PROJECT DESCRIPTION:

Part 1: Results of the Phase I Project. 1.1 Background: Phase I demonstrated the merits of biological waste management within a recirculating aquaculture system (RAS) to grow by-product crops on a commercial aquafarm. The objectives were met, establishing a firm foundation for Phase II. Acadia Harvest, Incorporated (AHI; previously known as RAS Corporation (RASC)) had shown that deposit-feeding sandworms (SW, Nereis virens) consumed all fecal and feed waste that flowed across their sandbeds from a tank of black sea bass (BSB, Centropristis striata). Approximately 1.0 m2 of sandbed containing SW were found to remove the particulate waste generated by 60 kg of BSB. With particulate waste accounted for, investigators decided to determine if the dissolved waste (e.g. including mainly ammonia, nitrates, phosphate and carbon dioxide) of both fish and sandworms could be utilized to culture microalgae that in turn would nourish a crop of oysters. Microalgae (Isochrysis galbana and Tetraselmis suecica) were inserted and cultured with SW and oysters (OY, Crassostrea virginica) along with California yellowtail (YT, Seriola lalandi), the latter a substitute for BSB as initially proposed.

Integrated multi-trophic aquaculture (IMTA) has been tried for years as an adjunct to salmon cage culture and now AHI is importing the concept on-land and indoors for use in Recirculating Aquaculture Systems (RAS). Hence, IMTA-RAS is a novel approach that has practical merit, enabling a high degree of monitoring and control. Moreover, ecological principle suggests diversity enhances overall system stability. Our man-made ecosystem is a mix of several trophic levels with multiple species sharing a common flow of seawater, energy and nutrients (Figure 1). System stability and health are critical to success in commercial aquaculture, the more so in highly intensive aquatic operations, but they are often elusive in monoculture of any kind.

Figure 1. Phase I/IB Experimental System – (1) Fish Tank; (2) Header; (3) reserved for Macroalgae; (4) Biofilter; (5) Open photobioreactor; (6) Sandbeds.

1.2 Conclusions of the Phase I/IB findings and how these conclusions support a Phase II proposal. Finding #1 - Growth and Health of Integrated Species: Phase I/IB demonstrated biological feasibility of basic IMTA-RAS in several significant ways. The five integrated trophic levels (including bacteria as the 5th) were mutually supportive, forming an ecological system where the whole was greater than the sum of the parts. YT and SW grew to market size with minimal and wholly acceptable mortality rates. Once the algal system was established, the OY grew rapidly and would have been of a marketable size by April 2014 had the trial continued. Two species of microalgae were also introduced as an external input from closed photobioreactors (PBRs). Finding #2 - Observation and Modeling: Once balance was achieved with adequate growth and excellent survival, a sampling routine was initiated, tracking key physical and chemical parameters. While the dataset has provided some insight into system dynamics, a more sophisticated, real time monitoring system was needed to predict instances when balance became somewhat precarious, thus allowing for improved husbandry response by a commercial operator. Moreover, “balance” in the Phase I set-up was under largely static conditions whereas the commercial operator will be harvesting, re-stocking and even adding external feeds and nutrients whenever appropriate. This could involve, for example, adding SW feed to expand production when worm prices rise or supplementing the algal nutrients to meet increased demand for OY. The Phase II plan will include major improvements in system monitoring intended to expand understanding of system dynamics and to increase the power of predictive modeling.

Finding #3 - Foundation for Phase II: The 1/20th scale Phase II pilot plant will include full infrastructure capacity for IMTA production with revenue from initial crop sales projected to exceed $250,000. The overarching goal will be to refine husbandry protocols in order to gain investor and management confidence for Phase III full scale production. Land-based, indoor IMTA-RAS was shown to be feasible in its most basic form, and is worth pursuing at an advanced level in Phase II. AHI and its subawardee, the University of Maine’s Center for Cooperative Aquaculture Research (CCAR), are qualified to design, install and operate the Phase II Pilot Plant. As a man-made ecosystem, it has and will continue to offer biological insights into intra- and inter-species interaction.

