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Sustainable production of biologically active molecules of marine based origin

Final Report Summary - BAMMBO (Sustainable production of biologically active molecules of marine based origin)

Executive Summary:

BAMMBO screened and identified target marine invertebrate organisms from diverse global locations for their potential to serve as sustainable producers of high value added bio-molecules (HVABs). Innovative solutions to overcome bottlenecks associated with developing economically sustainable, environmentally friendly and scalable culturing methodologies designed to produce high yields of value added products from marine resources for the pharmaceutical, cosmetic and industrial sectors were created. Novel analytical methods for the extraction, purification and enrichment of targeted bioactive compounds were developed. Life cycle analysis of the production pathways were undertaken to attain an environmentally holistic perspective of the sustainable production potential of HVABs from marine organisms. Knowledge and technology developed during the project in the form of know-how, new discoveries and novel inventions was documented as foreground IP and is progressing through the stages of IP protection. Non-IP sensitive outcomes were widely disseminated to target groups including researchers, policymakers and industrial stakeholders via a wide range of media routes as well as BAMMBO coordinated activities and participation at BAMMBO attended events. The European Strategy for Marine and Maritime Research encouraged capacity-building and promoted integration and synergies across all marine sectors. BAMMBO research has enabled involved RTD and SME partner participant’s to build their research and production teams and further facilitate the mobilization of project participants to exchange technical experience and knowledge. BAMMBO’s outcomes will serve in part to increase the competitiveness of the EU economy based on the capacity to create high value added knowledge based goods and services and foster the sustainable economic development of the marine sector.

Project Context and Objectives:
Summary description of project context and objectives

Europe is well positioned to maximize its marine biotechnology potential not only due to its extensive European coastline but also due to its outermost regions and territories of Europe in diverse zones providing a high marine biodiversity. The maritime economy of the EU comprises a number of different industrial sectors one of which encompasses blue biotechnology. Growth of this sector is anticipated in the next three years with the market projected to surpass €3 billion by 2015. The overall driving objective of BAMMBO is to develop a harmonious commercial relationship with the sea by applying sustainable environmentally kind practices to the valorisation of high value added biomolecules (HVABs) from marine organisms. The consortium hopes this approach will permit a conscientious means to enhance both human and economic benefits from the marine environment while creating new knowledge, processes, products and employment.
Marine ecosystems are biologically diverse competitive environments which have nurtured organisms capable of surviving in a niche existence. Taxonomic groups of interest to BAMMBO span macro-algae, micro-algae, sponges, bacteria, fungi, yeasts and epiphytic bacteria are as varied as the environmental conditions in which they reside. The unique ability of these organisms to cope with harsh environments renders these organisms as ideal candidates for novel sources of both pre-existing and novel HVABs with potential for providing sustainable economic and human benefits. A consistent supply of marine derived HVABs is one of the major limiting factors for biotechnological development. Unless there is a feasible alternative to wild harvesting, promising bioactive molecules are under threat of being undeveloped.
Irrespective of the marine organisms selected within BAMMBO sustainable cultivation methods are essential and in development within this three year project. A lack of understanding of growth life cycle parameters, symbiotic relationships and nutritional requirements for cultivation of marine organisms has hindered progress of this area in the past. Enclosed bioreactor systems for targeted organism cultivation having minimal environmental impact are essential to overcome bottlenecks associated with sustainable commercial culturing of marine life (See Table 1 Bottlenecks to commercial exploitation of sustainable marine derived HVABs). In terms of novelty marine life forms from diverse environments such as the Antarctic, Arctic White Sea Coast, Mediterranean and Atlantic Coasts were evaluated within BAMMBO as sources of both known and novel HVABs. Selection of organisms and HVABs were aligned with industry stakeholder needs based on feedback and many years of applied research with industrial partners. Within BAMMBO existing bio-banks and waste streams were bio-prospected with the intent of minimizing environmental impact and destructive in situ harvesting. Applied culturing and capture technologies were developed which were industrially sustainable and scalable. Target compounds progressing through BAMMBO were considered in a life cycle context to attain a holistic evaluation encompassing environmental, health and economic aspects. A comprehensive life cycle assessment (LCA) was applied to ensure the environmentally conscientious sustainability of entire production pathways from organism to final product. Where applicable LCA also permitted an effective economic comparison to be made between HVABs produced by advanced technologies and alternatives currently in the market place.

Main BAMMBO Project Objectives
1. To target known marine organisms, and screen and identify novel marine organisms, from diverse global locations for potential as sustainable producers of high added value compounds for use in the pharmaceutical, cosmetic and industrial sectors.
2. To overcome bottle-necks associated with developing economically sustainable and scalable culturing methodologies for marine organisms, designed to produce high yields of high value-added biomolecules while avoiding exploitation of natural marine resources.
3. To extract, isolate, identify, purify and enrich high added value compounds from marine sponges, algae, bacteria and fungi and to establish bio-active screening procedures enabling high-throughput screening of bio-bank samples.
4. To conduct a full socio-economic, environmental and sustainability assessment on the use of marine organisms for industrial scale production of high added value compounds, to ensure sustainable production pathways.
5. Exploit knowledge/technology developed during the project and effectively manage their transfer to achieve maximum impact for partners and stakeholders.

BAMMBO’s overall aim was the design of sustainable culturing technologies for novel marine organisms. This was in conjunction with bio-prospecting, purification and analysis to develop strategies for industrial scale production and commercialization of novel bio-products from marine ecosystems in an environmentally conscientious manner. The approach taken to address these objectives was to structure a project into one which comprised 9 inter-related workpackages with associated tasks, milestones and deliverables. Each workpackage was headed up by a Workpackage Leader within the consortium partner group. The BAMMBO workpackages were inter-related and focused on key stages of the project which ran for different time periods over the project three year period (Figure 1). The main research activities were categorised into workpackage 1 (MGT), workpackages 2-5, 9 (RTD), workpackages 6-7 (Dem), workpackage 8 (OTH).

Project Results:
Description of the Main Science and Technology Results/Foregrounds

The main science and technology outcomes for BAMMBO are outlined below in the order in which they were addressed within the BAMMBO description of Work (Annex I).

Workpackage 2: Marine Organism Bio-Prospecting

Workpackage 2 was led by UNS, France which focused on the screening of marine invertebrate species belonging to micro-algae, macro-algae, sponge, fungi, bacteria and yeast which were targeted for initial investigations that focused on the bioactivity guided screening and selection of target organisms (Table 2.0). Choice of species for initial analysis was based equally on BAMMBO culturing and extraction expertise as much as the need to use both reference and commercially viable strains for sustainable high value added biomolecules (HVABs) production. The selection of specific species of invertebrates and the targeting of HVABs was supported by a strategic market analysis and IP data assessment which was compiled by LIT based on past experience of industrial stakeholder supports offered through the Shannon Applied Biotechnology Centre, Ireland. The main bottlenecks to the production of targeted HVABs in the various marine taxonomic groups were also identified and considered in developing the research approach (Table 1 BAMMBO Bottlenecks).
An inventoried virtual repository of these organisms was generated as was a bio-bank of detailed information relating to the sustainable culturing and subsequent processing and screening of selected targeted organisms for HVABs (see www.bammbo.eu). A standardized protocol for the classification of the BAMMBO bio-bank was established. The database is accessible via the BAMMBO internet and/or intranet portals. An easy to use set of instructions for the use of the database system was developed which allowed for the capture of information relating to the taxonomic classification of the organism, collection date, country of collection, GPS coordinates of sample collection, number of samples taken, sample code generated, associated partner holding sample and a picture of the sample site and organism.

Sponges
Two target Mediterranean sponge species were targeted for the isolation and structure identification of HVABs from (i) Crambe crambe for the presence of bioactive guanidine alkaloids and (ii) Sarcotragus spinosulus for the presence of bioactive quinone- or furanoterpenes (Figure 2). The Laboratoire de Chimie des Molécules Bioactives et des Arômes (LCMBA) based at the University of Nice-Sophia Antipolis (UNS) first decided to investigate the secondary metabolome of Crambe crambe species in order to obtain the standards required for the quantification and analytical processes which were to ensue within the time course of the BAMMBO project as part of workpackages 4 and 5. Altogether 11 guanidine alkaloids were purified from this sponge of which 8 were novel and previously undescribed. Purified and structurally characterised standard reference samples generated by the UNS group served as the basis of extensive cytological bioassays performed in collaboration with the USC partner as detailed in workpackage 5.
Two families of crambescins (A, B and C) and crambescidins were identified. Structural elucidation was achieved using NMR and mass spectroscopies which identified two families of crambescins (A, B and C) and crambescidins. Due to the high complexity of the mixture to be quantified UNS developed a process based on a 2D UHPLC-qToF quantification which proved to be highly efficient in terms of time, sensitivity and resolution of the target compounds.
Sarcotragus spinosulus is a horizontally flattened blackish sponge with regular finely conulose surface. It is a southern species found around Atlantic and Mediterranean Coasts. Cytotoxic compounds comprising a variety of polyprenylhydroquinones and furanoterpenes including Sarcotin A were reported and identified by UNIGE during the course of BAMMBO research of extracts from this sponge.

Sponge Associated Bacteria
Sponges host large populations of symbiotic/epiphytic/associated microbes which have received considerable attention recently since specific species of these bacteria were shown to be producers of bioactive metabolites previously ascribed to their hosts. A key query of BAMMBO was to resolve whether or not epiphytic/associated bacteria of these hosts contributed to the generation of an otherwise regarded sponge bioactive molecule. Answering this question was more to do with commercial exploitation than with scientific curiosity as destructive harvesting of slow growing sponges represented a bottleneck to the commercial exploitation of the sponges. In contrast bacterial species with a demonstrated ability to produce a sponge HVAB could be fermented at a scalable level with enhanced commercial exploitation potential. Thus an assessment of the origin of bioactive metabolites from sponge cells or symbiotic bacteria was pursued which entailed co-culture and cell separation studies of isolated and mixed populations of sponge liberated bacteria. The study demonstrated that the guanidine alkaloids and furanoterpenes were not derived from the sponge co-habitants.

Microalgae
Standard protocols for growth, harvesting and screening of microalgal HVABs were developed by UGent. Phaeodactylum tricornutum, Cylindrotheca closterium and Haematococcus pluvialis were chosen as models for this purpose as these species were known to produce bioactive molecules with a defined and immediate market application, i.e EPA, fucoxanthin and astaxanthin, respectively. It is accepted that both harvest method and harvest time within the growth curve influences microalgal biomass composition. Standardised protocols were needed for potential large scale HVAB production (WP6) and LCA (WP7) studies as BAMMBO progressed. Growth curves were assessed using growth trial replicates assessed by microscope counting as well as absorbance based microtitre standardised accepted methods. Lipid, protein, carbohydrate and specific targeted HVAB analyses were performed on culture filtrates harvested across the microalga periods of growth. The specific properties and main findings of work with these microalgae are outlined.

