Integrated Sustainable Algae (InteSusAl)Summary of activities

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InteSusAl has received funding from the European Union’s Seventh Programme for research, techno-logical development and demonstration under grant agreement No 268164.

Authors

Andrew Kenny - CPI, UKChris Hainsworth - CPI, UKVictória del Pino - Necton, PortugalYago Del Valle-Inclán - Necton, PortugalInês Povoa - Necton, PortugalLucas J. Stal - Nioz, The NetherlandsDorinde Kleinegris - WUR-FBR, The NetherlandsBen van den Broek - WUR-FBR, The NetherlandsTom Bradley - ORE Catapult, UKVinicius Valente - EUREC, Belgium

Editor

Vinicius Valente - EUREC, Belgium

Table of Contents

Introduction 04

Microalgae yield optimization 09

Biomass Recovery 15

Design of a Heterotrophic Unit 19

Design of an Autotrophic Unit 25

Sustainability 32

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Introduction

This publication sumarises all performed tasks and main findings of the Integrated Sustainable Algae (InteSusAl) project. The document gathers input from all pro-ject partners and is intended to provide a snapshot from the extensive tasks and main outcomes. More detailed information can be found on the project website: www.intesusal-algae.eu

Biomass

Harvesting and oil recovery

Phototrophic units

Heterotrophic units

Glycerol

Sunlight

Nutrients & WaterBiodiesel

Feedstock

Oil

Biomass nutrients& CO2

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InteSusAl is a European collaborative project co-funded by the European Union’s Seventh Framework Programme for research, tech-nological development and demonstration under grant agreement No 268164.

Active since 1st May 2011 and running until 31st July 2016, the project has the objective to demonstrate an integrated approach to produce microalgae in a sustainable manner on an industrial scale.

InteSusAl’s approach optimises the produc-tion of microalgae by both heterotrophic and phototrophic routes and demonstrates the integration of these production tech-nologies (Raceway, PhotoBioReactor and Fermentation) to achieve the algae culti-vation targets of 90-120 dry tonnes per hectare by annumannum (see figure 1).

The project selects algae species and cul-tivation technologies to attain algal oil with a suitable lipid profile for biodiesel production and will validate this selection through conversion of the extracted oil into biodiesel to meet standard specifications.

It is expected from InteSusAl results to be able to significantly contribute to increase the security of energy supply for European Transport needs and greater penetration of Renewable Energy Resources (RES), reducing therefore, EU’s GHG emissions.

The sustainability of this demonstration, in terms of both economic and environmen-tal (closed carbon loop) implications will be considered across the whole process; including optimum use of algal biomass resources to enable commercialisation.

Budget : € 8.6 million

The Project

Figure 1- The InteSusAl approach.

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Centre for Process Innovation (CPI), United Kingdom - www.uk-cpi.com

CPI provides enabling technology and IP related to low cost heterotrophic algal cultivation and experience in collaborative project management. CPI is also the project coordinator.

Necton, Portugal - www.necton.pt

Necton has prior knowledge of microalgal phototrophic cultivation. The demonstration aspects of the project are located at their premises, in the Algarve region of Portugal

Royal Netherlands Institute for Sea Research (NIOZ), The Netherlands - www.nioz.nl

NIOZ provides expert knowledge on the optimum algae strain selection.

Wageningen UR Food & Biobased Research (WUR-FBR), The Netherlands - www.wageningenur.nl

WUR-FBR provides a wide ranging knowledge in the industri-alisation of algae based processes and separation technology. For InteSusAl, the centre focuses on optimum techniques to harvest microalgae.

Offshore Renewable Energy (ORE) Catapult, United Kingdom - www.ore.catapult.org.uk

ORE Catapult bring its expertise in the validation of environ-mental and economic sustainability

The Association of European Renewable Energy Research Centres (EUREC), Belgium - www.eurec.be

EUREC will enable, through established mechanisms, widespread dissemination of the project results.

The Consortium

The InteSusAl consortium is composed of six partners coming from four European countries, whose complementary expertise will enable to successfully deliver the expected results.

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The consortium demonstrates the feasibility of the approach in a one-hectare pilot unit built in the municipality of Olhão, in the Algarve region of Southern Portugal.

The demonstration unit is located in the facil-ity of the company Necton (www.necton.pt). The technology set in situ is composed of 4 m3 heterotrophic fermentation units, 60 m3 tubular PhotoBioReactors and 200 m3 raceways. Demonstration trials started in July 2015.

One-hectare demonstration site

Figure 2- The InteSusAl one-hectare demonstration site.

