was obtained for initial stocking with 100 mg fresh (Table 1.3.3.E). Whereas the DGR for stocking density >150 mg ranged between 17 and 18% but showed an overall declining DGR with increasing stocking density from 50-200 mg fresh. These findings revealed that the production turnover has a direct bearing on biomass density and the volume of culture medium used for their propagation.
It has been often questioned the use of protoplast technology for feeding of the ponds is economically feasible. The suggestions made include the use of technology for initial start-up feeding of ponds, and as backup material bank in case biomass crashes in the ponds. To establish the function of backup material bank, a bench scale model was prepared wherein protoplasts seeding on both sides of acrylic sheets accomplished. For this purpose, two types of tanks were constructed, horizontal tank with dimensions of 60×40×30 cm (Fig. 1.3.3.D) and vertical tank with 60×40×55 cm (Fig. 1.3.3.E). The horizontal tank can house one acrylic sheet for initial protoplast seeding purpose while vertical tank used for propagation of seeded protoplasts and can house upto 6 sheets. Protoplasts were initially seeded on a plate kept in horizontal tank filled with 12 L sterile seawater and incubated for 2 days. Afterwards, plate was reversed upside down and seeded on to the other side and incubated for another 2 days. After a total of 4 days of incubation in horizontal tank, sheet was transferred to vertical tank. The vertical tank was containing about 55 L sterile water enriched with MP I medium. On the same day, another sheet is kept in horizontal tank and seeded on one side. Likewise the cycle was continued. This model would work well as seed bank for the supply of biomass to the floating tanks.
The lab scale propagation model investigated in this study lead to design a layout for scaled up production of seed material which can effectively be used for periodical feeding of culture system throughout the year regardless of its availability in the nature (Fig. 1.3.3.F).
Demonstration of protoplast isolation, seeding and regeneration from Ulva in Siciliy
The findings investigated in India during the study period were replicated for the species of Ulva collected from Sicily coast. Following the above described methods, protoplasts were successfully isolated from Ulva laetevirens collected from Sicily, Italy. After three days of culture more than 90% of isolated protoplasts were viable, cell walls were well developed and visible at the light microscope (Fig 1.3.3.G a-c). Each regenerated cells had a typical cup shaped parietal chloroplast and some already initiated the first asymmetrical cell division (Fig 1.3.3.G d). Germlings continued their growth following the typical filamentous type development already reported for Ulva species (Fig 1.3.3.G e-h). Protoplasts seeded in the culture tanks developed in regular plantlets settled on acrylate sheets.

1.3.4. Studies about biowastes and emission recollecting and sorting
WP3 goals were
• Selecting adequate biowastes to be used for algae growth, based on its availability and collection system and reliable biowaste suppliers,
• Studying and carrying out a piping system for CO2 transport from steam generator, CHP and upgrading plant to open ponds.
First of all, a research on animal manures available in the Syracuse area was carried out in 2010-2011. This research led to the identification of five possible manure in the area: three from cattle, the fourth from pig manure, the fifth from caged laying hens. With its 45-48% of dry matter, more than 60% VOC and 3.6% of Ammonia, hens manure perfectly fitted with the need to balance biomass with high water content like seaweeds. Moreover, manure from laying hens may represent an important source of organic carbon, with a production of almost 900,000 tonnes in Italy and over 6,000,000 tonnes per year in Europe.
The second job has been more complicate: it started in late 2011 and ended at the end of the project. A pipeline was built to feed the floating ponds with the exhausts from the steam generator of the neighbouring factory Maxcom, but the gas injection operations have been affected by the delay in biogas and electricity production. The results demonstrated that the algal cultivation, in the transition from laboratory scale (tens of liters) or pilot plant (a few thousand liters) to an "industrial" small scale plant, (hundreds of thousands or millions of liters of water), suffers enormously of the energy spent for transportation of flue gas from the production site to the place of cultivation.
Summary of results of work
The results arising from the activities allow us to make some important considerations:
1) considering the methane yield not on dry matter but on the as such, the hens manure proved to be an excellent feedstock for anaerobic digestion processes, at the level of corn grain, especially in combination with other residual biomass with high water content such as OMWW and fresh beached seaweed.
The mixing system used in the B4B preparation tank allows you to automatically manage this mix hens manure/OMWW/algae.
The spread on the territory of laying hen farms lends itself to create small installations of AD with cogeneration of electricity and heat;
2) The effect of air bubbling increases 12-15% algae production in floating ponds, compared to that obtained with only the up and down movement of the water caused by the waves of the sea, for the same nutrients provided.
3) The cultivation campaigns of May 2013 and September/October 2014 have shown that the production increase obtained with the air bubbling caused by the supercavitating propeller of the harvesting system RoboHARV, is strictly consistent to that obtained by direct insufflation of air through tubes, but with a very low energy consumption of only 750 Wh per hour of operation. The comparison refers to a cultivation of 900 m2 floating ponds .
4) The effect of air bubbling obtained by insufflation of industrial flue gases, in an open seaweed cultivation ponds, needs an efficient and reliable automatic system to avoid a rapid drop of pH in culture medium, due to the slowness of the photosynthesis process and to its strong dependence on the sky conditions.
The slowness of the seaweeds photosynthesis process is the reason why CO2 injection or flue gas injection leads to identical results for algal growth, with an increase of algae production estimated at around 10%, lower then the CO2-biomass conversion obtained by means of microalgae cultivation.
5) COD, Ammonia and Phosphorus content quickly decrease both in the open ponds and laboratory scale tests carried out in the project. In this case, the results obtained in the laboratory and in pilot plants have been replicated with great fidelity.