P. tricornutum is a fast growing diatom which has emerged in recent years as a model system for molecular studies due to its suitability to genetically engineering. It is of particular interest biochemically since it contains high concentrations of polyunsaturated fatty acids (PUFAs) especially those classed as ?- 3 fatty acid including Eicosapentaenoic acid (EPA). The exploitation committee decided to cease work with these microalgae at the 12 month stage of the project due to the relatively non-competitive yield of EPA achieved as compared to other fermentable microbial sources supplying the market.
C. closterium is a common coastal marine diatom occurring in planktonic and benthic states with the ability to generate fucoxanthin with reported potent antioxidant, anti- inflammatory, anti-cancer, anti-obese, anti-diabetic, anti-angiogenic, and anti-malarial activities. Due to the benthic nature of this organism the consortium decided to cease further studies on this organism as it was not deemed a sustainable source of fucoxanthin.
H. pluvialis is capable of accumulating high levels (0.2-2% w/w dried biomass) of the carotenoid astaxanthin. Astaxanthin is used in the aquaculture industry as a natural pigment for farmed fish. It is also a powerful antioxidant with an activity surpassing that of ß-carotene and Vitamins C and E. Production of H. pluvialis at large scale was done via a two-step approach (i) vegetative cells grown over several days under nutrient sufficient conditions and low irradiance to build biomass and allow sedimentation and (ii) cells were exposed to low nutrient medium ± salt and temperature manipulations in combination with high average irradiance exposure. It was decided to continue work with H. pluvialis due to its commercial relevance to the SME partners. Optimal growth conditions for H. pluvialis astaxanthin production were; Medium (BBM), Salinity (2.5g/L= 4 PSU), pH 7.0 Temperature (23°C), Photoperiod (Continuous), Light intensity (60 µmol.photons.m-2.s-1) Aeration/mixing (Constant aeration at 0.1L/min of mixture of CO2 (1%) and air). In terms of growth H. pluvialis was generally viewed as a problematic species due to its slow growth and vulnerability to contamination which added additional cost to scaled-up processes. It was determined that production of H. pluvialis should be managed as a semi continuous or batch cultivation two stage process. Although nitrate deprivation in combination with high light intensity proved to be the best stressor for inducing astaxanthin accumulation at small scale, the collection of the species before applying this stress was crucial. H. pluvialis was deemed to have a high intraspecific variation in astaxanthin production. Growth conditions were further optimised and enhanced by the BAMMBO SMEs Algae Health and Greensea using their own commercial scale bioreactor unit systems (see WP6).
Gambierdiscus toxicus is a dinoflaggelate habitant of warm shallow reefs in tropical and sub-tropical zones. It is the source of the lipid-soluble ciguatera (a common seafood intoxicant derived from tropical marine finfish) toxins. G. toxicus contains the potent polar ciguatera toxin, maitotoxin, which blocks voltage dependent ion channels leading ultimately to cell death. Maitotoxin is the largest molecule in nature and its mechanism of action is unknown. It has been clearly linked to calcium increase mechanisms which are sustained and cytotoxic. Due to the large size and potency of the molecule it offers the potential to be a lead molecule for drug development.
Lesser known microalgae
UGent in collaboration with Algae Health and Greensea performed an extensive screen of lesser known microalgae species with potentially interesting fatty acid and/or pigment profiles. This work was not destined for extensive progression through BAMMBO but as possible microalgal alternatives on the longer and future terms. The species of microalgae screened for the quantified presence of targeted HVABs were Phaedactylum, Chaetoceros, Tetraselmis, Nannochloropsis, and Porphyridium, Ankistrodesmus sp., Desmodesmus sp. and Monoraphidium sp. The range of HVABs screened included EPA, DHA, lutein, aand ?-carotene, fucoxanthin, zeaxanthin, phycobiliproteins, chlorophillide a, chlorophyll A, B and C2, neoxanthin, violaxanthin, astaxanthin, diadinoxanthin, antheraxanthin, alloxanthin and diatoxanthin. Standard accepted methodologies were applied to quantify total pigments (as percentage total pigments/dry weight) and saturated, monounsaturated and polyunsaturated fatty acids (as percentage of total fatty acids) at both early exponential and late stationary phase growth. HVAB profiles for selected microalgae species were obtained. The overall conclusion was that both age of the culture (exponential and stationary) significantly affected the chemical composition of Ankistrodesmus, Desmodesmus and Monoraphidium. Secondly UGent showed that within the same species, two strains could also differ in their chemical composition.

Antarctic Fungi and Yeast
Selection of targeted Antarctic fungi and yeasts for lipases and ligninases were performed by UNICAMP in two steps; (i) solid medium (qualitative screening) and (ii) liquid medium (quantitative screening). Samples were collected from both the terrestrial environment including Lichens, Penguin soil, and Whale bones and from the marine environment such as sea Urchins, Sponges and Marine Sediments. In total 259 fungi and 99 yeasts were collected. Fungi and yeast were grown to pure culture in the laboratory using potato dextrose agar. The majority of these grew at 15oC. Sixty three percent of the cultures were isolated from the marine samples while 37% came from the terrestrial environment.
The first screening of lipases was carried out using a high-throughput screening (HTS) approach, where 24 - 96 microorganisms were screened per plate, resulting in a faster and lower cost methodology. Altogether 45 yeast and 68 filamentous fungi produced lipases at 15°C (Figure 2). A filamentous fungus (isolated from marine algae) and yeast (isolated from sea urchin) were selected as the best producers of lipases and submitted for taxonomic identification, in order to verify their identity and absence of pathogenicity. Taxonomic identification was performed using a polyphasic approach, where molecular and morphologic characteristics were evaluated.
Screening of ligninolytic fungi was carried out using “one by one” screening method (Figure 3). Results showed the presence of 29 fungi able to produce laccase with one fungus isolated from Antarctic marine sediment selected as the best ligninase producer.

Arctic Microbes
The White Sea is located in the north-west of Russia and is part of an Arctic Ocean region which deeply penetrates the continent. It is separated from the adjacent Barents Sea by what is referred to as bottom rise. Average depth of the White Sea is 60 m but the deepest parts reach 343 m. The climate at the White Sea region is continental with an average air temperature of about -10°? in winter occasionally falling below -30°?, and an average air temperature of 15-18°? during the summer months. For about five months a year (December to May) the White Sea is covered with ice, all except the central region of this area. The White Sea fauna and flora consists of Arctic and Arctic-Boreal species. It is rich in zooplankton, phytoplankton, micro-phytobenthos, macrophytobenthos and macro-zoobenthos organisms. The sea hosts more than 700 species of invertebrates.

Some 500 microbial samples were collected by Genetika along this Coast using standard microbiological approaches. Samples included: (i) filtered sea water (ii) dried sea foam (iii) the samples of sludge (iv) plant and insect samples which had naturally fallen into the water, (v) intestines and gills of fishes and (vi) fragments of macro-algae. These were prepared and subjected to targeted HVABs screening. For DHA screening different dilutions of marine samples were cultivated for 10 days at 18-22oC on a rich media containing anti-bacterial antibiotics to suppress the growth of bacteria resulting in yeast and fungal colony cultivation. Colonies were isolated putatively belonging to the Labirinthulida order and subjected to further cultivation in rich media prior to DHA quantification by GC. Four of the most promising producers were isolated.

Ubiquinone Q10 producers were initially screened based on the Genetika`s background know-how. Ubiquinone Q10 producing bacteria had an appearance which could be characterized as being yellow, red or purple in colour in more than 90 percent of evaluations. Isolated microorganisms were cultivated and the amount of biomass accumulated ubiquinone Q10 was evaluated by a developed high-performance liquid chromatography (HPLC) method. Four ubiquinone Q10 producers were isolated and taxonomically identified.

A culture enrichment technique was applied to isolate phytase producing organisms where (i) samples were cultivated and screened in minimal media with sodium phytate as a single phosphorous source to obtain a culture enriched with phytase producers followed by (ii) where different dilutions of enriched cultures were cultivated on minimal solidified media containing calcium phytate, which decreased media transparency. Colonies with different zones of clearing were formed due to calcium phytate disruption by phytase. The diameter of the zone was directly correlated with enzyme activity. Approximately thirty thousand colonies were analyzed and thirty-four colonies from this with the largest clearance zones were isolated and then cultivated in minimal media with sodium phytate. Thereafter, phytase activity of isolated strains was determined in the culture liquid by Fiske-Subbarow method and four strains with the highest enzyme activity were isolated. Identification of DHA producers (totaling 4) relied on the determination of 18S rRNA gene sequence and comparison of resulting sequences with known 18S rRNA genes. These strains were identified as Thraustochytrid Ulkenia, but were attributed with an equal reliability level to Thraustochytrium aureum and two Ulkenia species: U. radiate and U. visurgensis.

Ubiquinone Q10 and phytase bacterial producers were identified using 16S rRNA sequencing. One ubiquinone Q10 producers exhibited a high homology to Paracoccus marinus, but they were different from each other by the ability of the isolated strain to grow in 6% NaCl and possession of urease activity. Based on these facts, a new strain was attributed to an unknown species. To identify species attribution of one of the phytase producers PCR was applied. Genetika developed primers in the project background on the basis of gyrA gene (alpha subunit of DNA Gyrase). As a result, this strain was attributed to Bacillus amyloliquefaciens.

Macroalgae
Recent trends in drug research from natural sources suggest that macro-algae are one of the major producers of bioactive secondary metabolites with high biomedical potential including anti-oxidant, anti-microbial and anti-tumor activities. F. spiralis is a perennial brown alga found mainly in the higher marine intertidal region and subjected to tidal cycles enduring daily desiccation periods which are coincident with the highest levels of phlorotannins produced against this ecological pressure. Phlorotannin phenolic compounds are polymers of phloroglucinol which has been identified in brown algae. Phlorotannin’s health benefits are associated with their antioxidant, antibacterial and antitumor activities potential. The dark brown-red S. coronopifolius grows between low to deep water levels and produces halogenated metabolites with biomedical potential mainly related with antibacterial and antitumor activities. Several interesting di-, tri, or tetracyclic skeletons, most of which possessing one or two bromide atoms (bromoditerpenes) are produced by this macroalgae.

IPL, Portugal targeted two main macro-algae species, Fucus spiralis and Sphaerococcus coronopifolius which were later shown to produce phlorotannins and terpenoids with the high targeted bioactivities, respectively. Twelve other macro-algae species were also screened including Halopteris filicina, Saccorhiza polyschides, Stypocaulon scoparium, Asparagopsis armata, Plocamium cartilagineum, Ceramium ciliatum, Codium adhaerens, Codium tomentosum, Codium vermilara, Ulva compressa, Padina pavonica and Bifurcaria bifurcata. Antioxidant activity of macroalgal solvent extracts was assessed using the Total Phenol Content and DPPH radical scavenging assays. Anti-microbial activity of solvent extracts was assessed against B. subtilis, E. coli and S. cerevisiae. Anti-tumor potential of solvent extracts were measured against CaCo-2 and HEPG2 cell models. Cell viability and proliferation were assessed using Calcein-AM and MTT methods. Based on these screening assays both F. spiralis (for antioxidant) and S. coronopifolius (for anti-microbial and anti-tumoral) were selected for continued studies in BAMMBO. Specific other species of macroalgae were identified at this stage with promising activities for future continued research beyond BAMMBO. As reported in workpackage 4 and 5 IPL purified 5 bromoditerpenes from macroalgal extracts of which one bromoditerpene was novel and not discovered previously.

Workpackage 3: Sustainable Culture

Sustainable culture led by the Unit of Bioengineering at the Catholic University of Louvain at Louvain-la-Neuve (UCLouvain) was dedicated to overcoming bottlenecks associated with the development of economically sustainable and scalable culturing systems for marine organisms. Specifically systems were designed by UCLouvain to produce high yields of HVABs while avoiding destruction of wild marine resources. UCLouvain designed and produced four photo-bioreactor (PBRs) prototypes to develop economically sustainable and scalable culturing devices for microalgae (3) and macroalgae (1). Information relating to the specifications of these was transferred to industry partners. Two such initial systems were a 45 L Vertical Raceway PBR (Figure 4) and a 60 L Annular Plate Airlift PBR system (Figure 5) for microalgae which were supported with dedicated bioreactor control monitoring systems to optimize production parameters set to increase yields of HVAB production of interest to BAMMBO. The control systems helped overcome microalgal culture bottlenecks such as suboptimal illumination and suboptimal concentrations of dissolved O2 and CO2.
The control system had three mass flow meters, a dissolved CO2 probe, high and low concentration gas phase CO2 analyzers, infrared side-scattering flow-through turbidity cell for monitoring cell density, one submersible and two peristaltic pumps, four rotometers, two three-way solenoid vales, three three-way valves, and a complete interface and software control system from Qubit Systems. The system can run in continuous mode with constant cell density, with constant off-gas CO2 concentration or constant dissolved CO2 concentration.

The third microalgal system (UCLouvain patent application to be filed) designed by UCLouvain provided solutions to enhance the culture of the fastidious microalgae Gambierdiscus sp. (Figure 6) A fourth novel PBR was designed specifically for the continuous cultivation of macroalgal tissue cultures. High cell density phototrophic macroalgal tissue cultures of Ochtodes secundiramea which produces halogenated monoterpenes were cultivated (Figures 7 - 9).