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and environmental implications, including optimal use of algal biomass resources to enable commercialisation. Within each Algae Cluster project, a detailed Life Cycle Assess-ment (LCA) is currently being undertaken, strictly following the standards ISO 14040 and 14044. The three projects are following the same detailed methodology, which they developed together, and the LCA practi-tioners from each project are in constant contact. This ensures that the LCAs of the three projects is comparable.

Algae Cluster

The European Commission is also participat-ing in the funding of two other large-scale industry-led projects aimed at demonstrating the production of algal biofuels along the whole value chain, covering strain selection to algae cultivation and production, oil extrac-tion, biofuel production and biofuel testing in transportation applications. The projects are named BIOFat and All-Gas, which, together with InteSusAl, have received a total amount of € 20.5 M funding.

The three projects, known together as the Algae Cluster, consider sustainability across the whole process, in terms of both economic www.algaecluster.eu

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Microalgae yield optimization

Responsible experts from NIOZ:

Prof. dr. Lucas J. Stal : lucas.stal@nioz.nl Mr. Abolghasem Hedayatkhah (PhD student) Mrs. Anita Wijnholds Mr. Michele Grego (curator CCY)

Royal Netherlands Institute for Sea ResearchKorringaweg 7 - 4401 NT Yerseke, The Netherlands

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Photosynthetic microorganisms

Cyanobacteria and microalgae are pho-totrophic microorganisms that perform a plant-like photosynthesis. They possess two photosystems that are connected in series and that harvest light with which they gen-erate electrons from the splitting of water. This generates oxygen (therefore this mode of photosynthesis is called ‘oxygenic photo-synthesis’. With the aid of light energy, the electrons are transported to low-potential electron carriers that are mainly used for the fixation of carbon dioxide (CO2) to synthesize structural cell material for growth and stor-age compounds such as glycogen, starch, chrysolaminaran, and lipids. Photosynthesis also generates a proton gradient over the thylakoid membranes (in cyanobacteria and in the chloroplast of the microalgae) that is used to produce ATP to provide the energy

for the metabolic processes and growth of the photosynthetic microorganisms. A small part (10%) of the electrons is used to reduce other nutrients, mainly nitrate that serves as a source of nitrogen.

Storage compounds

While photosynthesis is the main mode of growth of most cyanobacteria and mi-croalgae, it should be realized that this is only possible during the day. In order to survive the night, phototrophic organisms usually store reserve compounds when they photosynthesize and mobilize them during the night. The most important storage compounds are polymers of glucose such as glycogen, starch and chrysolaminaran. These are mobilized and respired during the night, providing at least energy for maintenance purposes, but often also to take up and store nutrients and even for uninterrupted growth of the organism. In the latter case, the carbohydrate storage compounds also provide the organic build-

INTRODUCTION The main objective of this task was to identify and to select cultures of cyanobac-teria and microalgae that are good candidates for mass cultivation with the aim of the production of neutral lipids that serves as raw material for biodiesel. This work comprised the selection of strains with promising neutral lipid contents and subse-quently optimizing the growth (growth rate and yield) and to increase the content of neutral lipids by identifying the optimum culture conditions. One specific task was to investigate the possibility to feed the cultures with glycerol. Glycerol is a waste product from the biodiesel production. It is known to be toxic to some phototrophic microorganisms but in those that tolerate it, glycerol could be used as a carbon source that might increase the production of neutral lipids.

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ing blocks for the synthesis of structural cell material (proteins, cell wall, nucleic acids). While all phototrophic microorganisms contain carbohydrate reserve polymers, many of them also contain more or less neutral lipids. These lipids are supposed to be storage compounds but their func-tion is unsure. It is therefore also unclear why some microalgae and cyanobacteria produce more than others. Since its func-tion is not certain, it is also unclear what conditions would influence the amount of lipid that is stored in the cell. There is no doubt that the carbohydrate reserve compounds serve in the first place as energy storage (and to a lesser extent as carbon storage). It is therefore unlikely that lipids would fulfill the same function. However, neutral lipids may very well provide the organic building blocks for the synthesis of structural cell material, e.g. when growth continues during the night or when CO2 fixation is not taking place. An alternative role for the neutral lipids could be storage of electrons (redox buffer).

PERFORMED TASKS

Strain screening

The tasks that were planned and performed for this work package were the screening of our collection of cyanobacteria and mi-croalgae, CCY – Culture Collection Yerseke (https://ccy.nioz.nl). This collection comprises ~500 strains. The screening was done by using a staining of neutral lipid bodies using Boron-Dipyrromethene (BODIPY) and subsequent analysis by microscopy. The second screening was done by meas-uring lipid content chemically by using the sulfo-phospho-vanillin (SPV) assay. The combination of the outcomes of these assays were combined by other culture properties such as the growth rate and growth yield (should both be as high as possible).