Phase II in Brief: We propose to re-design and enlarge the system to simulate an actual indoor aquafarm. This 1/20th scale is the crucial next step before commercialization. We will simplify the layout for efficient use of space, maximum interchangeability of containments and optimum inter-connectivity. AHI has cultured fish, microalgae, invertebrate deposit-feeders and filter-feeders together and hereafter will add macroalgae as well. Moreover, where we have cultured a single fish at a time, Phase II will trial two fish crops (YT and BSB), at least three shellfish (American and European oysters (Ostrea edulis), soft shell clams (Mya arenaria), two bottom dwellers (SW and perhaps a Holothurian) and an expanded suite of algae species. If diversity conveys stability and health – as demonstrated in Phase I – we believe more diversity will be advantageous within the limits of practical husbandry. Finally, the work of Phase II will proceed in two stages over 24 month. Stage 1 will establish the ecological balance previously achieved in the largely static Phase 1 system. System balance will then be stressed in Stage 2 by inducing a variety of changes as would occur in Phase III: routine harvests and restockings with a variety of external energy inputs as well as crop substitutions. As we have already shown, a mix of species embedded in a shared flow of energy and nutrients can be self-managing. That alone suggests a paradigm change to aquafarming. Aquaculture has its critics, but with the growing demand for protein, the industry is here to stay. It does, however, behoove developers for both economic and ethical reasons to recognize and address shortcomings within the field. Streamlined infrastructure designs, sensible waste management, avoiding reliance on unsustainable forage fish, and spreading expenses over several crops will all tend to increase return on investment. In related projects, AHI has joined with numerous others to formulate and trial alternate aquafeeds that eschew fish meal and fish oil, a major opportunity for aquafarming. As these innovations reach the market aquafarmers can boost the economics of their businesses and enhance competitiveness, while offering consumers good value and variety in traceable and sustainable seafood.

Part 2: Phase II Technical Objectives 2.1 Specific Objectives Introduction: The overarching goal of this Phase II effort is to develop practical and proven husbandry protocols to demonstrate to stakeholders and investors that IMTA techniques will work in a commercial RAS aquafarm. Meeting this goal requires setting up and operating a 1/20th scale IMTA-RAS pilot plant, including the full spectrum of production factors from plant design to harvesting with an improved grasp of how the integrated species can be mutually supportive. This can be achieved through five main objectives:

(1) Optimize use of space at minimum cost, requiring a wholly novel placement, configuration and utilization of crop containments.

Space and cost considerations are paramount when production moves indoors. As shown in Figure 1 above, the original Phase I set-up was assembled from a variety of tanks and raceways, cylinders, and containers -- whatever was inexpensive and convenient. It worked well and demonstrated that a landbased, indoor fish farm employing IMTA techniques would succeed, but made poor use of expensive space. In preparation of Phase II, staff revisited the design, evaluating all aspects of the operation. Reduced to functional requirements, it became clear that IMTA would work with circular tanks for pelagic fish like YT and, with minor exceptions, raceways for everything else (Figure 2). The four rectangles depicted are 3-level stacks of fiberglass raceways (Figure 3). The raceways are identical in order to lower costs and to enhance flexibility. They will accommodate bottom-dwelling fish, by-product invertebrates and both macro- and micro-algae cultures. Moreover, they can double for some of the necessary recirculating equipment. The proposed system is simple and highly inter-changeable (Table 1). If this system functions as intended in Phase II, it will be used in Phase III, albeit with modifications based on 24-months of experience. (2) Utilize the Pilot Plant as operational mock-ups in preparation for Phase III.

Commercialization of an aquatic production system is driven by stocking and harvesting. Because an integrated aquatic system is being proposed, i.e. with shared sea water, every change in one trophic level will have an impact to one degree or another on all other trophic levels. While the basic-IMTA-RAS external inputs are mainly fish feed and light (sun and/or artificial), it may be necessary to add other external energy sources as required to boost harvests from one or another trophic level. In advanced- IMTA-RAS, additional fertilizer and/or CO2 may be added to boost microalgae productivity or to increase the size of the SW or OY crops by supplementing the particulate waste they feed upon. As detailed below, (2.3 Work Plan), AHI will initially establish a balanced, static system and once achieved begin introducing changes in order to document and manage responses in anticipation of full commercialization.