Microalgal Culture and Abiotic Stressors
The production of astaxanthin by H. pluvialis is stimulated by inductive conditions such as nutrient deficiency, high NaCl concentrations and other factors, under which the green vegetative cells gradually convert into red non-motile resting cells, producing astaxanthin. The effect of nitrogen and phosphorous limitation, salinity and presence and type of reactive oxygen species (ROS) and abiotic stresses on production of astaxanthin by H. pluvialis was investigated. Total astaxanthin concentrations increased with decreasing nitrate concentrations. There was a trend for lower phosphate concentrations to induce astaxanthin synthesis. High salinities (0.8g/L) induced production of astaxanthin. Microalgal culturing of this organism was further progressed and enhanced by the Algae Health and Greensea partners for industrial applications and exploitations (WP6) and further details are note reported here.

Abiotic stressors influencing elicitation of maitotoxin and ciguatoxin from G. toxicus were also assessed by USC using 4 Gambierdiscus strains G. beliceanus-A, G. australes, G. caribeus, G. beliceanus-B. Growth conditions were selected after assessing different eutrophic factors: Salinities (33%, 37% and 42%), culture media (K, L1-Guillard, GSe, F2-Guillard), light intensity (2000 to 4000 lux), soft aeration (O2) and mechanical agitation. Influence of light/dark cycles of 14/10, 16/8, 24 hours was also assessed. From these results, K media with 33% salinity, without agitation or aeration in a light/darkness cycle of 14/10 hours were selected as culture conditions for further sustained cultivation work by UCLouvain. The effect of temperature and pH evolution in the culture media was studied in relation with cells proliferation and also with toxins production. Gambierdiscus spp. growth was shown to be more efficient under alkaline conditions at 24 °C. Abiotic stressors had an influence on the elicitation ratio of maitotoxin to ciguatoxin.

One of the main bottlenecks for the growth of Gambierdiscus sp. is the lack of information about its physiology and ecology. Currently vessels in which Gambierdiscus sp. can be grown are limited to the use of Erlenmeyer flasks filled with K-media depleted of Si and the use of fluorescent light positioned on the side of the vessel with an intensity < 15 µEm-2s-1. UCLouvain have developed a bioreactor system for sustainable cultivation of Gambierdiscus. Further details of this system are not detailed further here as an Invention Disclosure Form and an Intention to patent this technology has been registered by UCLouvain to advance exploitation.

Macroalgal Culture, Abiotic Stressors and Stable and Immortalized Cell Lines
Work to create novel, stable and immortalized macroalgal cell tissue cultures of Fucus spiralis (for polyphenols) and Sphaerococcus coronopifolius (for halogenated terpenes) was conducted through collaborative efforts of UCLouvain and IPL. Some indications of F. spiralis and S. coronopifolius regeneration was evident in initial trials but these macroalgae eventually lost viability S. coronopifolius (20 days) and F. spiralis (2 Months). Work did continue to achieve stable cultures resulting in a stable immortalized culture of F. spiralis.

Macroalgal tissue cultures (explants, callus culture and regenerative shoots) of F. spiralis and S. coronopifolius were cultivated at bubbler flask scale. After having obtained a stable immortalized culture of F. spiralis, several fragments were exposed to different light intensities, namely 1000 and 2000 lux and the production of phlorotannins production was compared with the wild F. spiralis. The wild type presented the highest phlorotannin content followed by the tissue cultured at 1000 lux and finally by the tissue cultured at 2000 lux. The IPL partner demonstrated that it was possible to cultivate F. spiralis in lab environment; however, due to a low rate of growth, it was not a sustainable option to produce the targeted HVABs. In vitro culture of F. spiralis produced lower contents of phlorotannins than the wild type.

Sponge Culture and Abiotic Stressors
C. crambe and S. spinosulus were cultured within enclosed aquaria and by mariculture. UNIGE, Italy determined the optimal conditions for farming sponges which considered location, depth and various environmental conditions on growth rate and enhanced HVAB production. Mariculture was assessed in the Ligurian and Sardinian Seas (Italy). Performance data from sea based cultures were compared to those under wild and aquaria conditions. Shallow water USAMA® modules (3-10 meters depth) were selected to minimize the use of boats and underwater operations (Figure 10): 1.5 x 1.5 m2 modules of stainless steel, assembled on quite flat sandy or rocky bottoms and (ii) PVC square/rectangular modules, assembled in order to be anchored to pre-existing underwater structures. Four substrata typologies were tested: travertine tiles, nylon lines, cups and nylon meshes. UNIGE results showed C. crambe to grow better on tiles than on nylon meshes. In the Sardinia site the C. crambe explants settled on travertine tiles showed a very high survival rate in the long term (about 90%) and an extraordinary annual growth rate near to 1000%. The survival of S. spinosulus was high (over 90%) and growth rate was near 150% per year with the nylon lines method. Transferred target sponges to aquaria indicated it was a good ex-situ system to maintain sponge explants but not a suitable system for sponge growth. Responses to abiotic stressors were species specific showing C. crambe to tolerate harsher environmental stressors than S. spinosulus. Based on these research outcomes UNIGE have proposed to apply for a national patent to protect intellectual property pertaining to sponge cultivation.

UNS applied aquaria based feeding experiments using radiolabelled amino acids to identify the precursors of the metabolic pathways leading to these guanidine alkaloids. Until now only chemical hypotheses were present in the literature and experimental work was needed to identify the origin of the different part of the guanidine molecules. Because molecular biology is still not ready to explore sponge biosynthetic enzymes UNS started with feeding experiments to trace the metabolic pathways. UNS observe incorporation of labelled arginine into the targeted compounds and proposed a biosynthetic pathway for this family of compounds. Arginine, fatty acids and environmental condition effects on the metabolome were assessed in aquaria. Light and increasing temperature adversely affected alkaloids production.

Sponge and Macroalgal Associated Bacteria Culture
Regarding the isolation and characterization of macroalgal epiphytic bacteria the IPL partner obtained 129 epiphytic bacteria from four different macroalgae. Altogether 34, 26, 30 and 39 epiphytic bacteria from F. spiralis, S. coronopifolius, A. armata and B. bifurcata were obtained, respectively, all of which were purified and identified. IPL cultured all the isolated bacteria in 1L flasks in Marine Broth, at 20°C with light and constant aeration (optimal conditions). Additionally, cultures of C. crambe and S. spinosulus liberated surface associated bacteria in marine broth were created initially by UNIGE in collaboration with partner IPL and UCLouvain. These bacteria were revealed to be amenable to scale up for high biomass production for further bioactive compound extraction applicable to industrial scale. Extraction procedures for the bacterial biomass were developed and relatively potent bioactivities observed as outlined in workpackage 4.


Optimized Fungal Enzyme Production and Scale-Up
Optimised conditions for the production of lipase by Geomyces pannorum AL1-1B were established at 50ml scale by UNICAMP. Scale up (1.2L) fermentation of the Geomyces in olive oil media at LIT for fungal lipase yielded 750ml of culture supernatant (0.3 IU/ml lipase) after 14 days following processing. Harvested cell biomass was used for bioactive screening for anti-elastase, anti-hyaluronidase, anti-microbial and anti-oxidant activities as part of work package 5. Sustainable production of this lipase using Geomyces and Cryptococcus was not possible using conventional fermentation bioreactors as the inducing lipid interfered with oxygen transfer and availability following its enzymatic hydrolysis by induced lipase. In this unique case the bottleneck to sustainable production resides with the inducing substrate and not the organism per se. LIT have commenced circumvention of this bottleneck through the development of a recombinant expression system for the specific lipase gene derived from these yeast and fungal species of the Antarctic which does not rely on lipid induction or use as a substrate.

Scale up of Lipase Production by the Yeast Cryptococcus laurentii L59
Cryptococcus laurentii L59 lipase producing yeast were cultured (1.2L scale) at LIT using optimized conditions of UNICAMP. Cells and culture supernatant containing enzyme were harvested following five days of growth on 2% sunflower oil media. After processing lipase enriched culture filtrate (0.54 IU/ml lipase) was collected. Biomass was also used for bioactive screening.

Scale up of Manganese Peroxidase Production by the Fungus Cadophora sp. P1
Cadophora sp. P1 manganese peroxidase producing fungus was up-scaled (1.2L) at LIT using optimized conditions provided by UNICAMP. Cells and culture supernatant containing enzyme were harvested following 9 days of growth. After centrifugation of the culture, cells and 850ml of culture supernatant (69.6 IU/ml manganese peroxidase) were collected. The harvested cell biomass was used for bioactive screening for anti-elastase, anti-hyaluronidase, anti-microbial and anti-oxidant activities as part of work package 5.

Recombinant Phytase Generation and Culture
The approach to the construction of the recombinant phytase producers was based on the usually used methods of molecular biology. In this approach the phytase coding gene was cloned between the promoter and terminator region of expression cassettes for E. coli and B. subtilis, and the resulted expression vectors were transformed into the corresponding host strain. The strain of Bacillus amyloliquefaciens was chosen as a phytase gene donor after several unsuccessful attempts to amplify phytase coding genes from other isolated organisms. The sequence of phytase gene from this strain was determined that revealed substitutions in 11 nucleotides from the gene of reference strain: 93 T?C, 183 G?C, 312 T?C, 442 A?G, 636 G?A, 705 C?T, 753 T?C, 828 C?A, 978 G?A, 990 C?T, 1134 T?C. Analysis of resulted protein revealed the 148 Asp?Asn substitution. The length of signal peptide (26 aa) was predicted for this protein with help of the “Signal P” program. The phytase producer based on B. subtilis host strain was chosen for further experiments, instead of E. coli, for its ability to secrete proteins, that makes easier the recovery and purification of targeted enzyme. This producer was obtained by transformation of protease deficient strain Bacillus subtilis AJ73 with expression vector pCB20-phy. The resulted strain was named Bacillus subtilis phyBam.

Sustainable Culture of Bacterial Ubiquinone Q10 and DHA Producers
The most promising producers of ubiquinone Q10 and DHA, Paracoccus spp. and Ulkenia visurgensis, respectively, were chosen at the cultivation in the flasks and were subjected to optimization of cultivation conditions in the fermenter. The standard approaches were applied for optimization of cultivation conditions: the influence of inoculum level, of pH control, of aeration level, of media composition and feedings were studied. As a result, the maximal yield for DHA and ubiquinone Q10 was obtained on the level of 322 mg/l and 22.76 mg/l, respectively. Only the composition of medium was optimized for the recombinant phytase producer, because Genetika have optimized the other parameters in background experiments with different enzyme producers constructed on the basis of strain Bacillus subtilis AJ73. The maximal phytase activity for recombinant producer was obtained on the level of 8.37 IU/ml.

As a result of collaborative research performed by UCLouvain, LIT, UNIGE and IPL four identified and culturable species of sponge (species SS-BE/CC30) and macroalgae (species F40/F52 from F. spiralis) associated bacteria were documented and ranked as comparable producers of Ubiquinone Q10. Optimized growth conditions for these bacteria were identified and they were cultured at 1.2L scale. All the bacteria were determined to be sustainable sources of Ubiquinone Coenzyme Q10.

Workpackage 4: Extraction and Purification

The focus of workpackage 4 was the development, optimisation and application of analytical methods and scalable processes to extract and purify targeted HVABs from selected sustainable cultured marine organisms from the previous workpackages 2 and 3. Targeted organisms (and in selected instances associated sponge waste streams) within BAMMBO were isolated/sourced by different partners and subjected to a wide and varied range of processing, extraction, enrichment and/or purification methodologies permitting HVAB analyses. The basic approach to the extraction of HVABs from targeted organisms is outlined in Figure 11 and summarised as:

(i) Target taxonomic species of organisms were subjected to standard in-house extraction processes as identified and practiced by partner research groups. These extractions were performed and analyzed in the partner organization.
(ii) The same taxonomic species of organisms were simultaneously transferred to the LIT partner for extraction. The samples were subjected by LIT to (a) supercritical fluid CO2 extraction (SCFE) and (b) to the extraction process utilized by the partner in question who provided the sample. The extracts derived from these processes were returned to the partner in question for quantification of HVABs (Yield & Activity). An overview of extraction and the concept of SCFE is outline below (Figure 11).

