Final report for GS19-204
The limited supply and high price of fishmeal are emerging as big challenges to the sustainable growth of the U.S. aquaculture farmers. Thus, there is a need to seek alternative feed sources for aquaculture production. The long-term goal of this research is to produce a sustainable and cost-effective alternative to fishmeal in shrimp feeds to improve the growth of the aquaculture industry. This project was accomplished through a research plan with three objectives: 1. develop an economically feasible fractionation process to produce high-protein feed (PP) from a brewery waste, i.e., brewer’s spent grain (BSG), 2. demonstrate the effectiveness of PP to replace fishmeal via shrimp feeding trials and shrimp quality evaluations, and 3) conduct techno-economic analysis to evaluate the economic feasibility of the conversion of brewer's spent grain to PP. BSG was first subjected to a wet fractionation process to produce PP using different chemical/biological treatments. Under the optimized conditions, the produced PP contained 46% protein, which is an ~100% increase in protein concentration compared with the original BSG. The effectiveness of using high-protein feed as a replacement for fishmeal was then evaluated by shrimp feeding trials. The shrimp diets were prepared by using PP to replace fishmeal at increasing levels (10–70%). The results showed that up to 50% of fishmeal in shrimp feed can be replaced by PP without affecting shrimp growth and feed utilization. The economic analysis showed that the production cost of high-protein feed was $1,044 per metric ton, lower than the market price of fishmeal. Overall, this research benefit aquaculture farmers by providing a low-cost protein alternative to fishmeal and support the brewery industry by providing an alternative way of managing and using brewer’s spent grain. The research findings have been widely disseminated through mainstream public media and have drawn much attention from the agricultural professionals and the brewery industry.
- Develop an economically feasible fractionation process to produce high-protein feed from brewer’s spent grain.
- Demonstrate the effectiveness of high-protein feed to replace fishmeal via shrimp feeding trials.
- Conduct the techno-economic analysis to evaluate the production cost of the high-protein feed from brewer's spent grain
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1. Extraction of protein-rich product (PP) from brewer's spent grain
The raw BSG was obtained from Parkway Brewing Company (Salem, VA, USA). Raw BSG contained 79% moisture (w/w, wet basis), 45% fiber, 22% protein, and 8% fat (w/w, dry basis) was milled directly to reduce particle size. The PP production from milled BSG followed a modified method adopted from our previous study (He et al., 2019). Briefly, the raw BSG was wet milled and then enzyme hydrolyzed by Alcalase 2.4L FG (Novozymes Inc. Franklinton, NC, USA) with a loading of 5 µL Alcalase/g dry BSG, solid content 9% w/w, pH 8.0, and under temperature 60 ℃ for 1 hour. The hydrolysate rich in protein was then separated with a cold press (X-1, Goodnature Products, Inc. NY, USA) followed by a sieve shaker (RX-29, W.S.tyler, OH, USA). The material that passed through the sieve was concentrated using an evaporator and dried to less than 10% of moisture content. This dried product, the high protein-rich product, was named PP and subjected to the proximate composition analysis.
2. Fish diet formulation and preparation
The control diet (PP0) containing 35% (w/w) of fishmeal was formulated based on previous studies (Bulbul, Koshio, Ishikawa, Yokoyama, & Kader, 2015; Hulefeld et al., 2018; Liu et al., 2012) The other four test diets were formulated by replacing fishmeal with PP at increasing levels of 10%, 30%, 50%, and 70%, and designated as PP10, PP30, PP50, and PP70, respectively. All ingredients were mixed thoroughly in a Kitchen AidTM (Pro 600, Whirlpool Corp., Benton Harbor, MI, USA) for 30 min. Distilled water was then added slowly with mixing for an additional 20 min. The final mixture was extruded using a grinder attached to the Kitchen AidTM stand mixer. Extruded feeds were subsequently cut into pellets of approximately 10 mm long and 2 mm diameter, and were air-dried in 5 mm layers for around 48 hours to less than 10% moisture content at room temperature. The feeds were stored at -20 ℃ until the shrimp feeding trial was completed (within three months). The proximate compositions of diets were analyzed in duplicate and the results were published in our manuscript (He et al., 2020). Gross energy was estimated based on the feed composition according to previous studies (Bulbul et al., 2015; Kuhn, Lawrence, Crockett, & Taylor, 2016).