(3) Install the latest water quality monitoring equipment in a novel manner so as to significantly reduce the number of monitoring units required. Water Quality Monitoring Equipment (Novel Installation): A 4-channel Fluid Flow Injection Analysis (FIA) system will continuously measure ammonia, nitrite/nitrate and phosphate in water sampled from a single, fixed “sampling chamber”. Several bypass loops (“short circuits”) will originate from various sources in the IMTA system and flow continuously to the system sump. By connecting these flows to a solenoid valve manifold, each loop can be diverted in a repeating sequence to this common sampling chamber (much as a fuel injection system works in an automobile). Configuring all monitoring instruments (FIA, Yellow Springs Instruments YSI 5200A and Fluid Imaging system) to sample concurrently from the same sampling chamber will enable a several-fold multiplication of analytic power. It will also minimize costly set-up and calibration time, and eliminate the need for manual, batch analyses or duplicate monitoring units installed throughout the IMTA-RAS system. With control computer handling/flow timing, data logging and indexing measured values to sample sources, a water source can be diverted, allowed to flush the sample chamber within two minutes and then sampled for all water quality parameters. This comprehensive, real time monitoring will also enable automated changes in system configuration to maximize production and safe guard system health. For example, when the ammonia level is acceptable, fish tank water goes directly to the open PBR and the biofilter and foam fractionator are bypassed, receiving minimal or no maintenance flows. When the ammonia level exceeds a pre-determined value, an increasing portion of system water is diverted to the biofilter/foam fractionator before sending it on to the PBR.

(4) Develop a predictive model utilizing water quality data combined with manually recorded energy input, growth and survival information

Predictive Model: In Phase I, land-based IMTA-RAS proved to be self-regulating when left alone, but practical aspects of for-profit animal and plant husbandry now require changes. Even hourly adjustments are conceivable to optimize production, but only if those are informed changes. Fortunately, the sensors mentioned above are available to closely monitor critical system parameters. What is required is a predictive, and adaptive model that uses data to guide the aquafarmer’s biological and economic decisions, ideally with an ever-increasing capacity to manage the system remotely. Moreover, the wealth of data collected will offer a rare opportunity to intensively study ecological interactions under controlled conditions in an enclosed space. At some future time, we can insert robotics to reduce operating costs while increasing biosecurity with only moderately more investment. (5) Reduce the model into the format of an operating manual for day to day use by non-technical IMTARAS aquafarm managers and staff.

A highly evolved predictive model will be an essential aid to understanding multi-trophic interaction in a man-made ecosystem. However, it will be of little use unless it can be simplified and summarized in the form of an instruction manual. The Phase III IMTA RAS farm manager and senior staff may not be technical persons so day-to-day guidance will be absolutely critical. When RASC commences operations in Phase III, there will be technically qualified persons already experienced from pilot plant operations on hand, but as soon as the first full production unit is a success, it will be cloned both on site and, by means of licensing others elsewhere. At that point, it is crucial the operation be wholly manageable by nontechnical persons. This is an economic consideration (lower wages and salary rates) as well as for licensing purposes (“Anyone can run it”). More to the point, however, a clear manual will aid in safety, reliability and efficiency. In 2015 AHI is targeting the financing and construction of a commercial YT-only aquafarm on our site in Corea, Maine. The aquafarm will be designed to have the infrastructure in place to later accommodate an expansion to accommodate IMTA-RAS production. At the conclusion of Phase II in June 2016, the pilot plant design will be adapted as appropriate with the Phase II-proven IMTA equipment and expanded 20 fold. With YT already in production on site, this will add at a minimum BSB, two by-products (SW, OY) and possibly marketable seaweed. We anticipate that other species with biomediation and/or marketable potential that can, also, be integrated in a practical husbandry sense are likely to emerge in Phase II.

Figure 2. Proposed IMTA-RAS pilot plant. Large circular tanks hold pelagic schooling fish (YT) and rectangular raceways hold benthic, solitary species (BSB, SW and OY). When fitted with lighting, raceways serve as open photo-bioreactors for micro- and macroalgae. All containments are part of a single seawater system. Multiple functions can be combined; for example, a raceway can double as a sump that contains biofilter media or a header raceway that is well-lit and stocked with macroalgae.

Figure 3: In the pilot plant, a simulated aquafarm, the raceways are maintained at three levels. The system water leaves the header tank (that doubles as a raceway for microalgae culture purposes) and flows to the circular fish tank where the outflow is split so that particulates from a bottom drain go to the sandworm raceways and the overflow containing dissolved waste directly to those top-level trays that contain oysters and/or microalgae. At full commercial scale, there may be as many as 5 or 6 levels.