Optimization of growth

A larger amount of work has subsequently been performed to optimize the culture and growth conditions. In the case of one promising strain we have attempted to increase the growth temperature optimum. The rationale was that the original growth optimum temperature was too low to be successfully used for mass cultivation in more sunny places such as the Algarve in Portugal. The approach was to increase the culturing temperature in small steps and keep the strain growing at its higher temperature limit. Other growth conditions that were investigated were salinity and light intensity as well as the addition of glycerol to the medium.

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Optimization of lipid accumulation

The lipid accumulation in selected strains was studied by manipulating nutrient levels (nitrate, phosphate, iron) as well as tem-perature, salinity, light, and glycerol. The lipid content was measured by BODIPY and SPV assay. The lipid composition of the cells was also determined by using gas chromatography. The fate of glycerol was investigated by using 13C-labeled glycerol and mass spectrometry.

OUTCOMES

Screening of the culture collection

About 500 strains of the CCY have been screened. Basically none of the cyanobacte-ria made it through the screening, although some cyanobacteria did produce neutral lipid bodies, in general the amount was low and the strains with lipid bodies did in general not meet our conditions of growth rate and yield. Many strains of diatoms and

CCY0033 + glycerol

+ glycerolCCY0438

CCY0913 + glycerol

Figure 3- Effect of lipid content in the presence and absence of glycerol in different strains of micro algae.

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green algae produced much higher amounts of neutral lipids. From all strains the diatom Phaeodactylum tricornutum CCY0033 was selected as our model strain because of its rapid growth, good growth yield and high lipid content as well as a high tolerance for glycerol. However, we did also experiments with a few other selected strains.

Cellular lipid content

We noted that BODIPY staining showed wide variations of the size of lipid bodies in the cell. Some cells contained very few and small lipid bodies while other cells were almost completely filled with lipid. Of course, the lipid content either per cell volume or per culture volume is an average value. We reasoned that some cells might have up to 90% of their cell volume filled with lipids bodies. We asked ourselves the question what could be the reason for these large differences in lipid content between cells in the same culture and we hypothesized that the cul-ture may contain different cell types. Because cells with different lipid content may also differ in specific weight we tried to separate these cells in a density gradient centrifugation (Percoll). However, this did not result in the expected separation and throughout the gradient the cells were composed of a mixture with different lipid contents. We tried to starve the cells in the dark for 24 h in order to deplete the heavy carbohydrate storage. This did also not result in a sharp band of cells with the same density. Subsequently, we sorted the cells with high lipid content using a FACS (fluorescent-activated cell sorting) flow cytometer and continued growing the cells with the highest lipid content. However, the resulting cultures again were a mixture of cells with low and high lipid content. It is possible that cells with very high lipid content are less fit than those with a lower content.

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Growth phase and nutrient limitation

The accumulation of lipid occurs main-ly in the stationary phase, which can be more than 5 times than during exponential growth. Phaeodactylum tricornutum grown in a continuous culture (in which the growth rate is constant and determined by the dilution (media pump) rate of the culture, did not accumulate lipid. Of all nutrients, nitrogen depletion was the only factor that resulted in high lipid accumulation. Phos-phate and iron had little effect on the lipid accumulation, but affected of course the growth of the organism. In order to grow Phaeodactylum tricornutum under nitrogen limitation we co-cultured this strain with the N2-fixing cyanobacterium Cyanothece sp. However, without added nitrate in the medium, the diatom lost the competition.

Temperature, salinity, and light

The highest temperature that we could grow Phaeodactylum tricornutum was 27oC. Attempts to grow this strain at higher tem-perature failed, although we cannot exclude the possibility that longer adaptation times

are required to increase this limit. High temperature does result in higher lipid accumulation. High light does not result in high lipid accumulation, probably because at high light more carbon is diverted to structural growth. Both aspects are benefi-cial for mass cultivation e.g. in the Algarve, Portugal because of the high temperature (especially in summer), while the light effect will allow denser cultures. Salinity also is beneficial for lipid accumulation, probably through growth limitation.

Glycerol addition

Phaeodactylum tricornutum tolerates up to 100 g/L glycerol which is high compared to many other microalgae. The uptake and metabolism of glycerol in this organism occurs both in the light and in the dark, but only in the light it leads to accumulation of lipid, which can reach up to 40% of the dry weight. Cells that contain such high amounts of lipid are significantly larger (up to 30%) than cells with a low amount of lipid. This is possible because Phaeodacty-lum tricornutum is a diatom that does not always make a silicate frustule, which would otherwise have confined its cell volume.