A variety of different processes may be applied to extract biomolecules from marine organisms. In some cases such methodologies may be disadvantageous to the goal at hand as they can result in the exposure of targeted molecules to microbes, oxygen and elevated temperatures with negative influence on product yield, stability, quality, safety and organoleptic attributes. For this reason BAMMBO employed both solvent (very wide, varied and HVAB specific solubilisation/partitioning variations and methods) based and Supercritical Fluid Carbon Dioxide Extraction (SCFE) based processes to marine biomass. The SCFE process uses a supercritical fluid as a solvent. This is created when a gas is taken above a critical temperature and pressure yielding a ‘supercritical state’ of existence. Carbon dioxide, once pressurized can act as an effective solvent of non-polar compounds such as PUFA’s, carotenoids and Ubiquinone Q10. Once depressurized, carbon dioxide loses its solvating power and the extracted material precipitates. The separated carbon dioxide is condensed and recycled. SCFE is regarded as a clean environmentally benign technology. From a cost point of view SCFE systems involve a once-off capital investment for a process that can extract compounds directly from starting materials. The SCFE research complemented the work of the SME partners and the environmental sustainability and conscientious approach of BAMMBO (workpackage 7). Conditions were optimised to sequentially extract targeted PUFAs and carotenoids were possible from the one intact starting material by using a ‘natural and environmentally benign’ non-alcohol solvent modifier. For sponge derived HVABs a novel extractive process was generated by UNS to trap the targeted molecules from the marine environment.
The above approach allowed comparisons to be made between SCFE and other extraction processes for HVAB liberation, enrichment and potential commercial production or licence. The analysis of extracts and purified HVABs was the focus of workpackage 5. Specific details of all extractions are not detailed in this report so as to prevent disclosure of intellectual property relating to potentially exploitable foregrounds. Additionally, the sheer scale of marine organism biomass samples, HVABs and extraction processes developed, optimised and applied in BAMMBO restricts its detailing in this final report document extensively. The reader is advised that a public deliverable outlining the ‘Enrichment of Extracts for HVABs’ was uploaded to the BAMMBO website.

The main science and technological outcomes from the extraction and purification workpackage were:
UNS successfully scaled up the sponge extraction process. The combining of mariculture and the UNS “sponge milking” processes could open the way for a sustainable production of sponge HVAB at g scales per year. UNS observed that mechanical stress could induce the release of sponge HVAB in the surrounding seawater. UNS developed a process to extract the compounds from the seawater, first in small quantity and then in larger amounts. UNS called this process sponge "milking" which is a scalable and competitive process to produce the sponge metabolites as compared to synthetic processes. UNS anticipate that 10-100 g of targeted compounds per year may be generated and available for in vivo bioassays for example in the pharmaceutical industry (Figure 12).
LIT performed fatty acid extractions using both solvent and SCFE processes. The LIT optimized SCFE process resulted in 73% recovery of DHA (79% EPA) as compared to a 100% recovery using solvent methods. This resulted in the SCFE extraction process being deemed a promising extraction method for these compounds. A patent application for the extraction of high value bioactive compounds (DHA, EPA, carotenoids and Q10) from marine organisms in an environmentally benign manner by optimisation of SCFE is planned by LIT.
SCFE optimization for Coenzyme Q10 from Parococcus sp. permitted LIT to attain 100% recovery of Coenzyme Q10. The optimisation of SCF extraction conditions allowed LIT to achieve equivalent Q10 quantities as solvent extraction, therefore, SCF extraction could be considered as a promising extraction method. Ubiquinone Q10 plays an important role in the production of cellular energy and in the basic functioning of cells. As an antioxidant, it has been widely used as an ingredient in dietary supplements, nutraceuticals, and functional foods as well as in anti-aging creams and is beneficial in the treatment of several human diseases. The provision of a greener method of Q10 extraction will greatly benefit its supply.
H. pluvialis was targeted as the main source of astaxanthin. Optimisation of SCFE conditions was performed by LIT for astaxanthin and a 93% recovery was obtained as compared to a 100% recovery by solvent extraction methods. This SCFE may be considered a suitable alternative and greener extraction method for the reasons outlined above earlier.
Twelve pure guanidine alkaloids (crambescins/crambescidins) from C. crambe were isolated and purified by UNS. This work led to the structural identification of 12 polycyclic guanidine derivatives, including 8 compounds isolated and described for the first time in a pure form. Two families of crambescins (A, B and C) and crambescidins were identified by UNS. These were screened for a range of bioactivities.
Four cytoxic bioactive compounds (2’-[34-hydroxy]octaprenyl-1’,4’-hydroquinone, heptaprenylhydroquinone, octaprenylhydroquinone and Sarcotin A) were identified in S. spinosulus sponge species by UNIGE. The structural characterized was performed by means of the following spectroscopic methods: IR, UV, 1D and 2D 1H- and 13C-NMR, HR-MS.
An aqueous extract of O. secundiramea was purified for ?-phycoerythrin. The macroalga was the best source for this fluorescent compound. This protein was found in a fraction of a solvent extract and has led to a patent application by LIT regarding a new extraction method for this compound from this organism. It has also resulted in a patent application for LIT regarding development and application of an activity staining system for the ?-phycoerythrin profile on a Native-PAGE gel. This fluorescent protein was partially purified. ß-phycoerythrin is useful in fluorescence-label based immunodiagnostics assays such as those employed in immunoassay kits.
Recombinant phytase was purified 77-fold over the culture supernatant with a 10% yield by Genetika. The phytase specific activity (20 U/mg) and characteristics were evaluated.
Lipase enzymes from Geomyces sp. and Crypotococcus sp. were partially purified by LIT.
S. coronopifolius extracts were purified by VLC through collaborative efforts of UNS and IPL. The most active fractions with the highest anti-tumor and/or anti-microbial activity were identified. Altogether 5 bromoterpenes were identified (RMN) and purified (preparative HPLC). One new and novel bromoterpene was identified and structurally characterized
Carotenoids and fatty acid components of a number of microalgae and other species were fractionated and identified successfully providing complete profiles of the specific (carotenoid) or fatty acid composition of various BAMMBO species extracts. This was facilitated by optimized SCFE, solvent and LC and GC-MS processes of LIT.
Co-enzyme Q10 was identified and extracted from a number of bacterial species associated with specific sponge and macroalgal species. This work involved close collaboration of LIT, IPL, UCLouvain and UNIGE.

Workpackage 5: Analysis of High Value Added Molecules and Bioactives

The main scientific outcomes derived from workpackage 5 were those which related to the bioactive screening and in-process monitoring of targeted BAMMBO HVABs progressing through the selection and work processes of workpackages 2, 3 and 4. One of the final goals of workpackage 5, i.e. analysis of high value added molecules and bioactives was defining the cosmoceutical, pharmaceutical and/or industrial application potential of the candidate compounds from sustainable sources of marine organisms. As outlined in the workpackage 4 summary description the sheer range of targeted marine invertebrates, HVABs targeted, extraction processes and analytical processes applied over the 36 month period of BAMMBO limits extensive detail to be included in a balanced format within this report. As such a description of the main science and technology results are summarised from the main findings only. Readers are advised that a wide variety of public deliverables are available on the BAMMBO website for review (www.bammbo.eu).

Extracts Affecting Cellular Activities and Mechanisms
Cellular activity analyses were performed by USC and UNIGE. The USC laboratory received purified sponges derived molecules from UNS which could be divided into two families crambescines (Crambescin-a, -b, -c, homocrambescin a, bis-(ethyl)homocrambescin a, dehydrohomocrambescin a, norcrambescin b, homocrambescin b, norcrambescin c, homocrambescin c) and crambescidines (crambescidine-800, -816, and -830). Each family of compound was assayed for their effect on membrane receptors, intracellular membranes, secretory mechanisms, electrical events and/or neuronal plasticity. To address effects on membrane receptor affecting compounds several assays were performed. These included: intracellular calcium determination by fluorescence microscopy, determination of ?-secretase 1 inhibition by fluorescence resonance energy transfer (FRET), determination of interaction with the muscarinic acetylcholine receptor M3 by competitive radio ligand assay.

To assay the effect of received compounds on intracellular membranes and secretory mechanisms, initially whole human transcriptome microarrays were used to analyze the effect of selected molecules from each family of compounds: crambescidin-816, crambescin-C, -A and homo crambescin C. The human derived cell line HEPG2 was selected for these studies. Based on the results obtained the effect of these compounds together with others from each of the families were studied using a variety of molecular and cellular techniques that included real time-PCR, imaging flow cytometry, western blot, invasion assays, confocal microscopy, electron microscopy. The effects of crambescidins were further studied on human derived cell lines: Hop92, OU31, OVCAR, HT70, MSF7 and PS3. The effect of the received compounds on electrical events and neurons plasticity was determined using patch clamp and automatic patch clamp technology. Effects on the cardiac potassium channel HERG were studied using a murine cell line transfected with the gene coding for the human HERG by automatic patch clamp. Effects on neurons voltage gated sodium, calcium, and potassium channels were determined using cultures of mice primary neurons by patch clamp. The main findings from this USC work are outlined below:

The compounds effect on the muscarinic acetylcholine receptor M3, on the ?-secretase 1 and on intracellular calcium concentration were assayed by USC. Intracellular calcium plays a crucial role in regulating neuronal excitability, release of transmitters, synaptic plasticity, and apoptosis. In a Ca+2 free medium, Crambescidin 816 (C816) did not produce any effect at the studied concentrations thus indicating that the intracellular Ca+2 stores were not affected by the toxin. However when 1 mM Ca+2 was added to the bath solution, C816 induced a Ca+2 influx in a concentration dependent manner. In other set of experiments USC further elucidated if the effect of C816 was dependent on the age of cortical neurons, since calcium homeostasis changes during neuronal development. Thus, USC evaluated the effect of 10 µM C816 in young neurons (4-6 DIV) and mature neurons (10-11 DIV). USC found that in a Ca+2 free media any of the groups affected the intracellular calcium stores. When 1 mM Ca+2 was added to the bath solution USC obtained similar results to the previous set of experiments indicating that young neurons were more susceptible than mature neurons to the C816 insult regarding its effect on intracellular calcium concentration.
USC evaluated the mechanism underlying the C816-dependent Ca+2 influx in cortical neurons. In neurons, the entry of calcium from the outside is regulated by voltage-gated channels or by receptor operated channels controlled by the ionotropic neurotransmitters (glutamate). When L-type voltage gated calcium channels were blocked by 10 µM NIF, 3 minutes prior 10 µM C816 administration, we obtained that nifedipine did not block the Ca+2 influc produced by C816, thus indicating that the C816-dependent calcium influx was not through L-type voltage gated calcium channels. In summary, since Glutamate receptor-induced Ca+2 accumulation is well known to be correlated with neuronal degeneration in neurons, we hypothesize that the neuronal death produced by 10 µM C816 could be the result of the Glutamate receptor activation produced by this guanidine toxin.
Beta secretase-1 inhibitors were investigated by USC for their potential as drugs to treat Alzheimers disease and other neurodegenerative diseases. The effect of crambescidines and crambescines on ?-secretase 1 was assayed in silico to isolate the effect of the compounds on this protein and avoid interferences that could be expected if whole cell cultures were used. All the compounds received were assayed showing that crambescin-A3, and crambescidines inhibit ?-secretase 1 when used at 10 ?M.
The effect of all received compounds on the cardiac muscarinic acetylcholine receptor M3, targer for several drugs and substances, was determined by USC. The assay was performed using a cell model the allowed the isolation the effect of the tested compounds on the M3 receptor. A competitive radioligand assay, using chinese hamster ovary cells (CHO) transfected with the muscarinic acetylcholine receptor M3 and treated with crambescines, crambescidines, and atropine (positive control) in the presence of [3H]NMS, showed that all compounds had no effect on this receptor.
To determine the effect of crambescidines and crambescines on intracellular membranes and secretory receptors the effect of these compounds on the human cell line HepG2 was determined by USC. MTT assays showed that C816 reduced cell viability of the cell line HepG2 at concentrations higher than 150 nM after 24 and 48 hs. The cytotoxic effect observed for HepG2 cells was also observed on the human tumor derived cell lines HOP-92 (pulmonary carcinoma), MCF-7 (mammary carcinoma), OVCAR (ovary carcinoma), PC3 (prostatic carcinoma), OU-31 (kidney carcinoma), SK-MEL (melanoma), and HT-29 (colon carcinoma). C816 induced apoptosis in HepG2 cells as determined by Annexin V staining and caspase-3 activity measurements. While no apoptosis was detected after 6 hs, after 24 hs, 500 and 1000 nM C816 induced phosphatidylserine translocation and an increase in caspase-3 activity. After 48 hs the three concentrations tested induced apoptosis. After cytotoxicity determination, a concentration of 150 nM C816 was selected for whole human transcriptome microarrays experiments, since it was non-cytotoxic after short incubation periods but induced cell death after 48 hs. This experiment was performed to obtain a wide insight of the metabolic pathways altered by this molecule. Microarray results showed that the toxin affected the expression of approximately 5% the genes in HepG2 cells at the three times tested (6, 24 and 48 h). Functional analysis of differentially expressed genes showed that short-term exposure to C816 produced the down-regulation of genes involved in cell migration, cell-cell and cell-matrix adhesion, together with genes involved in cell cycle regulation. Positively regulated biological processes and cellular components after 6 hs included those involved in cytoskeleton organization and transcription regulation. Venn Diagrams showed that 72 down-regulated genes were shared by the three incubation times selected. These were involved mainly in cell junction, protein transport and secretion.
To test the effect of crambescines and crambescidines on electrical events and neuronal plasticity the compounds effect on the cardiac potassium channel hERG, and on primary cortical neurons sodium, calcium, and potassium voltage gated channels was determined by USC. The cardiac potassium channel hERG is known for its contribution to the electrical activity of the heart that coordinates its beating. When this channel's ability to conduct electrical current across the cell membrane is inhibited or compromised, either by application of drugs or by rare mutations in some families, it can result in a potentially fatal disorder called long QT syndrome. An undesired side effect of a number of clinically successful drugs in the market is the tendency to inhibit hERG, and create a concomitant risk of sudden death, as a side-effect. Due to this, hERG inhibition is an important anti-target that must be avoided during drug development. The effect of received compounds on hERG was determined using automated patch clamp. Results showed that none of the compounds (10 µM concentration) affected the studied cardiac channel.
The whole cell patch-clamp technique in the voltage-clamp configuration was used to analyze the effect of Crambescin C1, N Crambescin A2, Crambescin A2 (compounds from Crambescin family) and C816 (from Crambescidin family) on voltage-dependent sodium channels (NaV), voltage-gated potassium channels (KV) and voltage-gated calcium channels (CaV) channels in mice cortical neurons. First the concentration-response effect of crambescines and crambescidines was evaluated on KV channels (concentration up to 1 µM). The three Crambescins blocked the total potassium current in a dose dependent manner whereas C816 lacked this effect. To evaluate the effect of the compounds on CaV, one representative member of each family was used at 1 µM as at 10 and 100 nM none of them had significant effect on potassium currents. USC chose NCrambescin A2 and C816 since they belong to different structural families and because NCrambescin A2 showed the most interesting effect in potassium currents among the compounds analyzed. The results showed that C816 but not NCrambescin A2 partially blocks High Voltage Activated (HVA) calcium channels (CaV) channels in primary cortical neurons. The results shown in this work demonstrate that Crambescin C1, Crambescin A2, Norcrambescin A2, partially blocked KV but not HVA Ca2+ channels, whereas C816 (from Crambescidin family) had the opposite effect. Both, C816 and Crambescin C1, blocked NaV with a similar potency. These effects suggest a structure-activity dependence in these guanidine alkaloids. USC further demonstrate that C816 produces its main antagonist effect on L-type Ca2? channels.
UNIGE assessed purified bioactive molecules from the sponge species C. crambe and S. spinosulus on aspartate and acetylcholine release from cortical and hippocampal synaptosomes. For the study on the effects of sponge-derived compounds on neurotransmission, UNIGE used the intact synaptosomes model. Synaptosomal preparations contain completely sealed cytoplasmic sacs of 1-2 µm diameter and retain all the microstructures and cell organelles present in the intact synapse. The main findings from this work were:
Synaptosomes showed for the first time that 2’-[34-hydroxy]octaprenyl-1’,4’-hydroquinone and heptaprenylhydroquinone (purified from Sarcotragus Sp.) were able to increase the release of [3H]D-aspartate (an unmetabolizable analogue of endogenous glutamate) and [3H]acetylcholine in the hippocampus and cerebral cortex, although to a different extent. Moreover, crambescidine 816 caused a marked increase of [3H]acetylcholine in both brain regions, whereas crambescin C1 was much less effective. In addition, crambescidine 816 showed a strong effect on [3H]D-aspartate release both in the hippocampus and cortex.

Antioxidant Compounds and Extracts
The Oxygen Radical Absorption Capacity (ORAC) assay was used to quantitatively measure antioxidant activity of the majority of BAMMBO marine derived extracts at LIT. IPL also assessed the antioxidant activity of 27 species of macrolagae derived extracts and macroalage extracts of bacteria associated with these hosts. The assays applied were the ORAC, DPPH and the total polyphenol content assay.
Altogether 129 associated bacteria extracts from S. coronopifolius, F, spiralis, A. armata and B. bifurcate were assessed.
Some of B. bifurcata associated bacteria extract denoted moderate to high anti-oxidant activity on the ORAC assay. Among all organisms analysed the extracts from F. spiralis demonstrated the highest anti-oxidant activity. The fungal and yeast cells displayed good anti-oxidant properties which would be useful as a high value bioactive from waste cells in addition to their enzyme production.
Antimicrobial Extracts and Compounds
Both LIT and IPL performed analyses to asses the anti-microbial activity of marine derived extracts generated in workpackage 4. For LIT a variety of analytical methods were applied including those based on disk diffusion and broth culturing to assess anti-bacterial activity. Assessment involved the growth of a selection of Gram positive and negative bacteria in the presence and absence of the generated extracts. Antibiotic controls ampicillin, tobramycin and chloramphenicol were used as positive controls and respective extraction solvents were used as negative controls.
C. crambe extracts showed potent antibacterial activity against both E. coli and S. aureus. F. spiralis, S. spinosulus and S. coronopifolius showed antibacterial activity towards S. aureus.
IPL evaluated anti-microbial activity against S. aureus, B. subtilis, C. albicans, E. coli, P. aureginosa and S. cerevisae from 129 associated bacteria extracts derived from S. coronopifolius, F. spiralis, A. armata and B. bifurcata.
Three of the bromoditerpenes including the novel bromoditerpene from S. coronopifolius revealed high antimicrobial activity against S. aureus.
IPL demonstrated that a bacterial derived extract from C. crambe (CC-1) showed the highest antimicrobial activity against B. subtilis and S. aureus in all analyses.

Anti-Elastase and Activities
Skin ageing processes may be due to skin elasticity changes over time or due to UV exposure. Components of the extracellular matrix are susceptible to proteolytic degradation by different enzymes including elastase which affects skin elasticity. Inhibitors of elastase offer the potential to maintain skin elasticity and thus reduce skin sagging. Anti-elastase screening performed by LIT used the EnzChek Elastase Assay Kit. In this analysis system the presence of elastase inhibitors in extracts can be assessed. Over two hundred different marine extracts from microalgae, macroalgae, sponges, protist and bacteria were screened for anti-elastase activity. These extracts were obtained by solvent extraction and by supercritical fluid extraction (SCFE).

Anti-elastase activities of extracts were graded according to their concentration and inhibitory affect. Of all the extracts analysed those from F. spiralis and C. crambe demonstrated the greatest anti-elastase activities in some cases approaching complete inhibition. These two organisms were selected for further anti-elastase activity work and purification. These activities was performed in Work Package 4. Further details of the extracts cannot be detailed further for IP reasons.

Anti-hyaluronidase
Hyaluronidases are enzymes produced by humans and some pathogenic bacteria. Hyaluronidase catalyses the breakdown of hyaluronic acid, a major constituent of the skin’s interstitial barrier. Hyaluronidase diminishes the viscosity of hyaluronic acid, thereby increasing tissue permeability. Inhibitors of hyaluronidase activity offer the potential to act as a cosmetic additive for the alleviation of wrinkles. LIT led investigations to assess and identify potential hyaluronidase inhibitors. Activity was determined using a modified colorimetric assay (Morgan-Elson assay) which is based on the reaction of the N-acetyl-D-glucosamine (GlucNAc) at the reducing ends of hyaluronic acid with p-dimethylaminobenzaldehyde resulting in a purple coloured product, which can be spectrophotometrically measured at 586nm. The presence of an inhibitor in the extract results in loss of colour product in the reaction.

Among all tested marine organisms both F. spiralis and S. coronopifolius were the most promising candidates for anti-hyaluronidase activity. Additionally, four non-polar extracts from the enzyme producing fungal and yeast cells showed anti-hyaluronidase activity also. This inhibitory inhibitor presence makes this additional activity worthy of further characterisation if these cells are to be used for enzyme production.

Anti-fouling
UNIGE assessed the antifouling activity of purified bioactive molecules from the sponge species C. crambe and S. spinosulus. The crude extracts (C. crambe and S. spinosulus) and pure molecules from the sponge S. spinosulus were tested as antifouling agents on the settlement and mortality of A. amphitrite larvae.
Results indicated that C. crambe was highly successful in chemical defense demonstrating a well performing anti-settlement toxicity against barnacles. S. spinosulus did not show any significant antifouling activity.
Enzyme active Extracts (Lipase and Phytase)
Four strains of the most promising phytase producers sampled from the White Sea Coast, in Russia by Genetika were isolated and targeted following the screening process of 500 marine samples.

Genetika constructed two recombinant E. coli and B. subtilis bacterial phytase producers. Apart from their species differences these organisms differed in their ability to secrete the phytase expression product. E. coli does not secrete phytase and stores the product within the cell inside inclusion bodies. B. subtilis secretes the product into the growth medium. The yields of active phytase generated and the need for additional downstream processing steps to enrich and purify for phytase based on the use of these two organisms needs to be considered from a commercial production point of view. The maximal phytase activity observed for the recombinant B. subtilis producer was 2.86 IU/ml at the shake flask scale. Fermenter cultivation conditions were optimized for this strain which allowed the organism reach a value of phytase activity of 8.37 IU/ml. Unfortunately, the phytase activity of this recombinant producer was significantly lower in comparison with common industrial producers, which belong to fungal/yeast strains which are able to synthesize up to 5000 IU/ml of phytase. This data do not allow Genetika to refer this strain as a competitive commercial producer of phytase. In parallel with the BAMMBO project however Genetika are developing a competitive phytase producer by an additional modification of the present B. subtilis producer and an expression of phytase in other host strains, for example the yeast Yarrowia sp.

Ubiquinone Coenzyme Q10
Ubiquinone Q10 is ubiquitously found in organisms. It is an obligatory cofactor in the electron transport chain and involved in disulphide bond formation, control of redox flux, detoxification of ROS, gene expression and the generation of cell signals. Ubiquinone Q10 is orally administered for cardiomyopathy, diabetes, Parkinson’s and Alzheimer’s. Ubiquinone Q10 is popular in cosmetics owing to its antioxidant properties. Increased demand has resulted in a variety of non-natural commercial processes including chemical synthesises and microbial conversions.

In BAMMBO four strains of the most promising ubiquinone Q10 producers sampled from the White Sea Coast by Genetika were isolated and targeted following the screening process of 500 marine samples. Genetika targeted one of the most perspective ubiquinone Q10 producers and optimized cultivation conditions for this strain to reach an elevated yield of ubiquinone Q10. However, limitations to the culturing of the target species develop during the culturing process and alternative strains such as those of Altererythrobacter species is a potential industrial optional producer of ubiquinone Q10. LIT compared solvent and SCF extraction as methods for obtaining Ubiquinone Q10 and results were analysed by LC-MS. LIT achieved equivalent SCFE Q10 yields as solvent extraction processes. Ubiquinone Q10 solvent extraction of macroalgae and sponge associated fermentable bacteria by UNIGE, UNS and IPL which were analysed at LIT revealed that some of these bacteria had Q10 levels comparable to targeted Paracoccus sp. organisms of Genetika.