Uniform Raceway Design -- Multi-Stack Configuration Stack Levels A B C D Top Header/Macroalgae Oysters Oysters/PBR PBR Center Black Sea Bass Black Sea Bass Sandworms Sandworms Lower Sump/Biofilter Sandworms Sandworms Sandworms

Table 1: This basic IMTA-RAS loading represents four trophic levels (also to include the bacteria populations resident in the sand and on biofilter media as a vital 5th trophic level). The pelagic yellowtail are contained elsewhere in the set-up in circular tanks. Given more stacks and more levels per stack, advanced IMTA-RAS may accommodate even greater variety, the limits being practical, economic farm management and market demand.

2.2 Technical Approach Pilot Plant Operation: Phase II will begin with two species of fish, American oysters, sandworms and at least two each of microalgae and native macroalgae. Phase II will later explore greater biological diversity, albeit within the limits of cost effectiveness and market potential. Advanced IMTA-RAS might allow a second deposit feeder (perhaps a Holothurian or a lug worm) and a second or third filter feeder – probably European oyster and soft shell clams -- as long as they do not over-burden practical husbandry. Given that caveat, experience thus far encourages maximum diversity. Juvenile YT, SW, OY, and BSB will be stocked in their designated tanks and raceways immediately following installation of the tanks and raceways (month 4). They will be harvested, sold and restocked when they reach market size, which will be in the 9th to 12th months for the former three and the 14th to 18th months for BSB. AHI continues to operate an YT Pilot Plant at CCAR, funded by the Maine Technology Institute (MTI) and Coastal Enterprises Inc. (CEI). This resource in an adjacent building will allow staff to transfer 3-month old stock to tanks in the IMTA pilot plant, providing substantial fish biomass at the outset, short-cutting the time to market and allowing several YT crop harvests within the designated 24-months. However, OY will be stocked as 3.0 mm spat and SW as nectochaetes (worm larvae).

Basic IMTA-RAS demonstrated that a natural balance can be sustained across trophic levels with minimal external inputs. Up-take of particulate and dissolved waste, i.e. “zero waste aquaculture” with minimal or no make-up water and discharge, can be sustained with fish feed and light as the major energy inputs. This has appeal as a semi-closed emulation of nature, but must be refined for commercial production. As an example, an aquafarmer may decide to expand the OY crop beyond the limits of the microalgae cultured with internally generated dissolved waste, by supplementing CO2 and/or nutrients. There may even be an opportunity to add a supplemental oyster feed such as hydrolyzed squid waste or other finely macerated organic materials (AHI plans to undertake oyster feeding trials, separately funded, utilizing the partially grown Phase I OY stock).

Another external input that has already been included as a factor from the outset is batch or continuous inoculation of microalgae by means of an off-line closed PBR. Small bubble columns were employed for batch purposes in Phase I, but the PBR of choice for Phase II will be significantly larger with a capacity to operate continuously (Sea Caps Continuous Flow Algal Culture System). It will utilize 20 columnar 2.0’ diameter bags 65’’ high. Several algal species can be cultured simultaneously. The overall cell production can be calculated at 4 x 106 cells/ml of Isocrisis (for example). Densities as high as 2 x 107 cells/ml are possible with intensive lighting. Artificial lighting is a significant expense, so we anticipate in Phase III the closed PBR will be situated in a greenhouse (probably on the roof) to reduce costs. We anticipate algal yield will exceed the need for an inoculant at which time OY space and spat can be added.

Ecological Comprehension: Ecological principles suggest that greater diversity equates to greater stability and overall system health. The apparent chaos depicted in Figure 4 is more likely to have global stability. This was demonstrated in Phase I when a “balance” was achieved, with minimal staff adjustments. In Phase II, the balance, once achieved, will be deliberately stressed to simulate practical farm operations. Such applied science is especially satisfying where there is a clear objective such as RASC envisions for Phase III. However, the experience of managing – and being managed by – the Phase I simple ecosystem was intriguing and stimulating. The AHI and CCAR teams share a sense of curiosity and wonder when contemplating the Phase II system. This work will provide biological insights well beyond our targeted objectives for enhancing IMTA. To what extent can the multi-trophic and interspecies interactions be followed? Precisely what are the energy flows if indeed they can be quantified? Such basic understandings are of interest to ecologists and critical to the success of a commercial aquafarm alike.

Figure 4: Graphic showing the advanced IMTA-RAS. All trophic levels are contributing to and extracting energy and nutrients from a shared body of seawater.