Lipid composition

The major neutral lipids accumulated by Phaeodactylum tricornutum are C14:0, C16:0, C16:1ω7c, and C18:1ω9c. The C16 lipids are most abundant, especially C16:1ω7c. These lipids are particularly suitable for biodiesel production.

100% N 50% N 10% N 0% N

Figure 4- Increase in lipid content in cells that are starved for nitrogen.

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Biomass recovery

Responsible experts from WUR-FBR:

Dr. Dorinde M.M. Kleinegris: Dorinde.kleinegris@wur.nl

Dr. Lambertus A.M. van den Broek: Ben.vandenbroek@wur.nl

Wageningen UR Food & Biobased ResearchBornse Weilanden 9, 6708 WG Wageningen - The Netherlands.

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PERFORMED TASKS

The first task performed in the InteSuAl-pro-ject was a literature inventory of the different flocculation methods. The main methods are chemical flocculation by addition of e.g. metal salts or polyelectrolytes (such as chitosan, cationic starch and various polymers), elec-tro-coagulation, pH induced flocculation and bioflocculation. In the case of bioflocculation other good-flocculation microorganisms are used to flocculate the microalgae of interest. Apart from electro-coagulation, all methods

were chosen for further testing. The second activity was to set-up flocculation experiments with Chlorella protothecoides. In order to avoid that the biomass concentration or growth phase influences the flocculation efficiency C. protothecoides was grown in turbidostats to provide for a constant supply of biomass for the various experiments. In the next stage Phaeodactylum tricornutum was included to study its flocculation behaviour. Different methods were investigated and also the re-use of media was taken into account. The last task was the research on Nannochlorop-

INTRODUCTION The production cost of microalgae is still high and using microalgae for low value products as biodiesel the operational cost should be drastically decreased. The pro-duction cost is mainly due to high energy inputs required for e.g. water pumping, CO2 transfer, mixing and for harvesting the microalgal biomass. Here we focus on the harvesting of microalgae by flocculation.

Due to the small size of microalgae (µm-scale) and their low concentration in medium (0.5−5 g L-1), harvesting is a major challenge to reduce the production costs. The aim is to obtain a sludge concentrate of 200−250 g L-1, which means that concentration factors of 100-500 are required. Centrifugation is an effective method, but it is also cost and energy expensive. To reduce the cost of harvesting the microalgae biomass can be (pre-)concentrated by flocculation. Flocculation is used to aggregate microalgae cells in order to form flocs with a bigger size and higher sedimentation velocity. The first stage of flocculation is the aggregation of suspended cells into larger particles, resulting from the interaction of the flocculants with the cells (coagulation), the sec-ond step involves the coalescing of aggregates into large flocs that settle out of the suspension (flocculation). Flocculation combined with flotation or sedimentation and subsequent further dewatering by centrifugation or filtration is the most promising cost and efficient alternative for concentrating microalgae and to be able to re-use the medium. An excellent flocculant should be cheap, available at industrial scale, safe, will not modify the quality of biomass, and will not compromise the quality of the remaining medium.

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sis sp. and its flocculation behaviour. After testing many different flocculants with the various microalgae in small tubes, the best performing flocculants were further tested and optimized in lab-scale photobioreactors (Algaemists).

OUTCOMES

A laboratory flocculation protocol was set up and optimized for all three algae strains (Figure 5). Flocculation efficiencies and concentration factors were determined:

thus how much algae were harvested and what was the size of the remaining volume. In Figure 6 the flocculation of P. tricornutum is shown using a polyelectrolyte. The results of cationic polymers for successful floccu-lation of marine microalgae is published in Bioresource Technology1. Direct re-use of medium after addition of polyelectrolytes and subsequently centrifugation was not possible, as the freshly grown microalgae immediately flocculated. However, treat-ment of the medium by e.g. filtration before re-use decreased this problem. Further optimization of this process is still necessary.

Figure 5- Flocculation protocol scheme.

1. ‘t Lam GP, Vermuë MH, Olivieri G, Van den Broek LAM, Barbosa MJ, Eppink MHM, Wijffels RH, Kleinegris DMM (2014) Cationic polymers for successful flocculation of marine microalgae. Bioresource Technology 169:804-807.