Carotenoids and Spectral Activities of Extracts
Two candidate marine algae were identified as best producers of astaxanthin and ?-phycoerythrin phycobilipigment production.

H. pluvialis is a major source of astaxanthin followed by ß-carotene and lutein. Astaxanthin content determined by LIT varied with sample source and stress conditions applied. Astaxanthin content of H. pluvialis from Greensea was 1.8% (DW biomass) while the astaxanthin content of H. pluvialis from Algae Health was 4.7% (DW biomass, A1) and 4.9% (DW biomass, A6). Optimisation of SCFE of astaxanthin was performed by LIT achieving a 93% recovery as compared to solvent extraction (100%) methods. The SCFE process is a favourable alternative greener extraction method.
High-throughput spectral analysis was conducted at LIT on various BAMMBO extracts to assess the presence of coloured or fluorescent compounds. Extracts were scanned for their absorption and fluorescent spectra properties. The macroalga O. secundiramea was the best source of the fluorescent phycobilipigments ß-phycoerythrin. This pigment was partially purified in workpackage 4.

Essential Fatty Acid Extracts or Compounds
Benefits of polyunsaturated fatty acids (PUFAs) are widely accepted. PUFAs play key roles in cellular metabolism, membrane fluidity, transport and eicosanoid synthesis. Over the last thirty years the major source of PUFAs was fish oil. Declining fish stocks and contamination from the environment has led to search for of alternative oils sources. Microalgae offer a sustainable non-polluted source of such oils. However, the commercial exploitation of microalgal PUFAs is at an early stage. Efforts are needed to identify optimal production strains with abilities to produce high PUFA yields, high cell densities and grow on cheap carbon sources.

Four strains of the most promising DHA producers sampled from the White Sea Coast were isolated and targeted following the screening process of 500 marine samples. Based on the growth performance a Ulkenia sp. was recommended for DHA production. Accordingly, a SCFE protocol for DHA extraction from these species was optimized at LIT. During solvent extraction of lipids and fatty acids also performed at LIT an addition of a step of saponification improved the detection of EPA and DHA in lipid samples tested. The optimized SCFE conditions at LIT permitted a 73% recovery of DHA and 79% recovery of EPA making SCFE a promising extraction method for these compounds. Based on analyses by UGent the microalgae Nannochloropsis sp. and P. tricornutum may be considered an alternative source for the essential fatty acid EPA.

Extracts or Compounds with Anti-Tumor Activity
IPL led an evaluation of the anti-tumor potential of different macroalgae from the Peniche Coast, Portugal against two “in vitro” carcinoma models, the human colorectal adenocarcinoma (Caco-2) and a human hepatocellular liver cell line (HepG-2).
IPL studied extracts of F. spiralis, Halopteris filicina, Saccorhiza polyschides, Stypocaulon scoparium, Asparagopsis armata, Plocamium cartilagineum, Ceramium ciliatum, S. coronopifolius, Codium adhaerens, Codium tomentosum, Codium vermilarae and Ulva compressa. Highest anti-tumoral potential was verified for and extract of S. coronopifolius.
Using identical assays the IPL partner further evaluated the anti-tumoral activity of 129 associated bacterial extracts from S. coronopifolius, Fucus spiralis, A. armata and B. bifurcata.
IPL through collaboration with UNS evaluated the anti-tumor potential of five bromoditerpenes (4 known and one novel) on the HepG-2 human cell line.
Overall, four macroalgae displayed very promising anti-tumor activity which was linked to bromidoterpenes which exhibited high anti-proliferative activity at sub-toxic concentrations. Furthermore, macroalgal associated bacterial extracts presented interesting and promising anti-tumor activities mainly also in anti-proliferative experiments.

Workpackage 6: Semi-Pilot Scale Production

This workpackage entailed the semi-pilot scale production of HVABs identified in micro-algae which have immediate market potential and exploitation capacity to the BAMMBO SME partners Greensea (France) and Algae Health Ltd (Ireland).

GreenSea
Although the green alga H. pluvialis can be obtained from different culture collections worldwide, Greensea isolated their strain from Etang de Thau (Mèze, France) using standard isolation procedures. Strains obtained from culture collections, quiet often considered as “pet lab” microorganisms, are amenable to large scale production with some difficulties as these strains are adapted to a clean and less stressful environment. Once isolated and purified, the strain was maintained in flasks containing 150 mL of BG11 media with continuous illumination (40 µE m-2 s-1). Air containing approximately 0.5% CO2 was continuously bubbled into the flask culture in order to supply sufficient aeration. A subculture was conducted every week by replacing 80% of the culture broth with fresh medium. BG11 media showed the best growth rate compared to other media tested and was then selected for cultivation at small and larger scale.

The strategy used for the production of astaxanthin in H. pluvialis was to sustain rapid growth under favourable culture conditions (e.g. low light and nutrient repletion) (“green stage”) and then stress the cells by introducing high light and/or nutrient depletion to induce astaxanthin production (“red stage”). Greensea tested 2 different production systems at 100L and 2.5 m3 scale (airlift system and tubular bioreactor respectively Figure 14). The effect of various stress conditions on the production of astaxanthin in H. pluvialis (salinity at 0, 5, 10 and 20%) along with nitrogen deprivation was also examined. Finally the effect of initial biomass density for astaxanthin production during the induction stage was studied in a tubular photobioreactor (0.5 1 and 3 g/L). Supercritical CO2 was used to extract the oleoresin and carotenoids and astaxanthin content were determined from the oleoresin.
It was found that the airlift system showed a better growth of the green stage of the alga compared the tubular photobioreactor. This was probably due to gentle agitation in the airlift system required by the flagellated cells. On the contrary, the tubular bioreactor gave better results in terms of astaxanthin accumulation (more light in the system and no shear stress since the cells are cysted and robust).
It was also found that 10% salinity in the media (for the red stage) improved astaxanthin accumulation in H. pluvialis. With respect to the initial biomass density, 1 g/L was optimal for a rapid astaxanthin accumulation.
Finally, by using supercritical CO2 extraction method (with no co-solvent), Greensea obtained an oleoresin containing 11% astaxanthin (more than 98% of the astaxanthin is in an esterified form (Figure 15).
Algae Health’s main focus was the development of technology for the cultivation of microalgae to overcome the bottleneck of sustainable and scalable culturing.
During the BAMMBO project, 2 pilot scale 1,000L PBR units were developed.
Algal Health’s first focus was to identify the various systems currently being used to culture microalgae and then complete a viability analysis on each design. This viability looked at the weakness in the designs, such as contamination control, running cost, capital cost and scalability.
Four different types of PBR were reviewed as part of this process, Vertical Annular System, Horizontal Tube System, Bag Type System and Algae Health’s unique Tank Type System. The focus of this process was to identify and further improve existing systems and to potentially develop a new unique system to overcome common bottlenecks. The results of this PBR design review identified only two systems that were scalable, the Horizontal Tubular System and Algae Health’s unique Tank based PBR. Design weaknesses were also identified in both systems, this initiated design enhancements to reduce and in where applicable remove the limitation/weakness of the system.
Once Algae Health received the laboratory scale-up data from workpackage 3 Algae Health commenced scaling from ‘Seed Culture’ to ‘Flask’ to ‘20L Carboys’ to ‘1,000L System’. Some months were spent refining the growth process and at times further seed back up from 20L Carboys scale was needed. Once successful growth conditions were obtained, growth rates of the Pilot System were recorded relative to controls. Similar growth rates were observed between the 1,000L Pilot Tanks and the Flasks, which was an excellent outcome. Once growth rates and sustainable culturing were achieved at 1,000L Pilot Scale, Algae Health focused on stressing microalgae to produce the desired commercial compound ‘astaxanthin’. The stressing cycle was more complex than the growth cycle so Algae Health needed to further modify the equipment. After a number of trials Algae Health were able to produce consistent stressing cycles and achieved an excellent yield of 4.9% astaxanthin in the biomass. This enabled Algae Health to achieve a further two parts of the overall BAMMBO objective of producing high yield of HVABs and also the transfer to Pilot Scale.
As part of the process Algae Health also looked at using an organic media as an alternative media source. It was found that the organic media provided better growth properties than the chemical media. But it was found that since the organic media was a premixed waste stream from the fish industry, Algae Health could not replenish the specific chemicals that were depleted by growth, hence it was not viable to use on a large scale. Tests were completed in the laboratory where culture could be centrifuged down and re-suspended in fresh media. The laboratory data showed enhance growth from the organic media which demonstrated that significant improvements could be made to the chemical media to optimise growth. Repeatability testing was also completed to ensure that the tanks performed in a consistent manner so that it could be determined whether the culturing process was sustainable. It was found that culturing was repeatable to within +/-5%. This was done over 10 cycles.
The stability of the astaxanthin bio-active was assessed over 180 days at various temperatures. It would found that apart for a few percent drop (4-5%) in the initial day of starting the test, there was minimal to no reduction in the bioactive over the subsequent 180 days. As a concluding deliverable, the cost of the cycle and the output in terms of the bioactive produced were collated into a commercial viability analysis. Based on the current market pricing for the product and the volume of product produced, it was possible to determine the gross margin (sales value less cost of production). This commercial viability analysis was completed for both SME’s Algae Health and Greenseas and the result are shown (Table 4)
Workpackage 7: Life Cycle Assessment of Biologically Active Molecules

The sustainability (Figure 16) assessment conducted in the framework of the BAMMBO project was the first study on the production of biologically active compounds obtained from marine organisms. Although the exploitation of these organisms to obtain other products such as biofuels had previously been evaluated through LCA methodology from an environmental perspective, the work performed in BAMMBO addressed specifically and for the first time the production of high value molecules for strategic sectors including pharmaceutical, nutraceutical or cosmetics, among others.

The environmental LCA was conducted according to the ISO 14040 standards (Figure 17). This approach includes four stages: i) goal and scope definition, ii) inventory analysis, iii) impact assessment and iv) interpretation of the results. To do so, information from all partners was collected using specific questionnaire templates for cultivation and extraction systems.

In the first stage, the aim of each study was clearly defined, including the selection of the functional unit (or reference unit for the whole assessment) and the system boundaries, that is, the processes included within the LCA, as shown in Figure 18. Other key parameters such as the level of aggregation and the quality of the data, the elementary flows or the cut-off criteria were also selected in this stage of the assessment.

Once each system was totally defined and the process flowchart was built, life cycle inventory (LCI) analysis stage was carried out. LCI consists of the collection of data associated with the use of resources, as well as energy consumption, emissions and products resulting from each activity in the production system. Since high quality data are essential to make a reliable LCA, on-site measurements provided by the involved partners were used. The information was completed with other data from the literature and databases, mainly Ecoinvent, that were considered for the background system (group of auxiliary processes for the production of raw materials or further treatment of output flows). The gathered information was used to model the evaluated systems in Excel simulators containing detailed mass and energy balances for each step of the processes (Figure 19). These models were especially developed for the processes designed in BAMMBO and had not been used before in the environmental assessment of high value molecules from marine biotechnology. Moreover, USC is currently applying analogous procedures for the evaluation of other systems involving the exploitation of marine organisms as valuable natural resources. Finally, the quantified input and output flows of each system (including water, nutrients for the preparation of culture media, solvents for extraction, electricity requirements of cultivation and extraction stages, as well as generated wastes to treatment or by-products, among others) were listed per subsystem in a specific inventory table, as the example given in Table 5.

The third stage of the LCA consisted of the determination of the potential impacts to environment by performing the classification of the quantified emissions into different impact categories and the use of characterization factors to convert those emissions into standardised indicators (e.g. kg CO2,eq in the case of global warming potential). In this case, the characterisation factors reported by the Centre of Environmental Science of Leiden University (CML 2001 method) were used. The evaluated impact categories are listed in Figure 20. The results included the total potential impact to each of the addressed categories, together with the breakdown of the different contributions grouped per subsystem (e.g. preparation of the culture media, cultivation, harvesting) and per involved activities (e.g. production of solvents, production of electricity requirements, transport of raw materials).