2.3 Work Plan: Task 1. Install New IMTA Equipment: The Phase I IMTA set-up was shut down at the conclusion of Phase I/IB. For Phase II, AHI will move into another, considerably larger space at CCAR, one that was designed and funded specifically to “incubate” innovative aquaculture projects. As mentioned, it will accommodate 1) four stacks of three 20’ raceways each, 2) two 22’ diameter fish tanks (70 m3 each) and 3) large, continuously (if need be) operating closed PBR. Fish feces and waste feed from both fish tanks can be directed to the stacks with the option of one tank’s particulate waste source being directed to an existing mechanical filter, thereby providing a useful comparison as an experimental reference. In parallel, AHI will be investigating ways to utilize the trapped waste as a possible supplement to algae and bacteria for oyster nutrition, whether macerated, hydrolyzed or otherwise made palatable and digestible. The design of our tanks and raceways will be studied for suitability. Potential enhancements include lighting, camera observation, automated feeding, O2 injection, in situ inventory monitoring, and selective, non-intrusive harvesting. Two 20’ raceways will be configured to accommodate BSB as this solitary, bottom-dwelling species exhibits widely different behaviors from YT. These necessitate careful consideration of water flow and particulate removal to culture BSB. Our initial choice of the containments is appropriate, but modifications will be considered as Phase II progresses. Two of the raceways will hold 15” of water and be stocked with BSB (Figure 5). These are fitted with five drainage inserts to remove the 15% water that contains most particulate waste and, five inert fences, each with an adjustable size aperture that allows the smaller BSB to escape from the larger, more aggressive individuals. Unlike schooling pelagic fish such as YT, sorting by size is essential for culturing BSB to prevent small individuals from being “bullied” when attempting to feed. Gating the sections as shown in Figure 5 will encourage self-sorting. Five of the 12 raceways are designated for SW culture and will contain an 8.0” deep layer of clean sand with 2.0” of water above the sand. The water is shallow to accelerate the lateral flow and enhance the spread of particulates.

Figure 5: Prior to initiating Phase I in April 2013, AHI had maintained BSB in the same 9.2 m3 circular tank in which it subsequently maintained YT. By comparison, over 18 months, BSB growth was highly variable and poor overall while YT grew much faster with a narrow J-curve. We concluded that in addition to genetics, behavioral differences associated with solitary, benthic existence versus pelagic schooling were overarching. Using a rectangular, gated raceway as shown will be better suited to BSB. Sizing gate apertures sequentially should encourage self-sorting by size thereby reducing aggressive behavior while encouraging uniform growth.

Task 2. Verify Near-Static Balance: Proper balance in an IMTA system is critical for the well-being of all of the organisms grown. It will require another 3 months for the crops (and several successions of mainly Nitrosomas sp. and Nitrobacter sp. bacteria populations) to mature until a mutually supportive balance is achieved as was the case in Phase I. Changes in any portion of the IMTA system are likely to have downstream effects, especially in terms of water quality. Continuous monitoring of nutrient values (ammonia, nitrite/nitrate and phosphate) and other water quality parameters (pH, dissolved oxygen, carbon dioxide), will provide a greater understanding of the linkages between system components and set the baseline for the predictive model.

Task 3. Induce Changes: Installation of the Phase II IMTA system is anticipated to take three months. Loading of juveniles of the animals and algae will take place in the 4th month – (October 2015. At least two species of microalgae and macroalgae will be introduced early with more species of the former added later, as long as they are small in size to facilitate OY digestion. RASC plans to include macroalgae as a component as it will absorb dissolved wastes including CO2 and contribute O2. These trials of species that tolerate 20⁰ to 22⁰C and are native to Maine’s coastal waters may result in a market opportunity that is not yet apparent. There are several native species of macroalgae that are tolerant of warmer water (220 C). Gracilaria tikvahiae is one option as it does very well in a tank based system (Yarish, C, Redmond, R., Jang K., 2013). Another is sea lettuce (Ulva lactuca), a green, flat-sheeted seaweed that is especially effective at taking up nutrients. (Ms. Sarah Redmond, a project consultant, is managing a separately funded project culturing macroalgae at CCAR).

The system should be operated in balance for another three months (to December 2015) with minimal external inputs other than fish feed, artificial light for the PBRs and continuing algal inoculations as required. In the 3rd month following installation, the largely self-sustaining IMTA system will be ready for other external inputs as would be appropriate for a full scale IMTA aquafarm. At this stage, we will begin the harvests and restocking as well as the introduction of external energy inputs whenever appropriate to maximize production. The overarching point is that this “green” approach to aquaculture must first be economically viable. Proof that these goals are wholly compatible will be the main pilot plant deliverable.