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on magnetic stirrer Weight tube + OD750

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Bioflocculation did not work under the tested condi-tions for C. protothecoides and P. tricornutum. The algae did flocculate, but the harvest efficiencies were very low. For all microalgae, pH-induced flocculation worked well, when tested via the protocol described in Figure 1. Increasing the pH results in precipitation of calcium and magnesium salts. The latter play an essential role in the flocculation process. In the lab-scale photobioreactors the pH induced flocculation worked even better. In Figure 7 the results for Nannochloropsis sp. are shown. The pH was increased to pH 10.5 and minutes after the airflow for mixing was stopped, the algae started to coagulate and settle. P. tricornutum was very fast and settled within 8 minutes. For Nannochloropsis sp. this process took up to 90 minutes before all microalgae were settled to the bottom of the reactor. Subsequently the pH of the medium (10.5) was adjusted to pH 7.5 by adding HCl and subsequently filter sterilized. The medium was supplemented with trace elements, phosphate and iron solution, together with nitrate, carbonate and buffer. It was observed that the growth of Nannochloropsis sp. in this or fresh prepared medium was equal.

Figure 6- Phaeodactylum tricornutum flocculated

with a polyelectrolyte. Magnification 100x.

Figure 7- pH-induced floccula-tion of Nannochloropsis sp. in a photobioreactor at pH 11.5. Pictures were taken at

different times after induction of flocculation. The white

arrow indicate the top of the algae pellet.

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Design of a Heterotrophic Unit

Responsible experts from CPI:

Andrew Kenny: andrew.kenny@uk-cpi.com Chris Hainsworth: chris.hainsworth@uk-cpi.com

Centre for Process Innovation (CPI)Wilton Centre,Redcar, TS10 4RFUnited Kingdom

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FERMENTATION DEVELOPMENT

To enable the use of glycerol as a feed stock into the heterotrophic microalgae produc-tion CPI designed, built and commissioned a media clarification unit. The system was designed to process sufficient glycerol containing media to support the hetero-trophic requirements on the 1 hectare site.

Fermentation development activities in-cluded studies to define the operating parameters which have included media development, inoculums seed strategies and glycerol feeding strategies.

Studies were performed using 2 L shake flasks to determine the growth profiles of Chlorella protothecoides. Media composition was varied to utilize technical and waste glycerol to understand the effects on the growth performance.

To ensure a robust process the inoculum volume were altered to understand the effects of using a lower seeding volume without compromising the growth rate.

Figure 8 shows the growth profile of C. protothecoides using varying inoculum volumes using technical grade glycerol. The 0.27 % volume was chosen as it repre-sents a manageable seeding volume (1.6 L) based on a 2 L transfer flask and gives an acceptable process time.

INTRODUCTION The main objective for this work package was to design a low cost heterotrophic fermentation system that could be integrated into the one hectare site, for the pro-duction of high algal biomass.

Carbon dioxide produced during heterotrophic production can be utilized in the autotrophic growth units to ensure a closed carbon loop.

Initial growth was performed using technical grade glycerol to determine the growth of the algae Chlorella protothecoides before running with waste glycerol.

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Figure 2

Figure 8- Growth profiles of different inoculation volumes using technical glycerol.

Figure 9- Growth profiles of different inoculation volumes using waste glycerol.

The inoculum volume experiment was repeated using waste glycerol. Figure 9 shows the growth curves of the three con-ditions which surprisingly found that waste

glycerol was not inhibitory to growth and

had a shorter lag phase. This shows that

waste glycerol could be utilized.

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PROTOTYPE DEVELOPMENT ACTIVITIES

Work package 3 (WP3) outlined the development of the heterotrophic system.

In 2012 the first heterotrophic low cost fermenter (LCF) prototype was designed and constructed. The LCF prototype was a single use plastic bag of 1,000 L volume, Figure 10.

The LCF bag prototype was operated during 2013 to con-firm the productivity targets, however due to robustness issues and poor yields, a redesign of the heterotrophic system was undertaken and a second fermenter designed and built in 2014.

During the period of redesign, fermentations were per-formed at 10,000 L scale at CPI using the basic operating parameters established for cultivating C. protothecoides This data established a benchmark for growth and also helped in the redesign of the LCF.

The new LCF prototype (2a) was a stainless steel design based on an intermediate bulk container (IBC) modified to incorporate an agitator, air sparge line and a cooling jacket, Figure 10.

Growth studies performed showed the fermenter could support algae cultures for a suitable duration to achieve an optimal cycle time for biomass production.

However, unexpectedly it also demonstrated a low biomass productivity when compared to the algal fermentation performed at 10,000 L scale (Figure 11).

Figure 10- Fermenter bag and stainless steel design.