3. Experimental shrimp and feeding trial
Post-larvae 12 to 15-day old (PL-10 to PL-15) Pacific white shrimp were obtained from Miami Aqua-Culture, Inc. (Boynton Beach, FL, USA). Shrimp were initially cultured in a 480 L recirculated seawater system containing 260 L of 25 parts per thousand (ppt) synthetic sea salt (Crystal Sea Marine Mix, Marine Enterprises International, Baltimore, MD, USA), equipped with mechanical and biological filtration. The shrimp were fed a starter commercial diet followed by a larger feed (2.4 mm diameter, with a minimum crude protein content of 35% and maximum crude fiber of 4%) twice per day until they grew close to 1 g.
The shrimp feeding trial was carried out in six aquatic habitat systems (A-habb, Pentair, Minneapolis, MN), each equipped with five 10-L tanks. Two hundred forty shrimp were randomly selected from the 480 L tank and distributed evenly to the new systems with 30 tanks. Eight shrimp were allocated to each tank which provided one extra shrimp per tank in case of mortalities occurring due to transportation or acclimation stress. Shrimp were acclimated to the new system for 3 days and fed the same commercial feed mentioned above. The commercial diet was then replaced with the control diet at a gradually increasing ratio of 20, 40, 60, 80, and 100% (w/w) in the following 5 days. During this eight-day acclimation period, shrimp were fed twice daily for a total feed rate of 8% (w/w).
After the acclimation period, shrimp numbers were adjusted to seven in each tank to start the feeding trial within a density of 107 shrimp/m2. The initial weight of each shrimp for the feeding trial was 1.10 ± 0.06 g (mean ± standard deviation). Each dietary treatment consisted of six replicates (six tanks) in the six aquatic habitat systems. During the feeding trial, shrimp were fed manually three times a day at 08:30, 15:30 and 22:00. Shrimp were weighted on a tank basis every early Thursday morning to track growth and feed efficiency. The weight gain data was used to estimate the daily weight gain for the following week. The feeding rate was adjusted weekly based on shrimp weight and remained consistent across all treatment groups on the same day within the range of 5.5-8.5% (g dry feed per g wet body) based on the cumulative tank shrimp weight. Mortality was checked several times a day. Any moribund or dead shrimp were removed from the tanks, and the feed weight was immediately adjusted based on the weight of shrimp remaining in the tank.
The water quality was monitored daily for dissolved oxygen and temperature, using the ProDO meter (Yellow Springs Instruments, Yellow Springs, OH, USA). The salinity of the systems was measured daily with a refractometer (WL0020-ATC, Agriculture Solutions LLC, ME, USA). Total ammonia-N, alkalinity as calcium carbonate, nitrite-N, nitrate-N, and pH were monitored three times per week (APHA, 2012). Sea salt and sodium bicarbonate were used to adjust salinity and alkalinity, respectively, during the feeding trial. During the shrimp feeding trial, the values of the water quality parameters were within desired ranges for Pacific white shrimp (Kuhn et al., 2010; Schuler, Boardman, Kuhn, & Flick, 2010; Van Wyk et al., 1999) (mean ± standard error): temperature (29.6 ± 0.0℃), dissolved oxygen (6.2 ± 0.0 mg/L), salinity (25.2 ± 0.0 ppt), alkalinity (107.8 ± 1.3 mg CaCO3/L), pH (7.9 ± 0.0), total ammonia-N (0.3± 0.0 mg/L), nitrite-N (0.1 ± 0.0 mg/L), nitrate-N (13.3 ± 0.5 mg/L).
4. Sample collection and biochemical analysis
At the end of the feeding trial, all shrimp were fasted for 24 h after the last feeding. Final survival rates and the total weight of shrimp were recorded at the tank level. Shrimp from each tank were subsequently euthanized in an ice bath (< 4 ℃), then vacuum-packaged separately and stored at -80 ℃. The frozen shrimp were freeze dried and milled into powder. These 30 powder samples that respectively obtained from the surviving shrimp in 30 tanks were then submitted to composition analysis. Hence, six replicates of growth performance and composition analysis data were received for each diet treatment.