Task 4. Experiments: Much of the above is concerned with Phase II infrastructure because it is novel, integral with the 24 months pilot plan and essential to the projected economic viability of Phase III. Once the new design is stabilized the science comes from the questions asked of the man-made ecosystem it supports. Experiments to be conducted throughout Phase II are designed to answer the following questions: Question 1: What is the minimum daily water replacement rate required once an initial system balance is achieved? To what extent is this impacted by occasional external input of feed or fertilizer? By routine harvests and re-stocking? We will study practical considerations of raceway and tank size, height of tanks, water turnover rates, O2 input if any, degree of external feeding and fertilization, remote observation, remote control, and possibilities for robotic usage. Question 2: What are the roles of the several bacteria populations? What are their successions over time? Does the routine addition of probiotics alter those populations that naturally establish in sandbeds and biofiltration media and in what ways? What percentage of the oyster nutrition is comprised of bacteria? Question 3: Given that the sandworm deposit feeders and the filter feeding oysters both consume particulates, albeit of different size particles, what is their relative importance in RAS waste management? If particulate wastes are finely macerated or hydrolyzed, will the oysters consume more? Will the harvested oysters meet shellfish certification standards directly or will purging be required before human consumption? Question 4: Given excellent real-time sensors to monitor water quality and other key factors, to what extent can the interactions between trophic levels be monitored? Are the data sufficient to detect favorable or unfavorable impact for farm practice purposes? Are they sufficient for an enhanced understanding or natural marine ecosystems? Question 5: Is land-based, indoor IMTA-RAS overly complex for commercial purposes or does the presumed stability gained from complexity confer long term benefits on an aquafarm and to what extent? Will a predictive computer model assist an aquafarmer in managing the marine plants and animal husbandry? What sort of model would that be?

Task 5. Oyster Certification: There are currently no state or national regulations permitting the consumption of oysters grown to market size indoors under IMTA conditions. Ms. Lori Howell will be retained to guide AHI through the regulatory process to obtain appropriate certification before the OY crop can be test marketed. Ms. Howell is Vice President of Spinney Creek Shellfish Inc. She is an Attorney, has a Masters degree in Health Science, is a Member of the Executive Board of the Interstate Shellfish Sanitation Conference and Chairman of the Maine Shellfish Advisory Committee.

Task 6. Sensory Evaluation: Thus far, both YT and BSB have been well received by seafood distributors. Although these are professional appraisals, they are anecdotal. Before the conclusion of Phase II expansion, AHI will contract with UMaine’s Department of Food Science and Human Nutrition to conduct formal Sensory Evaluations (“taste panels”) of harvested fish and oysters. The latter will be tested in direct comparison with product from a well-recognized coastal oyster aquafarm.

Task 7. Data Acquisition: As mentioned under Systems Monitoring above, brand name instrumentation will be employed to collect and record data on key chemical parameter about every half hour. Since managing nutrient levels is critical to IMTA-RAS, it is essential to have accurate, real time sampling of these parameters that only a flow injection analysis (FIA) system can provide. A single, four-channel system can measure total ammonia, nitrate, nitrite and phosphate in parallel. This device will be configured to continuously cycle through samples from each tank in the IMTA system providing concurrent, high resolution data on system wide nutrient dynamics. This data will not only inform investigators of steady state and dynamic harvest conditions but will also allow us to tune mechanisms within the system such as flow distribution and nutrient supplementation to maintain system stability while maximizing efficiency and productivity.

Task 8. Develop transition to full scale IMTA-RAS: Technical planning for expansion will be a consideration throughout the 24 months. The sample list of questions referred to above is only indicative of the extent to which the prototype set-up will be probed for guidance. Numerous observations and experiments will be conducted within the time and budget allotted in Phase II. All will be relevant and inter-related with AHI’s focus on commercialization.

Task 9. Project Management: At the beginning of and throughout the period of performance, PI Tap Pryor will assemble the team, review the work plan, schedule and deliverables and determine due dates. He will also guide and oversee the day to day activities of the team. He will manage both the financial and administrative aspects of the program and be responsible for the interim and final reports. Working with the company’s business team, Chris Heinig and Ed Robinson, he will be involved in the company’s efforts to secure NSF Ph IIB match funding.