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A computational fluid dynamics (CFD) study of the fermenter design indicated that the combination of a turbine (pitched blade) that drew liquid down through the centre of the vessel and the lack of baffle plates, creating a vortex at higher speeds, resulted in inefficient mixing and therefore poor oxygen transfer to the cells. This led to the development of a second stainless steel fermenter.

The second fermenter (prototype 2b) was a replica of the first but with the addition of baffle plates and an improved impeller. CFD and kLa studies showed that the ox-ygen transfer had doubled. This would indicate that prototype 2b theoretically could support a higher algal biomass and so improve productivity.

DEMONSTRATION OF THE PILOT PLANT

Work package 4 (WP4) was designed to demonstrate the effectiveness of the fer-menter once it had been designed, built and delivered to CPI.

Figure 11 shows the fermentations per-formed to date in the 10,000 L CPI fermenter and the 1,000 L fermenter prototype 2a, with the projected values for prototype 2b. The projected value is based on the improvement to the oxygen mass transfer (kLa) and should enable the fermenter to support a greater level of biomass, bringing the productivity closer to the values displayed by the 10,000 L fermenter.

Figure 11- Comparison of growth in the 10,000 L fermenter and the InteSusAl fermenter.

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Productivity 10,000 L CPI

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Dry Biomass (g/L) 56.5 19.2 50

Dry Biomass (kg/day)* 66.75 3.264 54.3

Dry Biomass (Tones/year)** 22.03 1.077 17.9

Table 1 shows the actual results for the 10,000 L fermenter and fermenter pro-

Table 1: Dry biomass yields

totype 2a with the projected results for prototype 2b.

The objective of the project was to develop a system to produce 90 - 120 tones of dry biomass / hectare/ year of heterotrophic and phototrophic biomass combined.

The demonstrated yield for prototype 2a was 19.2 g/L with a feed rate of ½ vessel volume per day.

* Based on a fermentation duration of 28 days ** Based on 330 production days per year

Initial testing of prototype 2b for the pro-ductivity trails are a biomass yield of 50 g/L and a feed rate of 1.75 vessel volumes per day could be achieved.

Figure 12- Work package 6 productivity targets – total biomass from 1 hectare site.

During WP6, productivity trials, we are aiming to improve operability and pro-ductivity of the heterotrophic system to

increase yields and thus achieve the overall project objective from the 1 hectare site as shown in figure 12.

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Design of an Autotrophic Unit

Responsible experts from Necton:

Victória del Pino: vdelpino@necton.pt Yago Del Valle-Inclán: yago@necton.pt Inês Póvoa: ines.povoa@necton.pt

NectonCompanhia Portuguesa de Culturas Marinhas, S.A.8700-152 Olhão, Portugal

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PERFORMED TASKS

Task 3.2 Autotrophic unit design and build

Development of photobioreactor (PBR) pilot

Sub-task 3.2.2 Development of raceway (RW) pond pilot

Integration of operations

Deliveries and milestones were achieved with the development of prototypes vertical tubes air-lift and raceways.

OUTCOMES

Besides construction delays of DEMO Plant, due to bureaucratic issues, a great know how and improvements were gathered during experiments at prototype level. Based on their high potential for feedstock for oil convertible into biodiesel, Nannochlo-ropsis sp. and Phaeodactylum tricornutum were the selected microalgae to perform

InteSuAl assays. Both these strains are rich in Fatty acid methylesters (FAME) and are known to be relatively easy to scale with high densities.

Between 2013 and 2014, experiments were performed in prototypes of vertical tubes air-lift of 3 m3 and raceways of 500 L to extrapolate the data for the scale up in industrial tubular photobioreactors and raceways.

Experiments for the optimisation of the culture conditions were based on Inte-Susal assumptions towards sustainability and integration namely: i) culture media composition, ii) semi-continuous regime, iii) recycling of culture medium and iv) inoc-ulation rate (%). The use of cheaper and simpler culture media revealed no effect on the growth or in the productivity but a potential in the improvement of lipids profile. Several dilution rates were tested on semi-continuous regime according to production season for both species in order to set data for productivity trials on larger scale systems.

INTRODUCTION Under the WP3 framework, Necton aimed to design an autotrophic unit at proto-type level of raceway and photo-bioreactor technologies to be integrated within the heterotrophic unit of fermenters, to achieve high productivities of dry algae biomass per annum.

Prototype Development Activities

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The feasibility for medium recycling was tested and promising results were obtained. For both microalgae, culture media could be recycled without compromising growth rates or productivity, although further stud-ies are needed to test feasibility on long term runs and impact on biomass composition. The recycled culture medium was tested either by centrifugation or by filtration treatment. Clarification by centrifugation showed better results in terms of microal-gae cell content and colour. Although this is a more efficient process, it involves greater energy and time consumption. On the other hand, filtration process led to a less bacterial contamination, providing encouraging results, nevertheless it needs to be improved.