1.3.5. The LCA study carried out on the overall B4B production process
The LCA study carried out for the B4B process is an insight assessment of the environmental effects related to the implementation of the B4B system. Specifically a detailed analysis regarding the calculation of the overall environmental benefits, taking into account all the potential benefits of the system, is performed. The results of the study represent a useful tool to understand the overall environmental performances of the B4B novel system for the biogas production and thus representing a useful benchmarking instrument for stakeholder and investors.
The work has been divided in the following main sections:
• LCA of the B4B system – evaluation of the base scenario and preliminary comparative scenario. The study performed aims to figure out whether seaweed (macroalgae) cultivated in near-shore open ponds could be considered a beneficial aspect as a source of biomass for biogas production within the co-digestion with local agricultural biological waste and evaluate the level of sustainability respect conventional biogas production routes and use of fossil-based fuels (i.e. natural gas).
• LCAs of alternative feeding strategies for the reduction of the overall impact. Respect the B4B scenario presented within the preliminary study, a further LCA study has been performed in order to evaluate the reduction of the overall environmental burden based on a different mix of local biomass feedstocks. Namely three scenarios including a mix of of algae biomass, poultry manure, Olive Mill Waste Water (OMWW), citrus pulp, food waste (specifically from kinder garden canteens and meat industries as leftovers of butchered meat) have been compared.
The study would like to evaluate the more sustainable scenario of biomass feedstock only with a simple optimisation of the input of biomass mix in order to maximize the environmental performances of the B4B plant.
• Sensitivity analysis. The analyzed process within the LCA depends on a large amount of parameters and assumption made. For this reason a sensitivity analysis based on the variation of initial inputs has been proposed. Specific attention was devoted to the information and assumptions made on: average transport distance of the biomass, NH3 emissions from biomass storage, average biogas yield from biomass, Electric and thermal self-consumptions, electric and thermal efficiencies of the CHP.
• Operational guidelines. Based on the results of the sensitivity analyses specific operational guidelines have been discussed.

LCA of the B4B: base scenario and preliminary comparative scenarios
According to the ISO STANDARD 14044 and the ILCD Handbook (guidelines European Commission, 2011) have been firstly compared a preliminary base scenario (i.e. the biogas production system of the proposed novel production biogas scheme based on algae - scenario A) and 2 initial comparative scenarios (i.e. a conventional biogas plant using an agricultural mix as feedstock - scenario B), a non-renewable production pathway using natural gas instead of biogas as fuel - scenario C).