2.4 Performance Schedules Stocking & Harvest Dates: For simplicity in scheduling, AHI intends that the four main crops be routinely harvested over the two years as each crop comes of market size. YT, SW and OY share an average nine month growth cycle. AHI has limited experience with BSB (as compared to YT), but we project a likely growth period from 14 to 24 months, which is in accordance with published literature. However, such relatively slow growth and such a wide spread may not be attributable solely to genetics, but may instead be a function of the quality and character of the feed, as well as the nature of the containments and the sorting of the fish by size. In this task, BSB will be assigned two of the 12 raceways, so that different feeds and different sorting techniques can be compared for their impact on growth.

Once we are in full scale production, continuous harvesting and re-stocking will be routine to make optimum use of the facilities. Customers desire regular shipments, ideally on a weekly basis. A capacity to plan and to achieve such weekly delivery commitments is an important feature of IMTA-RAS. Partially grown YT should be available to the pilot plant at all times from RASC’s adjacent MTI-funded facilities; CCAR staff routinely spawns SW juveniles (nectochaetes) with few interruptions. OY spat is available seasonally (April through October) and BSB eggs will be available in June 2014 and 2015 only (from Milford Marine Laboratory). Harvest dates in Phase II have been adjusted accordingly.

2.5 Milestones: Milestones 2015 2016 2017 Quarters 3rd 4th 1st 2nd 3rd 4th 1st 2nd Set-Up Stock Inventory Establish Balance Dynamic Balance Harvest YT Harvest OY Harvest SW Harvest BSB Project Management Interim Report Final Report 2.6 Deliverables: Simplified Farm Design: The experience with the Phase I set-up suggested numerous design improvements for Phase II. This entails selecting auto-feeding devices and regimes for YT and BSB, in tank sorting methods for both, and ideally non-intrusive ways to monitor rates of growth and total biomass. Moreover, a level rectangular raceway holding fish (or oysters) will accumulate waste. It can be removed by multiple drains which may be aided by multiple inflow taps. For SW, stocking the sandbeds and flowing particulate waste to them as in Phase I led to non-uniform growth (best at the inflow point) whereas there are a variety of possibilities to capture the particulates for separate spreading by introduction of the flow at multiple points. With OY, the obvious choice was to route microalgae through their trays, but oyster trays placed directly in the open PBRs thrived and this technique will be tested on a larger scale over longer periods. The deliverable will be a report presenting IMTA-RAS in a much advanced form. It will serve the Phase III staff that follow, and will be a basic document to facilitate licensing or franchising this technology.

Integrated-Multi-Trophic Production: Phase I demonstrated the feasibility of integrating several trophic levels with multiple species. Ecological principles support diversity as a natural way to achieve stability. Polyculture is far from new; freshwater hydroponics makes full use of it. However, it is rarely applied in marine systems and not at all to the extent proposed in IMTA-RAS production. One goal will be to identify the practical limits to additional species within each level. One limit is farm husbandry since 3 or 5 species of fish crop could be problematic versus 1 or 2. Over many decades, large scale agriculture has gradually evolved to monoculture, despite some obvious problems, and to date, large scale aquaculture has followed the same path. Organic farming has offered an alternate, as have IMTA pioneers setting up kelp and mussel rafts near ocean salmon cages. The upside is that diverse crops find diverse markets; hands–on experience with complexity yields surprise solutions; apparent chaos emerges as self- correcting. While the Phase II goal is to gain confidence in IMTA-RAS in order to justify a substantial investment in a 20-fold expansion, it also is an opportunity to explore the limits of co-cropping in an aquatic system. The Phase II Final Report will consider limits to diversity with further recommendations thereto, probably the second most important deliverable after large scale IMTA facility design.

Predictive Modeling: While system health and stability combined with additional by-product revenues are sound goals, they come at a cost. Managing diverse and multiple crops could become problematic to system management if not taken into full consideration at the outset. Real-time knowledge of water quality is vital to prevent and respond to emergencies. Other means of remote observation will yield data on energy and nutrient inputs that will help predict crop harvest dates and the ordering of new stock. With the sophisticated data acquisition proposed for Phase II, a useful model should result, ideally yielding an instruction manual for practical IMTA aquafarm management.