Regarding the interpretation of the results, the order of magnitude of the impacts associated with BAMMBO processes highly depended on the target organism and products. Although some systems presented significant impacts in several categories, their economic and especially their social value justified their development, since in many cases the evaluated processes are currently the only existing route to obtain these compounds. Among the subsystems of BAMMBO processes, the cultivation was found as the key stage with the highest environmental impacts for most BAMMBO products, as shown in the example given in Figure 21. The extraction step also had significant contributions to the impact in most cases. Other subsystems, such as the cleaning of the reactors and the preparation of culture media caused limited impacts. Regarding the involved activities, the most relevant contributors were the production of electricity (especially related to the cultivation stage due to the consumption of energy by lights, pumps and compressors, stirrer) and the production of chemicals, mainly solvents used during the extraction stage and, to a lesser extent, nutrients for the culture media.

The socio-economic assessment was carried out in a similar way to the methodology described for the LCA. Thus, a specific questionnaire template applied to BAMMBO context was prepared and sent to the two SME participants for the collection of socio-economic information. The selected indicators (based on the UNEP/SETAC Life Cycle Initiative Guidelines for Social Life Cycle Assessment of products published in 2009 and applied for the first time to the sector of marine biotechnology) were classified into several subcategories associated with five stakeholder categories (workers, consumers, local community, value chain actors and society). Economic data included investment costs, raw material costs or labour costs, among others.

According to the type of process and involved companies, three main stakeholder categories were selected: workers, consumers and society. For each of them, several indicators were selected and grouped into subcategories that reflect different social benefits. Two types of indicators were measured: quantitative indicators and semi-quantitative indicators. In both cases, an index of 1 (lowest benefit) to 4 (highest benefit) was assigned to each indicator. For quantitative indicators, the indexes were calculated according to minimum and maximum levels in the world and an average value of the determined indexes was obtained for each subcategory. For semi-quantitative indicators based on Yes/No questions, a value of 1 was assigned to negative response and a value of 4 was considered for affirmative response. Some specific indicators were scored according to expertise knowledge.

The results (Figure 22) allowed identifying several strengths of the involved companies according to the measured indicators, mainly related to workers (fair salary, balanced working hours) and consumers. The evaluation also found some key issues that could potentially improve by adopting the appropriate management strategies. These measures focused on the weakest aspect of the two companies: society. The reason for this weakness was related to the small size and resulting low potential market share of the SMEs. However, the development of codes of conduct and the efforts to obtain environmental and safety certifications might lead to important improvements in this area.

For the economic evaluation, a cost-benefit analysis (CBA) approach was considered, including investment costs, overhead costs, variable production costs (materials, energy, operating labour, disposal) and research and development costs. Two indicators were determined: total net present value (NPV) and pay-back time. NPV reflected the net profit of the process, whereas the pay-back time showed the time needed to recover the initial investment (Figure 23). The results suggest that the evaluated process could constitute a very profitable process that would probably compensate the initial investment in a short period of time. The calculated NPV was widely positive. Regarding the operating costs distribution, most of the cost was associated with the staff, whereas energy consumption constituted the second most relevant issue.

The last stage of the sustainability assessment was the comparison of novel BAMMBO processes with existing commercial systems for the production of the addressed compounds. Since most of the evaluated processes were only implemented at lab scale and in many cases were the only available route for the production of the specific compounds, the comparison between existing and novel pathways was applied to the single pilot scale process developed in BAMMBO, which corresponded to the production of the red carotenoid astaxanthin.

The natural and synthetic pathways were studied according to the methodology and impact categories used by Baker and Saling (2003). Three conventional scenarios were evaluated: synthetic production from petrochemical raw materials, natural production by yeast P. rhodozyma and natural production by microalga H. pluvialis. The impacts for two BAMMBO scenarios (initial pilot scenario and optimised scenario, both producing biomass with 5% astaxanthin) were calculated with respect to the conventional microalgal system. This conventional system consisted of open ponds to cultivate H. pluvialis with concentrations of 2% astaxanthin. The considered ratios included the effects of different reactor configurations in each specific category (e.g. different land use or energy efficiency of open ponds, tubular PBRs and flat-panel PBRs).

The obtained results (Figure 24) showed that yeast fermentation was the conventional route with the highest impacts in most categories, whereas the synthetic processes seemed the most efficient option from an environmental point of view. However, synthetic astaxanthin is not recommended for human consumption, so its range of application is more restricted than the natural compound, especially linked to nutraceutical and pharmaceutical uses. Among the evaluated BAMMBO scenarios, the initial system showed high energy consumption associated with the cultivation step, although it had lower contributions to most impact categories than the two conventional natural sources. On the other hand, the optimised BAMMBO system was found a competitive option with respect to synthetic astaxanthin that presented remarkable improvements in most of the evaluated categories. In addition, this route would provide natural astaxanthin, which can be applied in a wider range of sectors to obtain more valuable products.

Potential Impact:
Expected Final Results and Their Potential Impact

Research and the Innovation Union Flagship Initiative are at the top of the EU's agenda for growth and jobs with future living standards dependent on our ability to drive innovation in products, services, business and social processes. BAMMBO’s aim was to identify cheap, sustainable and abundant sources of bioactive compounds for cosmetic, pharmaceutical, nutraceutical and industrial uses. There are strong end user driven demands or trends for natural agents in these markets and the majority of research and development in these spaces is focusing on naturally derived agents as those identified in the ‘Sustainable Production of Biologically Active Molecules of Marine Based Origin’ KBBE project. This increasing trend is driven by (i) lifestyle changes driving increased demand for convenience foods; (ii) technological advancements improving sensory attributes and widening application scope; (iii) growing consumer interest in natural products; (iv) globalization of bioactive market and (vi) new product development.