Goal and scope
The main aim of the proposed study was addressed to the evaluation of the potential environmental impacts caused by biomethane production from macroalgae and its combustion in a CHP unit with 40 kWel capacity for the final production of electrical and thermal energy. For this specific case the selected LCA inventory included all the main steps: from the algal biomass cultivation and harvesting (in open ponds), followed by anaerobic co-digestion with manure and other type of biowastes for biogas production, its final combustion within a CHP unit. The construction phase has been taken into account together with the extraction and transportation of resources (but not the dismantling of the facility).
According to the main function of the system (i.e. production of heat and electricity) joint with the use of the digestate as organic compost for soil fertilization the functional unit was defined as: 1.02 kWh of electrical energy, 10.92 MJ of thermal energy, 1.86 kg of compost.
The overall scheme of the adopted LCA is reported in figure 1.3.5.A starting from the initial seaweed cultivation till the final CHP unit. All the principal information for the inventory were collected from the information received by all B4B partners taking into account the technical design and the experimental part. Thee resting of the the input data has been selected from literature and directly from the Ecoinvent database. Within the study the effects of different allocation criteria methods have been evaluated.

Life Cycle Inventory
The main results from the inventory are reported in table 1.3.5.A and 1.3.5.B taking into account all the materials used for the realization of the plant components, the respective amounts and considering a life span of 15 years for the plant.
The total amount of the algal biomass produced has been calculated taking into the inputs to the cultivation, the consumptions, the emissions and outputs. These calculatione are affected by the time of the algae growing within the cultivation phase, the initial mass of algae placed in the ponds, the daily growth rate (DGR). This last parameter has been considered affected by: the type of seaweed supposed to be cultivated (i.e. Ulva Enteromorpha prolifera and Ulva lactuca), the amount of CO2 injected, the addition of inorganic nutrients, the photoperiod, and the solar irradiation. The scheme is reported in the figure 1.3.5.B.
The chemical compositions of the Ulva lactuca assumed for the calculation is reported in the table 1.3.5.C. The seasonal variation of the growing rate due the temperature has been considered on a range of 15-30% with a biomethane yield approximately around 280-560[lCH4/kgV.S]..
The mass balance of the CO2 injected within the pond was also evaluated taking into account the inlet CO2 in the open pond defined as
CO2 in=CO2rel+CO2bm+COd.p.+CO2wat
The overall scheme is reported in the figures 1.3.5.B and 1.3.5.C.
The inlet CO2in was calculated taking into account the CO2 equivalents from a crude oil-based boiler. In this way was possible to define the CO2 release in the atmosphere from the ponds.
The inventory was completed by the transportation distances assumed depending on distances locally defined and the number of trips of the transportation vehicles.
The phase of the plant construction has been considered within the overall impact, but at this stage of the study the dismantling and land filling was not included within the study. The overall summary of the main values for the inventory are reported in Table 1.3.5.A 1.3.5.B 1.3.5.D where the plant components, matter and energy consumption are reported, including all the steps of the whole system.
The values used within the biomass transportation unit process have been evaluated with reference to the weekly transportation of biomass from local suppliers providing the requested plant feedstock.
The considered main biomass contributing to the co-digestion of algae biomass for the base scenario A is poultry manure, Olive Mill Waste Water (OMWW) and citrus pulp. The feedstock is stored separately in special containers and then sent to the next shredding stage prior to the correct evaluation of the required amount. The storage period of approximately 7 days with production of GHG emissions, together with acidification and eutrophication compounds has been evaluated and (conservatively) considered within the modelling phase.
For this case study, emission from the poultry manure storage (principally CH4 and H2S) have been considered negligible, due to the limited duration of the storage; while N2O and ammonia emissions have been considered since they take place in the very first days of storage, and the values have been obtained according to biomass composition from analyses performed on biomass used in the plant.
The environmental impact of Olive Mill Waste Water (OMWW) is related to the release in ground waters of polyphenols, and COD (Chemical Oxygen Demand). Polyphenols content from specific analysis on local OMWW have been used. Gaseous emissions for the storage of Citrus Pulp are gathered from literature data.
Within the biomass pretreatment process unit, the following steps have been taken into account: collection, dewatering and desalinization of the algae, manure and citrus pulp pre-treatment (chopping and handling), biomass feeding to the preparation tank and residence time in the homogenization tank, where a better homogenization of biomass over time is performed prior submission to the AD processes.
The main steps of the 2-stage anaerobic co-digestion have been considered, including the following processes: biomass injection to the hydrolysis and methanisation reactors, biogas collection and digestate extraction. The conversion process is performed at mesophylic conditions; the amount of the required heat was evaluated as equivalent to 1 MJ per m3 of biogas produced.
To estimate the annual production of biogas within the installation, the following calculation and assumptions have been performed: evaluation of the carbon content within each feedstock biomass, evaluation of the biogas yields from each feeding biomass, efficiency of bio-degradation activity within the digesters equal to 70%; decrease in carbon content due to volatilization of CO2 during the storage phase were taken into account.
The positive environmental impact due to the anaerobic digestion process is mainly related to the avoided emissions that would occur if biomass, instead of being used as feedstock, would be composted or spread on land according to conventional methods. With regard to poultry manure and citrus pulp, the overall avoided emissions have been calculated mainly according to IPCC factors, specifically in terms of ammonia (NH3), hydrogen sulphide (H2S), nitrous oxide (N2O), biogenic carbon dioxide (CO2) and biogenic methane (CH4) (with regard to climate change impacts). Only the avoided emissions of methane and carbon dioxide were allocated to the process of anaerobic digestion while the rest have been associated with nitro-denitro and the biogas upgrading process.
At the basis of this choice, there is the fact that during the digestion step there are neither nitrogen nor sulfide removal dynamics. Avoided methane emissions were calculated using the model proposed by IPCC. Within the impact given during the cultivation phase, the results show that nutrient supply and water refilling in the ponds play a crucial role in terms of energy within the unit processes.