3.2 Current Staffing Profile: Technical: The pilot plant will be managed by three marine biologists on AHI’s staff and located at CCAR. Two are part time for IMTA-project purposes: UMaine PhD postgraduate Dr. Kevin Neves, and UMaine PhD candidate Mr. Patrick Erbland and one full time trainee. The pilot plant requires 24-hour surveillance and these three persons will be supported by CCAR staff. CCAR will also be responsible for spawning BSB and SW. CCAR also maintains ~100 yellowtail broodstock, and has the option of obtaining eggs from hatcheries in Chile. CCAR staff has long experience in spawning sandworms and have provided nectochaetes (worm larvae) to the project whenever required since October 2011.

3.3 Managerial and Technical: The founders of AHI, Chris Heinig and PI Tap Pryor are each working 30 to 50 hours a week respectively for the company and have had extensive experience with the operation of oyster hatcheries, on-land oyster and algae aquafarms, as well as farming steelhead trout, tilapia and milkfish commercially. Chairman and Chief Business Officer Ed Robinson, a seasoned executive who retired early from a successful career in life sciences, also spends an average of 40 hours per week on sales, marketing, legal and financing activities. Accounting is the responsibility of Margurite Kelly. In addition, AHI has one consultant for marketing, Tony Barrett. All are key personnel responsible for Phase II are identified above, and expected to participate for the term of the award.

3.4 Future Staffing Plans: As Phase II winds down, the team will be planning for Phase III aquafarm expansion at Corea. Due to the demands of the latter, a general manager will be recruited as well as five semi-technical laborers from Gouldsboro, a township of four coastal villages where fisheries is the main source of employment. The site at Corea was a former Naval Station and this part of Hancock County is an economically depressed area with chronically high unemployment where the 2010 census counted 1,533 residents, a loss of 234 from 2000. In 2000, 10.4% of the population was below the poverty line with a per capita income of $18,203. Anecdotal opinion is that income levels are even lower today due to the ongoing decline of the fishing industry. Town officials have welcomed AHI’s proposal to operate in Corea.

Part 4: Consultant and Subaward Agreements. 4.1 Consultants Bacteriologist: AHI is in the process of recruiting a bacteriology consultant. In two years of fish and sandworm trials it has become clear that bacteria in the sandbeds are a beneficial system component, especially in converting ammonia to nitrite and nitrite to nitrate. In addition to providing biofiltration services, this 5th trophic level within IMTA probably serves in part as an oyster feed. It may also be desirable to add probiotics to the system. With the assistance of a bacteriologist, investigators will be able to identify the bacterial species, their location, the several successions of their populations over time, and perhaps comprehend and maximize their role. Costs of consulting time and laboratory analyses have been included in the budget.

Other Consultants: Three other consultants will be hired for their expertise: (1) Ms. Lori Howell (Attorney) re preparing AHI for meetings with state and/or federal regulators, to assure that sale of IMTA-grown oysters is permissible; (2) Dr. Joseph Van Ryzin (Ocean Engineer) to assist staff in the development of comprehensive and economic modeling software; and (3) Ms. Sarah Redmond (Phycologist) will plan for and manage the single raceway devoted to macroalgae. (Brief CV’s are included in Biographical Sketches).

4.2 Subawards: A subaward to CCAR in Franklin Maine is budgeted entirely for staff support, including the Director, Stephen Eddy, who is designated as Co-Principal Investigator. CCAR has one of the most extensive facilities available to aquaculture businesses anywhere. The facilities are available to test out processes and new products for aquaculture. Unlike other business parks, aquaculture business incubation clients need supplies of filtered fresh water and sea water, discharge capacity, water treatment, storage and distribution systems and various types of buildings designed specifically for aquaculture systems equipped with complex climate control system and mechanical filtration. CCAR has an experienced staff with knowledge of rearing technologies, holding systems, grow-out and hatchery operations. In addition to its Phase I/IB and proposed Phase II activities, CCAR has a shared interest in the outcome of IMTA RAS investigations. Therefore, it is providing the larger room (100’ x 75’ x 10.5’) for 24 months at no charge for rent, power or heat. Currently, AHI is launching a 5,000 fish pilot plant at CCAR for its YT development program.

A second subaward will be issued to the University of Maine’s Department of Food Science and Nutrition for the services of Dr. Mary Ellen Camire who will plan, organize, implement and report on sensory evaluation tests of the several forms of produce cultured under IMTA conditions, including YT, BSB and OY.

Part 5: Equivalent or Overlapping Proposals to Other Federal Agency. (Not applicable).

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