Tests on raceways prototypes revealed that Phaeodactylum tricornutum can have a limited growth performance due to signif-icant thermal amplitude on these systems.

On the other hand, Nannochloropsis sp. showed a good adaptation and robustness. Evaporation rate on these systems had a negative impact on growth performance during trials, reaching maximum values of 9% per day.

Figure 13- Victória del Pino presenting at the 1st InteSusAl

Workshop on Sustainable Microalgae production on

21 May 2015.

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Demonstration Pilot – Build and Commission

PERFORMED TASKS

Task 4.1 Site Preparation

Task 4.2 Integrated pilot phase 1

Orientation and Geometry of tubular photobioreactors (TPBRs)

Technical project management Topography Simulation of deformation of acrylic tube (1)

Simulation of pultruded fiberglass profiles (2)

Simulation of deformation of TPBR frame (3)

Continuous flowcharting, 2D technical drawing and 3D designing

Task 4.3 Integrated pilot phase 2 (1 hectare)

OUTCOMES

Between 2013 and 2016, under the scope of WP4 (Engineering), several analysis were performed for development and design of InteSuAl DEMO Plant (1ha). This included project management, topographic services, simulations of protruded profiles, deforma-tion simulation of tubular photobioreac-tor frame, and deformation simulation of acrylic tube from TPBR unit, 3D design and technical drawing including building and operation set up of pilot plant.

For the selection of the best configuration, an extensive data analysis of total radi-ation for several orientations and TPBRs disposition were performed. The preferable configuration was the one that provided more productive days throughout the year and was estimated considering optimal conditions for microalgal production. For a set of 4 TPBRs, the best disposition was shown to be East-West due to shadowing effect. The best layout for tubes distribution was 16 horizontal tubes x 5 vertical tubes, which allow an increase of productivity, although the area occupation is also higher.

INTRODUCTION Under the WP4 framework, Necton aimed to build a single pilot line of autotrophic unit to be integrated within heterotrophic unit, to achieve high productivities of dry algae biomass per annum. Deliveries and milestones were achieved with the site preparation and design of single pilot line.

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Productivity trials

PERFORMED TASKS

Task 6.1 – Operation of the initial pilot line

OUTCOMES

During 2015, on the framework of WP6, both strains were cultivated in InteSuAl DEMO Plant (1ha) composed by a total of 60 m3 Tubular Photobioreactors (4 units x 15m3) and 200 m3 raceway (1 unit). The inoculum was grown in Green-wall systems, with total capacity of 8 m3, composed by plastic transparent bags. TPBR´s were inoculated at the end of 2015 and culture conditions used on were as follow: pH set point: 8,5 controlled by carbon dioxide injection diffuser submersed in culture; temperature set point: 26º C controlled by heat exchanger inside the TPBR tank and water sprinklers. Nutrients were fed daily keeping culture at 2 mM nitrates/day. A semi-continuous regime was used with aprox. 20 % daily renewal.

INTRODUCTION Under WP6 framework, Necton aimed to perform productivity trials on autotrophic unit. Deliveries and milestones were achieved for Tubular photobioreactors (TPBR) and data was gathered from growth performance of two microalgae on TPBR. Ongoing tasks will provide data for Life Cycle Inventory (LCI) and business case development.

Figure 14- Tubular Photobioreactors.

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Table 2: Nannochloropsis sp. and Phaeodactylum tricornutum growth and cultivation parameters on TPBR

Nannochloropsis sp.

Phaeodactylum tricornutum

Growth rate / d-1 0,13 0,20

Biomass Productivity / g∙L-1∙d-1 0,3 0,4

Biomass Yield / g∙L-1 4,0 3,1

Areal Productivity / g∙m-2∙d-1 18 19

CO2 consumption (kg CO2/ kg DW biomass) 2,3 2,9

Estimated Biomass Production (t DW/year) 3 2

Nitrate consumption (kg NO3 / kg DW biomass) 0,4 0,4

Table 1 aims to illustrate the data obtained from Nannochloropsis sp. and Phaeodac-tylum tricornutum growth and cultivation parameters on TPBR.

Table 1 aims to illustrate the cultivation data for each strain. It is important to point out that data consider the maximum profitability of the InteSuAl production unit and for that would be necessary to consider two shifts (14h working process). For each strain and concerning the production period within the autotrophic regime, using only

TPBR, it is expected to obtain 3 and 2 ton biomass/year for Nannochloropsis sp. and Phaeodactylum tricornutum, respectively (Table 2). TPBRs have proven to be very effective in CO2 sequestration and biomass productivity.