Results from the impact analysis
The results of LCA for Scenario A (see figure 1.3.5.D) show that the most beneficial effect on the process considered is related to the avoided impact from the use of a locally produced biofuel. From a quantitative aspect, this represents 74% of the overall beneficial impact on environment (82% of overall balance). While the effects of disposal of poultry manure and OMWW represent 11% of positive impacts (12% of overall balance).
One environmental hot spot of the plant is related to gathering and storage of poultry manure: in fact the weekly transportation represents a relevant impact in terms of fossil fuel consumption and climate change effects. Ammonia emissions in the storage phase are responsible for impacts on respiratory inorganic compound emissions and on acidification and eutrophication effects with a share of 35% in respect to the total negative impacts of the plant (5% of overall balance). The other transport processes and biogas upgrading allocate 31% of the total negative impacts (4% of global result).
In regard to the impact category “respiratory inorganics”, the results obtained are connected to emissions of NH3 in the storage phase of poultry manure and the use of trucks for transportation of biomass. Within the mid-point category, there is an evident effect of the avoided emissions due to the substitution of electrical energy from conventional sources (shares 50% of the total reduction), substitution of compost from conventional composting facility (17 % of total reduction) and avoided emissions of NH3 due to the treatment of poultry manure and citrus pulp in the plant (30 % of total reduction).
The impact due to biomethane production presents a relatively low share of the overall impact. The substitution method used to evaluate the beneficial effect on the use of AD digestate as a by-product is reflected as a positive impact in terms of avoided production of compost. The overall assembly of the plant facilities, considered with a technical life-span of 15 years, represents the most substantial impact mainly due to the energy consumption and use of fossil fuels necessary for raw material extraction and their treatment for building the main plant components. This affects mostly the impact category of use of non-renewable resources and respiratory inorganics.
The main normalized results among the 3 scenarios are presented in Figure 1.3.5.E reported in terms of final damage categories. The results confirm that both scenarios A and B present an environmental performance which is much better than that achieved with conventional non-renewable-based technologies (Scenario C). Due to better insight into the results, it has been noticed that the process of cogeneration provides the most evident beneficial contribution to scenarios A and B with regard to the impact category of fossil resource depletion. The reason why scenario A gives the worst result in this category is that, given its pilot scale and pre-industrial development level, the examined system has higher levels of self-consumption.
Moreover, the production of compost as a by-product is beneficial in terms of decreasing impact. That is mainly relevant to Human Health and Ecosystem quality impact categories, which, specifically for both scenarios A and B, are accounted for a share of approximately 45%. Scenario B presents a higher beneficial effect for depletion of fossil based-source due to a scale effect (Scenario A is based on a pilot scale plant).
It is observed that the implementation of algae cultivation, and thus the use of algae biomass, provides an improvement in environmental performance at around 10% with respect to Scenario B and 38 times lower impact than in Scenario C. One important aspect should be devoted to the Land use damage category. In fact, the results highlight that Scenario B (conventional biogas) results in the worst overall performance: this is mainly due to the higher rate of land occupation needed for dedicated energy crops. This aspect in turn highlights the beneficial effects of having an exploitable biomass, with high photosynthetic efficiency, in the near-shore open ponds. On the other hand, the overall beneficial effect should be evaluated in a comprehensive way; in fact, the use of a Pressure Swing Adsorption (PSA) upgrading unit technology (considered in the analysed system) consumes 29% of the gross electricity produced.
The study proposed at the first stage highlights that biogas production from fresh algae co-digestion with manure and/or agricultural waste seems to be a valuable bioenergy root from an environmental point of view, if compared to the conventional techniques. The implementation of a scale-up system of the proposed concept would be beneficial in order to better understand the real environmental hot-spots at a larger scale of production and thus to study the real economic feasibility within an industrial market.