Proximate composition analysis of the diets and shrimp whole body was determined following the standard AOAC methods (AOAC, 2016). The moisture content was determined gravimetrically by oven drying at 105 ℃ to constant weight. The freeze-dried shrimp were also dried in an oven to determine moisture due to the incomplete moisture evaporation during freeze drying. Ash was determined by weight difference before and after the incineration of samples in a muffle furnace at 550 ℃ for 12 h. Crude protein was determined by following the Kjeldahl procedure, multiplying the total nitrogen by 6.25. The Foss TecatorTM Digestor and KjeltecTM 8100 Distillation Unit (FOSS North America, Eden Prairie, MN, USA) were used to determine the total nitrogen content. Crude fat was determined by Randall/Soxtec/Hexanes Extraction-Submersion Method using petroleum ether as an extraction solvent, and FOSS 2055 Soxtec Avanti Manual System (FOSS North America, Eden Prairie, MN, USA) was used. Neutral detergent fiber (NDF) contents were quantified by using the ANKOM Filter Bag System (ANKOM 2000 automated fiber analyzer, ANKOM Technology, Macedon, NY, USA). High-performance liquid chromatography (HPLC, Agilent Technologies, CA, USA) combined with modified performic acid oxidation was used to analyze amino acids. Mineral determination using a modified method in a previous study using microwave-assisted acid digestion (Farzad et al., 2019). Briefly, 0.5 g dry diet or shrimp body sample was transferred to a polytetrafluoroethylene tube. Four milliliters of nitric acid (69% w/v), four milliliters of distilled water, and two milliliters of hydrogen peroxide (30% w/v) were added. The samples were then heated to a temperature of 180 ℃ and held at this temperature for 15 min using a microwave power of 800 W. After digestion, samples were diluted and sent to inductively coupled plasma mass spectrometry for mineral analysis (Agilent 7900 ICP-MS, Santa Clara, CA, USA). For the fatty acid determination, the lipids in diets were extracted using chloroform/methanol (2:1, v/v) following the method of Folch, Lees, & Stanley (1957). The extracted lipids were transformed to fatty acid methyl esters (AOCS method Ce 1b-89), which were analyzed using gas chromatograph-mass spectrometer (QP2010, Shimadzu, Kyoto, Japan), equipped with column ZB_WAXplus (60m x 0.25mm, thickness 0.25 µm), an FID detector, and an auto injector AOC-20i. The injection mode was split. The carrier gas was helium. The temperature program included a gradient from 175 up to 225 ℃ with an increased rate of 2.0 ℃ min-1.
5. Shrimp performance indicators
The shrimp growth performance was analyzed using percent weight gain, specific growth rate (SGR), feed conversion rate (FCR), shrimp survival percentage, and protein efficiency ratio.
6. Process simulation and techno-economic analysis (TEA) to quantify the protein production cost from brewer's spent grain
The TEA model for PP production from BSG included a process flow diagram, rigorous process modeling based on the mass and energy balance, and economics of materials, utilities and equipment using SuperPro Designer 11.0 (Intelligen, Inc., NJ, USA). Given the USA has a high number of large breweries to provide a centralized BSG resource, and the BSG itself is highly perishable and costly to ship, it is assumed that the BSG processing plant is co-located at a large brewery. The co-location strategy has certain advantages, including avoiding the shipping cost of BSG and preventing BSG from spoiling. The wet fractionation process was designed to handle 590 t of wet BSG per day, which is the average BSG generation rate by large breweries in the USA (TTB, 2019). The process runs 330 days (7,920 hours) per year with three shifts per day; the remained 35 days are scheduled for equipment maintenance and cleaning (Davis et al., 2011). The cost of maintenance is included in the category of fixed operating costs. PP was assigned as the main product, and fiber residue (FP) after protein separation was a coproduct to be sold for credits. The detailed process modeling and TEA were published in He et al. (2021).
1. Nutritional Composition of the high-protein feed
Under the optimized processing condition, the high-protein feed (PP) had 45.8% crude protein, 9.5% crude fat, 0.8% neutral detergent fiber, and 6.7% ash. The total carbohydrates in PP were calculated by the weight difference between the total dry matter and the sum of crude protein, total fat, and ash according to the reference (Merrill & Watt, 1973). The calculated total carbohydrates in PP was 38.0%. It is important to mention that the protein concentration in PP is doubled compared to the protein concentration in the original brewer's spent grain, indicating that the developed fractionation process was not only able to separate protein from the brewer's spent grain but also able to concentrate protein in PP.