Monitorisation and control of photobioreac-tors is accomplished by SCADA software in order to optimise productivity and achieve a fully monitored microalgae production. This integrates data from sensors, pH, tem-perature, flow rate, water level, and many others and displays all status parameters of all production plant components.

Data acquisition from 1 ha DEMO Plant (Fig. 1) will allow us to perform Life Cycle Assessment (LCA) for algae biomass pro-duction based on real data and extrapolate for larger-scale facilities. Next step, under the scope of WP5, include the development of a business plan for 10 ha demonstration facility to attract investors.

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Figure 15- InteSuAl DEMO Plant.

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Sustainability

Responsible expert from ORE Catapult:

Tom Bradley - Senior Project Engineer: tom.bradley@narecde.co.uk

Offshore Renewable Energy CatapultNational Renewable Energy Centre Offshore House, Albert StreetBlyth, Northumberland - NE24 1LZ, United Kingdom

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The tasks undertaken were:

1. Goal and Scope Definition – Define the methodology used and the assumptions within the LCA study.

2. Life Cycle Inventory – Gather data on the construction and operation of the facility, and of the technologies it will be compared against.

3. Life Cycle Impact Assessment – The collated data was used to create detailed LCA models to understand not only the impacts compared with other biofuels and with fossil fuels, but additionally to understand where the main sources of impacts are and suggest remedial actions.

4. Interpretation – Detailed assessment of the results of the LCA and their im-plications.

The final objective of this Work Package was to understand the impacts of the In-teSusAl methodology of algae production in comparison with other technologies.

INTRODUCTION There is much debate over the environmental impacts of biofuels, including those of algae based biofuels. In order to understand if algal biofuels truly do offer an environmental benefit over other biofuels and fossil fuels, a detailed analysis had to be undertaken. The methodology used to quantify this was Life Cycle Assessment (LCA). Within the project, an LCA analysis compliant with ISO 14040 and 14044 was undertaken to measure the impacts of both the construction and the operation of the InteSusAl facility.

PERFORMED TASKS

One important element of the LCA study was to ensure that it is comparable with other European demonstrators of algae biofuels. One common issue with algae biofuel LCA, and in fact LCA in general, is that there are many choices on the pa-rameters of the study which can impact the results. Issues can include the boundary conditions, databases, quality of data, impact methodologies used, how co-products and recycling are considered.

In order to ensure that the three projects within the Algae Cluster (InteSusAl, All-Gas and BIOFAT) produced LCA studies which could be compared, a common methodology was agreed between the projects, taking into account the Renewable Energy Directive, ISO 14040/14044, the ILCD handbook, and the methodologies used by current prominent studies.

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Data was gathered on both the phototro-phic and heterotrophic production of algae from the main InteSusAl site in Olhão, as well as fermenters operating at CPI in the UK. This data included all construction materials and products used, as well as all inputs for the operation, and the algae yields produced.

This data was compiled into the industry standard LCA software GaBi, to create de-tailed models of the environmental impacts of the system. Impacts studied were the ReCiPe mid-points (incl. Climate Change, Eutrophication, toxicity, ozone depletion, particulate matter formation, acidification and fossil depletion) and the IPCC AR5 global warming potentials over 20 and 100 year periods. In order to include the latest climate change data within the stud-ies, InteSusAl worked with the company Thinkstep to input the Global Warming Potentials from the IPCC 2013 Synthesis report, as previously only the data from the 2001 and 2007 reports were available for GaBi or any other LCA software.

OUTCOMES

The agreed common methodology within the Algae Cluster was published within Applied Energy as:

Bradley, T., Maga, D., Antón, S., “Unified ap-proach to Life Cycle Assessment between three unique algae biofuel facilities”, Applied Energy, 54, 1052-1061, 2015

Initial results from the InteSusAl system itself show a promising comparison with crop based biofuels and diesel. However the facility is still running and gathering data, hence these results will be refined. Results from the detailed modelling will be previewed at the Algae Roadmap event, and published in full following the completion of the InteSusAl project.

Figure 16- Tom Bradley presenting

at the 23rd European Biomass Conference and

Exhibition in June 2015.

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InteSusAl

C/O EUREC E.E.I.G.

Place du Champ de Mars, 21050 Brussels, Belgium

Tel: +32 2 318 40 47info@intesusal-algae.eu

www.intesusal-algae.eu

InteSusAl has received funding from the European Union’s Seventh Programme for research, technological develop-ment and demonstration under grant agreement No 268164.