LCA OF ALTERNATIVE FEEDING STRATEGIES FOR THE REDUCTION OF THE OVERALL IMPACT
Goal and scope
Respect the B4B preliminary base scenario and the 2 comparative alternatives presented within the preliminary study a further LCA study has been performed in order to evaluate the reduction of the overall environmental burden based on a different mix of local biomass feedstocks and an optimization of the transport distance. Namely three scenarios including the use of algae biomass (ulva lactuca), poultry manure, Olive Mill Waste Water (OMWW), citrus pulp, food waste (specifically from kinder garden canteens and meat industries as leftovers of butchered meat) have been compared.
The study would like to evaluate the more sustainable scenario of biomass feedstock only with a simple optimisation of the input of biomass mix in order to maximize the environmental performances of the B4B plant.

Life Cycle Inventory
In the tables 1.3.5.E/F/G/H are reported the yearly amounts of the biomass used for each type of scenario considered.
All the emissions within the biomass storage phase (mostly in terms of biogenic CO2 and NH3) have been calculated from assumptions based on a literature review of several study.
Starting from the biomass composition of each feedstock (reported in the table 1.3.5.J) it has been calculated the theoretical biogas yield and thus the overall yearly amounts (table 1.3.5.K).
Within the evaluation of the biogas production from the citrus pulp, leftovers of butchered meat and kindergarden canteen leftovers has been considered the amount of the C emitted as CO2 during the initial phases of the storage. The estimation have been based on the studies proposed by Barrena (Barrena et al., 2008) specifically addressed to the leftovers of butchered meat, and by Beck-Friis (Beck-Friis, et al., 2001) specifically addressed to the food canteen leftover and the citrus pulp.
An important aspect considered was the evaluation of the thermal energy demand during the hydrolysis and methanation phases. Thus the overall energy balance has been calculated through the definition of the seasonal external temperatures at the plant site, the temperatures of the hydrolysis and methanation digesters (i.e. 70oC and 37 oC) and the geometry of the equipments. The thermal dispersions through all the external surfaces and the thermal resistance depending on the type of material of the digester have been also evaluated and considered within the calculations.
Depending on type of initial biomass mix the amounts of digestate (both liquid and solid fraction) have been calculated. This aspect was crucial to evaluate the overall amount of the avoided impacts given by the use of digestate for composting.
For all the 3 the scenarios was considered the internal electrical used within the preliminary base scenario A. The overall summary of the inventory proposed for the 3 scenarios is reported in tables 1.3.5.L/M/N.
As briefly mentioned the evaluation of the alternative scenarios considered as avoided impact within the LCA study was a crucial point of the whole study. In this light it was evaluated the alternative scenarios of the use of poultry manure as fertilizer on agricultural land, the avoided impact due to the use of OMWW associated to the release in the groundwater of Polyphenols, the avoided emissions from alternative use of citrus pulp, kindergarden canteen leftovers and leftovers of butchered meat. In the tables 1.3.5.O/P/Q/R a resume of the previous information is reported.
From the figure 1.3.5.F (comparison of results at mid point categories) and based on a more detailed analysis of the outcomes is possible to notice how the cogeneration steps is bringing the most beneficial benefit, where the transport together with the upgrading phases represent the most important impact for all the 3 scenarios. This aspect is related to the main environmental burdens linked to the CH4 and SO2 emissions considered and presented in the overall LCI. Results show the relevance of the transportation and thus the importance to create the optimal logistic scenario regarding the biomass transportation. The contribution of the transportation of the poultry has the lowest share for the Scenario 1.
The relevance of the impact related to the biomass storage are more evident and critical for the storage of the meat industry leftovers within the Scenario 1 mainly due to the important level of NH3 emitted by these leftovers during the storage phase.
From the analysis of the avoided impact is possible to identify the most relevant beneficial contributions given by the avoided disposal of the leftovers of butchered meat. Specifically it is possible to understand how for the Scenario 1 this effect is more relevant due to the higher amount of leftovers of butchered meat in the feedstock share.
The summary of the overall environmental profile at end-points categories is reported in the figure 1.3.5.G reflecting the best environmental performances given by the scenario 1 mostly in terms of Human Health and Depletion of Resources impact categories.