The overriding objective of BAMMBO was therefore to develop a harmonious commercial relationship with the sea by applying sustainable environmentally kind practices to the valorization of high value added biomolecules (HVABs) from marine life. This approach permitted a conscientious means to maximize both human and economic benefits from the marine environment while creating new knowledge, processes, products and employment (Figure 25). Through the bio-prospecting strategy employed in BAMMBO sources of identified and potentially novel bioactivities with defined and immediate market position were identified. In the majority of cases these bioactivities were supplied from established sustainable cultures of marine organisms based on developed processes that overcame pre-existing bottlenecks to their production. An overview of the main results and their socio-economic impacts and wider societal implications where relevant are outlined below:
The BAMMBO consortium comprises 11 partners of whom there are 9 RTD performers and 2 SMEs. From an educational point of view the RTD performers are located in Universities and Technological Institutes. BAMMBO achieved two major educational impacts as such (i) enhanced mobilisation and exchange of post-graduate students between research centres within the consortium (including ICPC country Participants) thereby providing an enhanced student learning experience and (ii) the research program has permitted the modernisation of 3rd Level teaching programs being offered at the undergraduate and post-graduate level.
An extensive array of scientific publications, book chapters, magazine articles, public deliverables, lectures, presentations and radio and television scientific reports were released by BAMMBO during the 36 months of the project. This had the benefit of promoting, informing and educating community stakeholders of the science behind the project. This work which was supported by the European Union was at the end of the day for the benefit of a wide range of community stakeholders.
An invertebrate marine extract bio-bank was created which is available to BAMMBO researchers and other prior-consortium agreed RTD and industrial performers. The bio-bank will promote continued research between the consortium partners and between BAMMBO and the wider scientific and industrial communities over the coming years. This will be especially relevant not only for the forthcoming Horizon2020 programme series of calls but also to national research and enterprise funding schemes of each partner.
In developing the bio-bank consideration was taken into account of the need to avoid unnecessary harvesting of marine organisms from the environment and as such avoid defeating one of the main aims of the project from its inception. BAMMBO partners drew on their pre-existing resources and in essence made available a larger virtual bio-bank for use within the project. Such pre-existing resources from Genetika (Arctic Bacteria and Fungi), UGent (Microalgae), UNIGE (Sponge), UNS (Sponge), UNICAMP (Antarctic Fungi, Yeast and Bacteria) and IPL (Macroalgae) are available for further research and utilisation by industries and RTD performers across Europe. This promotes social inclusion and interaction at the scientific level.
The BAMMBO bio-bank software applied during the course of the project was based on a system developed by the University of Nice. This has the potential to be modified for the needs of similar inventory systems and as such was identified as a Technology Package Offering by UNS, France. Availability of the system has the potential to avoid unnecessary expenditure of time and money to recreate such a system for similar purposes.
Prior to BAMMBO there was an identified need for photobioreactor designs which permitted the cultivation of shear sensitive microalgal organisms such as those belonging to H. pluvialis. Previously this organism demonstrated unacceptable rates of cell death in high throughput photobioreactor designs. Scalable cost effective industrially relevant photobioreactor systems were built in BAMMBO and further developed by Algae Health and Greensea for the sustainable cultivation of microalgae whilst permitting automated control of a wide range of culture parameters. The developed microalgal system technologies were optimised by the industrial partners which resulted in the fine-tuned stress optimisation of H. pluvialis for reproducible production of astaxanthin up to 1000L commercial scale. This 1000L system was demonstrated to be one of the most commercially viable systems in the market today. BAMMBO reactor designs will allow for economical and sustainable production of bioactives creating new products and ultimately supporting employment.
With the support of BAMMBO funding commercial viability analyses were performed by Algae Health. These results together with enhanced production capacities and cost savings have permitted Algae Health to create 6 jobs in 2013-2014. The company has since scaled their technology to a level which utilises multiple 20,000L photobioreactor modular units. These novel photobioreactor designs are in essence repeatable units, which can be converted into “bioreactor farms”, increasing efficiency, decreasing carbon footprint, and enabling the economic mass production of novel compounds. For Algae Health further projected business expansion indications are for 23 employees based on forecast export sales in the coming years. As such a significant outcome of BAMMBO was the commercial advancement of a ‘High Potential Start Up’ to an ‘Established Company’ state generating sales outside of Europe (exports from Europe) further increasing Europe’s competitiveness. Furthermore the Technology of Algae Health was identified as a Technology Package Offering available for National and European RTDs and SMEs alike wishing to perform microalgal growth trials for commercialisation.
BAMMBO research funding together with a 2013 Venture Capital fund secured by Algae Health permitted the company to retain expertise, expand facilities/resources and increase product production. This funding has a direct benefit on the Western Region of Ireland which was significantly affected by the economic down-turn of the last 5 years. From a market perspective both Algae Health (Ireland) and Greensea (France) are ideally placed for growth based on their project involvement. It is worth noting that the market for carotenoids was predicted to reach €1.2 billion by 2015. In the case of the carotenoid astaxanthin – while the market has been dominated by low cost synthetic analogues recent consumer analyses have noted that there is an increasing demand for natural alternatives for synthetic astaxanthin and associated carotenoids. Algae Health, Greensea and associated side stream support businesses are set to prosper in this developing economy by default.
Most of the astaxanthin production from H. pluvialis takes place in mainland China, Israel, India and USA and a few small producers in other countries. No European country has been shown to date to commercialise astaxanthin at the same level as Israel or USA. BAMMBO provided Greensea and Algae Health a real opportunity to demonstrate the feasibility of growing the H. pluvialis at larger scale and producing the high value carotenoid astaxanthin. Greensea are continuing the development of this product with a short term commercialisation objective.
Using an LIT developed SCFE technology a solvent free process capable of yielding 11% astaxanthin oleoresin (high value astaxanthin) was developed which may be applied to the above commercial processes to enhance revenue. This exploitable foreground SCFE technology is available not only to the BAMMBO SME partners but other producers in Europe intending to extract HVAB lipophilic compounds, by licence agreement. This technology while demanding a high initial capital expenditure has the capability to generate higher value ‘niche’ organic products which are free of chemical residues using a carbon neutral process. These are benefits for the environment, consumer and the manufacturer.
Three of the main bottlenecks to the commercial utilisation of sponges as sustainable sources of HVABs are (i) a lack of understanding of the biosynthetic processes utilised by sponges in the creation of HVABs (ii) the slow growing nature of some sponge species thereby resulting in limited production capacity resulting in the need for wild harvesting and (iii) selective extraction of targeted metabolites from seawater. Research carried out by UNS as part of BAMMBO has resulted in these queries and bottlenecks being answered in part and addressed, respectively. Through their research UNS identified that arginine plays an initial role as a precursor in the synthesis of the guanidine alkaloids of C. crambe thereby opening up the potential for synthetic and/or semi-synthetic production continuous processes for these potential lead drug molecules. Aquaria based systems to sustainably culture sponges for over one year were also developed thereby avoiding environmentally adverse destructive in-situ harvesting of sponges. It was also learned that selective modification of the environmental sponge conditions can be used to enhance the production of targeted crambescins and crambescidins. Novel extraction systems were devised and applied to selectively extract targeted sponge metabolites from seawater. Comparative analysis of culturing methods also indicated that greatest yields of sponge metabolites were derived from mariculture processes. Mariculture processes also lend themselves to the developed UNS extraction system. Based on the advances made by the UNS partner a spin-out enterprise ‘SeaRena’ is in development with a planned roll out for October, 2014. The company will be dedicated to the valorisation of HVABs produced by marine invertebrates such as sponges using the developed ‘Sponge Milking Technique’. This process has been disseminated in the Hi-Tech update of EuroNews in 2013 (See: http://fr.euronews.com/2013/07/02/cramb-cramb-l-eponge-medicament-du-futur/). This was broadcast to 300m people and produced in 13 languages. The UNS work has been the subject of seven publications and more are in preparation. Several invention disclosure forms (IDFs) have already been registered in La SATT Sud Est for the transfer of technology in order to assess the patentability of the compounds and the bioactivities evidenced. Ultimately the societal implications and socio-economic impacts encompass the potential development of new lead molecules for drug development, the creation of a spin out business offering employment in the South of France (Stage 1) and greater potential involvement of UNS with International RTD and SMEs through Horizon 2020 and other national and international funding programmes.
While true ‘epiphytic’ sponge and macroalgal bacteria were not discovered other species of bacteria ‘associated’ with sponge and macroalgae were identified with demonstrated bioactivities of interest to BAMMBO. These bacteria were cultured at both small and relatively larger scales. Due to the nature of bacteria that reside as mixed populations on and within sponges the capability to culture isolated species of such derived bacteria is no small achievement. A large array of bacteria were taxonomically identified with a demonstrated individual ability to produce a variety of targeted BAMMBO HVABs (Ubiquinone Q10, anti-microbial, anti-tumor and anti-oxidants). Within this group of taxonomically identified bacteria selected candidates were sustainably cultured at scalable levels. Overall, this is an exciting finding as the organism species identified have the potential for sustainable fermentation at large scale. Such organisms unlike microalgae are less susceptible to adverse shearing effects during culture. Knowledge and possession of these bacterial is not only scientifically valuable but also commercially relevant as the US and European Ubiquinone Coenzyme Q10 market alone has been projected to exceed €133 million by 2015. The benefits are scientific and commercial.
Six bromoditerpenes were purified from S. coronopifolius one of which has been identified as a novel bromoditerpene (not yet discovered or reported previously) displaying anti-proliferative activity against HEPG2 cells at sub-toxic concentrations. This has obvious societal benefits not only through the better understanding of cancer biochemistry but also the commercial potential from synthetic and or semi-synthetic processes to create this compound. Of course it needs to be highlighted that such research is at an early stage and limited potential impact can be made in the lengthy drug development process.
Overall, 11 guanidine alkaloids (8 New/Novel, not yet discovered or reported previously) were identified and characterised from C. crambe sponge by UNS, France. Like most C. crambe compounds they displayed potent biological activity in most analyses. But specifically through collaborative work with USC it was assessed that Crambescidin 816 and Crambescin C1 blocked voltage gated sodium channels and induced Ca2+ entry. This indicated that these compounds could decrease neurotransmitter release. Blockage of T-lymphocyte potassium channels indicates that Crambescidin C1 may act as a lymphocyte activation inhibitor.
Crambescidin 800, 816 and 830 displayed positive effects on human tumour cell lines. Crambescidins inhibited cell migration (at non-toxic concentrations) and was cytotoxic (at higher concentrations) for lung carcinoma (HOP92), colon carcinoma (HT-29), melanoma (SK-MEL), renal carcinoma (OU-31), prostate carcinoma (PC-3), ovary carcinoma (OVCAR), and breast carcinoma (MCF-7) cells. The production of HVAB’s in BAMMBO will have a positive social impact on human health down the line potentially expanding available therapies, and enabling a greater range of ailments to be treated. Likewise, the production pathway optimization of bioactive compounds will make existing therapies affordable and widely available. This will open new market opportunities for the European pharmaceutical industries, realizing the dual benefit of European job creation and enhanced European health. Environmentally, it is not acceptable to harvest bioactive compounds from their natural habitat because it causes ecosystem destabilization and has a negative impact upon biodiversity. The in vitro production of high valued compounds will produce high-valued molecules without disturbing such natural habitats. Production would be based on commercial enterprises such as those developed by UNS which has obvious social and economic benefits.
As a result of BAMMBO a better scientific understanding of the existence of cold adaptive lipases and ligninases was acquired. Lipases and ligninases have a variety of applications in the food processing, environmental remediation, detergent and biofuel industries. Culture conditions for these Antarctic yeast and fungal species were developed. While difficulties were experienced in the sustainable culture of the lipase expressing organisms as mentioned previously solutions are at hand to progress the scientific challenge. Such solutions reside with genetic engineering and the cloning of the Antarctic lipase genes in heterologous hosts such as the fungus Aspergillus niger or the yeast Yarrowia lipolytica which are used for the expression of industrial proteins. The initial scientific challenge to identify cold adaptive enzyme preparations from Antarctic species has been overcome. The next stage is the progression one step further towards the generation of genetically modified organisms for commercialization and/or licensing of cold adaptive lipases.
The macroalga O. secundiramea was identified as a good source of the fluorescent phycobilipigments ß-phycoerythrin. Methodologies were developed to extract this compound which has application sin the diagnostic and imaging industries. The O. secundiramea could be sustainably cultured in a bioreactor and as such this technology offers an alternative source of this protein. LIT have filed two IDFs for this new extraction and activity staining systems.
UNIGE, Italy research outcomes identified key features enabling the correct choice of sponge farming site, farming method and culture timing which has a high impact and influence on industrial sponge farming. UNIGE’s optimized sea based culture of C. crambe exceeded 1000% in annual production of biomass, encouraging the sustainable production in the field of this sponge species’ biomass. The added, environmental friendly, value in sea based sponge farming derives from the active filter feeding activity of these organisms. Ultimately 10 kg of farmed S. spinosulus grow c. 100% yearly, filtering more than 100 million liters of marine water per year, thereby removing up to 10-15 kg of potentially pathogenic bacteria.
The new sponge farming protocol (C. crambe on travertine tiles) developed by UNIGE is being investigated for an Italian Patent application as “A new method to maximize the biomass production in sea based, shallow water, sponge farming on natural substrate”. In the view of the UNIGE PI aquaculture is, in general, highly impacting (pollutant) on the marine environment, requiring high energy consumption. However, industrial sponge farming could have a positive impact on coastal waters resulting in a bioremediation effect as tested by several Italian initiatives involving the implantation of small sponge farms inside the turistic harbour of Santa Margherita Ligure, Italy and at an industrial floating cage fish farming plant in Lavagna (Ligurian Sea), Italy.
The assessments performed within the BAMMBO framework by USC, Spain addressed for the first time the three dimensions of sustainability applied to marine biotechnology. The study allowed establishing a procedure to collect inventory information, model novel cultivation and extraction techniques, and simulate different conditions for the characteristic stages of the processes associated with marine biotechnology. The results have identified the key activities and stages with significant impacts throughout the life cycle of the products. The outcome will contribute to focus future efforts towards sustainability on the most problematic stages and therefore, to undertake process design in a more efficient manner to achieve more environmentally friendly production systems while saving money.
Indeed, several suggestions and strategies were proposed to the involved partners in the published deliverables and other internal documents in order to optimize the developed pathways and improve the sustainability of these options. In most cases, this optimisation should focus on the reduction of electricity requirements by using alternative configurations such as outdoor photobioreactors or changes in light patterns. Recycling the culture medium to maximise the conversion of nutrients would also help to reduce impacts, whereas recovery and reuse of solvents for extraction seemed to constitute an essential strategy for most extraction systems. Additionally, some novel technologies such as supercritical CO2 extraction show a great potential to produce high value molecules with lower environmental impact. Therefore, the performed assessments will provide the participants with environmental and socio-economic information that may help to differentiate themselves from competitors, an aspect that is especially important in the case of the involved SME partners.
The results obtained from the life cycle assessment of BAMMBO processes were collated into 5 separate deliverables which were public in nature. The information generated as such is open to the public and can serve as a basis for other European and international researchers to measure sustainability in future projects and processes as well as for manufacturers worldwide. The outcome will be applicable not only to the organisms and products studied in BAMMBO, but also to other producers and compounds.
In the development of a fully-fledged knowledge based bio-economy from marine resources BAMMBO aimed to accelerate the transformation of the marine sector through implementation of a clear and realistic strategy for overcoming the current bottlenecks and barriers encountered in the culturing and biotechnological exploitation of marine biomass resources for production of high value added products for the pharmaceutical, cosmoceutical and industrial sectors. Upon consideration of the information reported within this report it is clear that the strategy employed demonstrates BAMMBO was effective at progressing the major activity blocks connecting strategic objectives with strategic impact through knowledge acquisition (Figure 25).

Main Disseminating Activities
Outcomes of BAMMBO research activities to target groups was achieved by way of several dissemination routes. These included a wide range of International Scientific Publications, Posters, Book chapters, Conference Presentations, Invited Speaker Presentations, Radio Interviews, Newspaper Articles and Web Podcasts/News Updates, a Television Report (via Hi-Tech Euro News - http://www.euronews.com/2013/07/02/sponging-off-the-sea/) Magazine Articles, Website Updates, Public Deliverable availability/distribution, Public Information Events hosted by Consortium, Workshops and Special Sittings of the BAMMBO Consortium, SME updates, Participation in EC surveys and Reports (STAGES Survey) following requests from their representatives.
The main dissemination metrics were:
105 BAMMBO deliverables of which 24 were Public.
37 Scientific Publications
41 National/International Presentations as Invited Speakers and Conference Posters;
5 Conferences/Seminars organised
4 Workshops organised.
950 Multilingual (7 languages) BAMMBO brochures circulated
9 Press releases
2 Popular Press or Magazine Articles
2 Bioreactor exhibition days

Exploitation of Results
In accordance with the BAMMBO exploitation plan all BAMMBO IP material were possible was gathered and collated throughout the project course. At specific time points BAMMBO IP information was collated and assessed (Months 15, 18, 33 and 36). Where necessary and following discussions with either the Consortium Members, Technology Transfer Officer(s), SME partners, BAMMBO Advisors and External/Internal Partner Organization Stakeholders the IP was positioned for one of several follow up processes namely:
o Retention of the Foreground IP by a Partner Organization as Know-How or Trade Secret
o Filing of an Invention Disclosure Form with the Organizations TTO and/or Coordinator
o Development of a Patent Application
o Development of a Technology Package Offering and Potential Licensing
o Spin out of research outcomes into a new Start-up company
o Use of developed Foreground IP in future follow on projects including Horizon 2020.
o Publish where4 possible non-protected outcomes
As IPR related matters follow a due process the IP situation of BAMMBO will continue to be monitored and updated as agreed in the BAMMBO Grant Agreement. The main IP related metrics were:
23 IP items documented comprising categories of New Discoveries/Compounds, Patents, Novel Inventions, Know-How, New and or Modified Production Processes
8 Invention Disclosure Forms filed and registered with the Coordinator
4 Patent Application Intentions
8 Technology Package Offerings



List of Websites:

Contact Details and Website
For an extensive listing of all the BAMMBO Participants and their full contact information please go to the BAMMBO project website at www.bammbo.eu
The BAMMBO Coordination Office is Located in the Limerick Institute of Technology, Limerick, Ireland
o Tel: +353 61 208 208 Ext: 526
o Email: Coordinator: daniel.walsh@lit.ie or Coordinating Office: bammbo@lit.ie
Postal Address: Limerick Institute of Technology, Moylish Park, Limerick, Ireland.