Overall Technology comparison
In the following part is proposed the overall comparison of the scenarios with the 3 different feedstock mix proposed and those proposed within the preliminary evaluation (i.e. conventional biogas production and the fossil based scenario using natural gas – figure 1.3.5.H).
The overall environmental performance of the conventional biogas is similar to those of the B4B scenarios even if there is an important effect given by the request of arable land for the cultivation of the energy crops.

Summary of results
The hypotheses assumed within the evaluation of the 2 alternative scenarios for feedstock mix within the B4B system have shown the effectiveness respect the base scenario 0.
It has been possible to quantify an improvement of the global environmental performances with reference to the Scenario 0 equal to 3% and 1% respectively for the Scenario 1 and 2. Globally it could be state that the best environmental performances is that one of the Scenario 1 where it has been assumed the hypothesis to maximizes the biogas production independently by the transport optimization.
Specifically the Scenario 1 presents important benefit in regard to the impact category of Resource depletion, while shows a worsening of the environmental of the environmental performances in regard to the Human Health category. Nevertheless it is important to observe that all the scenarios of the B4B show an overall beneficial impact.
It must be stressed that in the Scenario 1 it is possible to have the better environmental improvements in regard to the impact category of Resource depletion (i.e. 49% respect the Scenario 0). For the Scenario 2 this value is around 29%.
The reason of this difference is related to 2 main factors: the higher biogas production within the scenario 1 and the avoided impact given by the alternative way to use the leftovers of butchered meat.

Sensitivity analysis and operational guidelines
Also the effects of a sensitivity analysis to further provide specific guidelines in reference to a proper management and planning of the plant were investigated.
From the analysis of the most significant sub-processes the following parameters were the targets of the proposed sensitivity analysis: average transport distance of the biomass, NH3 emissions from biomass storage, average biogas yield from biomass, electric and thermal plant consumptions, electric and thermal efficiency of the CHP:
The sensitivity analysis is performed by the evaluation of the ratio of the percentage of variation of the output result as a consequence of a variation imposed to one of the inputs parameter (Njakou Djomo, et al., 2011) hereafter defined with Ci.

Results of the analysis
The results of the sensitivity analysis and the main parameters involved within this evaluation are summarized in the chart reported in the table 1.3.5.R. The values of the sensitivity coefficient are compared for variations of ± 10% of the analyzed parameters reported as well in the legend.
Results shows (see Figure 1.3.5.I) that the proposed model is generally robust for all the selected parameters object of the sensitivity (i.e. Ci<1) except for the electrical efficiency of the cogeneration. This exception is however justified in the light of the peculiarities of the process (aimed at the production of energy) and of the used allocation factors of the environmental impacts, which assign to the energy outputs the 85% of the environmental impacts. These, in turn, are divided between heat and electricity production, respectively according to the 63% and 37%.
Analyzing the results more in detail, it is clear that the parameter electrical efficiency of the cogeneration has, on the single score, the only sensitivity coefficient slightly greater than 1 (1.09 and 1.05 respectively for variations of + 10% and -10% compared to the original value).
The thermal efficiency of the cogeneration system has a higher impact than the electrical efficiency only in the Resource Depletion category. This is due to the fact that in the adopted model (for the B4B plant) the produced electricity replaces an equivalent amount of energy generated with the Italian mix of primary resources.
The study of the response of the system to the variations of the inputs can also be extended beyond the simple sensitivity analysis. This basically allows you to validate the used model and to determine the individual parameters which, in the first instance, should be kept under control to avoid compromising the overall result of the system.
Cross-correlating the parameter variations, you get a dynamic simulation of the system answers to the input variations.

Operational Guidelines
From the sensitivity assessment is evaluated the important role covered by the efficiency of the CHP.
In order of priority on the outcomes of the process, the electric self-consumptions and the thermal efficiency of the CHP follow. This means that a positive (or negative) change of one of these parameters is affected only by an opposite variation of a parameter which is hierarchically more significant.
The electric efficiency of the co-generator in the B4B plant, as certified by the suppliers, is equal to 28.5%. It is believed that similar plants can still implement co-generation engines with higher efficiencies than that used in this case, achieving even better results than those obtained with the B4B demonstration plant.
However, the considerations set out so far remain valid, requiring some attention when choosing a CHP, as well as to the maintenance schedule of the equipment: this must be such as not to allow the deterioration of the overall performance of the system, threatening to undermine the efforts made to improve other aspects or individual processes.

Potential Impact:
The most important result of the project is the production of biogas from a mix of algae and biowaste with a fully automated production system. In addition to a quantity of about 90Kg wet per day of algae, other substrates were used in the recipe of the Anaerobic Digestion phase, namely: an activation mix made of rumen and corn, then algae, chicken manure and Olive Mill Waste Water.
The project has clearly demonstrated the usefulness of algae in the input mix to the anaerobic digestion process, however the enormous difficulties encountered in the management of the algae cultivation system (see below), realized for the project in the Augusta bay, suggest the need to acquire this biomass from the environment, by placing such a facility in an area where its growth is widespread due to eutrophication phenomena. For this reason the B4B plant, developed at a pre-industrial scale, in the coming months will be moved to another location in order to carry on the energy production from algae (collected in the environment) and other biowastes.
Many are the potential impacts of what has been achieved in the B4B production process: from a sustainable and low cost biogas production, to the breakdown of costs and environmental impacts associated with the disposal of the algal biomass and of the other used biowastes, to the production of attested biological fertilizers, the use of which can replace the conventional ones thus saving to the environment all the impacts of their production. Last but not least, algae, in their growth in the environment, feed on pollutants, in this way reducing the environmental pressure. Many lagoons, lakes, sea coasts that suffers for nitrifications phenomena caused by an increasing level of nitrogen and phosphorus concentration could be “cleaned” by the B4B process because it has been demonstrated that it is completely sustainable and energetically independent.
The limited extent of the biogas production plant realized for the B4B project represents a high incentive to the spread of this type of facility. Moreover the process versatility assures also a certified waste water treatment in addition to the biogas production.
A feasibility study for the B4B demonstration plant take off in the market, with hypotheses for three new locations for the plant itself, was carried out.
In fact the Consortium is considering to transfer the plant in some Italian areas which are experiencing high levels of criticality in terms of the presence of algal biomass and biowastes from industrial and livestock activities.
The Coordinator of the B4B project, in the past months, has established contacts with stakeholders and local authorities and has carried out a technical-economic study concerning the feasibility of transferring the plant in other Italian locations, depending on the specific availability of biomass and biowastes in the investigated areas.
Based on information received from public institutions and private companies, three possible new locations have been identified:
1. Piombino Dese (Province of Padua, northern Italy);
2. Cisterna di Latina (Province of Latina, central Italy);
3. Goro (Province of Ferrara, northern Italy).
For each of these locations a technical-economic study has been carried out, based on the types and quantities of biomass and biowastes available in the areas, also considering a short supply chain procedure, characterized by a limited number of production steps.
The economic issues concerning the three sites previously exposed were analysed taking into account a few economic indicators in order to understand what could be considered the best scenario among the proposed three. The indicators were: NPV (Net Present Value); IRR (Internal Rate of Return); PAYBACK.
As gains of the plant, the incentives provided for the production of energy from renewable sources in the current Italian legislation were also considered: OMNICOMPREHENSIVE TARIFF (0,236 €/kWh); BONUS C.A.R. (0,01 €/kWh)
At the same time the transportation cost of the biomass was included in the overall evaluation; it varies as a function of the travelled distance, of the number of transports and of the kind of transported biomass (liquid or solid). The economic assessment has stated that the first scenario, Piombino Dese, is the best under the practical and, above all, economic point of view, due to the fact that 2 over 3 of the used biowastes are available on site. For this location the “payback” is the lowest of the three proposed sites, whilst NPV and IRR are the highest.
The studies about biowastes and emission recollecting and sorting have demonstrated that: 1) the spread on the territory of laying hen farms lends itself to create small installations of AD with cogeneration of electricity and heat; 2) the use of macroalgae in cultivation open ponds for CO2 capture does not seem highly efficient from the energetic point of view.
The complete LCA procedure, carried out with a very detailed approach for the whole production process, has studied in depth the environmental and energetic charge of the production system, pointing out the positive or negative generated impacts, even carrying out specific studies on alternative feeding strategies for the reduction of the overall system impact. Also a sensitivity analysis was carried out in order to define the guidelines for the proper management and planning of a plant for the production of biogas through co-digestion of a mix of waste biomass: this last research highlighted what are the most important parameters that cause impact variation (positive or negative) in the whole production process.
The drone Roboharv has fulfilled the task for which it was built. Beyond the capacity to harvest algae, which activities were severely limited due to the problems of the B4B algae cultivation system, the tests have demonstrated the capacity of the drone to move the algae inside the ponds up to one meter depth (with difficulty at greater depths). The release of air in the water also creates a cone that spreads up to 2 m; the propulsion system is sufficient for its intended purpose: up and down movement of algae; release of gas in water, deliberately inducing cavitations in it.
The realized amphibious system has also attracted interest for its versatility: the Coordinator has recently reached an agreement with a marine company in order to develop any technological improvements to the use of the Roboharv drone both for algae collection and for its use with echo sounder instruments in lagoons and coastal areas. The operation of the prototype will then be tested in a lagoon environment both as algae collector and as multisensory marine platform.
A research was carried out about protoplasts with application in laboratory both in India and in Sicily (University of Messina, member of Conisma). Following the successful demonstration of reusability of enzyme for protoplast preparation from green seaweed Ulva lactuca, a study was undertaken to investigate the biomass yield potential of germlings raised from protoplasts as well as fragments of chopped thallus used for protoplast preparation. The lab scale propagation model investigated in this study lead to design a layout for scaled up production of seed material which can effectively be used for periodical feeding of culture system throughout the year regardless of its availability in the nature.
The studies carried out by the Partnership on the different available biogas upgrading systems, especially related to their separation efficiency, will permit the take off of the bio-methane market in the automotive sector, guaranteeing lower emissions at the local and global level and a lower dependence on conventional fuels.
To this extent many studies were also carried out by the Partnership on European legislative tools on biomethane and renewable energies, in particular referring to the legislations of the countries participating to the project. Socio-economic studies were also realized on the same topics.
As affirmed earlier the difficulties encountered in the management of the algae cultivation system (ponds+docks for a total extension of 900 m2) have caused a huge commitment of the Partnership both for the activities necessary to its recovery and in economic terms, greatly increasing the difficulty of the overall management of the demonstration plant. Despite the problems it was possible to carry out both cultivation and harvesting campaigns of the biomass.
The experience gained from the B4B cultivation system has shown that it is possible to reproduce closed and semi-rigid system for research purposes, but not for an “industrial” cultivation of algae.
What happened has demonstrated the possibility to build closed basins with only floating ledges (that is, without the use of semi-rigid docks). In fact, the floaters in the perimeter of the system were effective for three years and have guaranteed the unsinkability of the ponds at the perimeter itself. The solution that is deemed appropriate in order to enable the staff to perform the control operations of the cultivation is to use small watercraft such as, for example, rafts to be constrained to the edges of the pools. In case a cultivation system was necessary for any purpose, it should be redesigned without any rigid walkway, as this element does not appear essential, in our experience. In case it would be indispensable for practical reasons, it would be technically possible to make the structure functional and unsinkable, but with a considerable economic burden for the plant.

List of Websites:
http://www.biowalk4biofuels.eu/