2. Shrimp feeding trial and its growth performance
The final weight, weight gain, specific growth rate (SGR), feed conversion ratio (FCR), survival, and protein efficiency ratio (PER) of shrimp fed diets with different levels of fishmeal protein replaced with PP protein after the eight-week feeding trial. There were no significant differences in survival (76.2 – 94.3%), PER (1.2 - 1.3) or FCR (1.8 - 1.9) among dietary treatments (PP0, PP10, PP30, PP50 and PP70). However, when 70% of fishmeal protein was replaced by PP protein in the shrimp diet (PP70), the SGR of shrimp were significantly lower than that in the control group (p < 0.05). To better monitor the effect of increased PP in shrimp diet on feed utilization, the FCR changes within and between dietary groups in each week were compared. In the initial 2 weeks, the FCR increased and fluctuated among the test dietary groups comparing with the control group. There were no significant differences in FCR among dietary groups in weeks 3 to 8, except for week 7.
As stated in the previous paragraph, the shrimp survival and feed utilization indicators, including FCR, survival, and PER, were not affected when as much as 70% of fishmeal was replaced with PP in shrimp diets. One reason might be the low fiber content and the high protein content of PP. Because fiber is not digestible by fish or shrimp, adding high-fiber ingredients to aquaculture diets increases fecal losses, thus negatively impacting feed utilization efficiency (Naylor et al., 2009). Unlike raw BSG which has 50–70% of fiber, the upgraded PP (from BSG) has less than 1% fiber (NDF) on a dry basis; therefore, the negative effect of the fiber in PP on feed digestibility is minimal. The high level of fishmeal replacement with PP could also be attributed to the balanced amino acid profiles obtained by supplementing lysine and methionine as well as the low antinutritional compounds in PP. The lysine in PP protein (4.04%, w/w) was 45% lower (relative basis) than in fishmeal protein (7.29%, w/w); however, this deficit was eliminated by mixing the soybean meal and krill meal in diets, which adjusted the final lysine content in the diets to a range of 2.4–3.0% (w/w). Direct addition of synthetic methionine (0.1–0.2%, w/w) into diets offset lower levels of methionine in PP. Moreover, unlike some plant-based proteins such as canola meal and soymeal, which contain certain levels of antinutrients (Gatlin et al., 2007), the PP from BSG (originally from barley) contains insignificant levels of antinutrients to fish and shrimp. Moreover, the small molecular size of PP protein could also be a contributor to the high-level replacement of fishmeal.
Although the FCR, PER, and survival were not affected, we observed the differences in shrimp growth performance among different groups. The shrimp fed PP50 did not show significantly different SGR compared to the control group (PP0); however, the shrimp fed PP70 showed a significantly lower SGR compared to the control group. The lower SGR was likely due to the delayed adaptation to PP diets at the beginning of the trial, especially during the first week. The high levels of plant protein could cause a change in the texture of diets (Alceste, 2000). The peptides and free amino acids in PP may also affect palatability (Song et al., 2014). Thus, the shrimp had to adjust to the new diets with different smells and tastes, and change their metabolism functions to more efficiently digest the new diet (Aksnes et al., 2006). Before starting the test diets, shrimp were fed increasing levels of the control diet (PP0) to replace the commercial diet. Therefore, shrimp that received the control diet (PP0) experienced a likely advantageous acclimation prior to the trial initiation and hence grew faster than shrimps in the other four dietary groups during week one. In this study, the amount of feed assigned to each group was calculated based on the previous week’s shrimp weight in that group, thus the treatment group that grew faster in week one received a higher amount of feed than the other groups. This exaggerated the weight gain difference among different treatments. Therefore, although FCR was similar among groups, the SGR of the shrimp fed PP70 was lower. Besides, the shrimp fed PP70 also had the lowest final weight among all groups. In the future, it might be better to feed all shrimp groups with the same commercial feed prior to trial initiation, in order to eliminate the growth difference caused by this acclimation issue. Penaeid shrimp have limited ability to synthesize highly unsaturated fatty acids such as eicosapentaenoic (20:5n-3, EPA) and docosahexaenoic (22:6n-3, DHA). The decreased weight gains in shrimp fed PP70 might be also due to the imbalanced fatty acids and lower content of EPA and DHA in the diet compared with PP0. It was reported that dietary fatty acids affected the growth and immune system of white shrimp (Zhang et al., 2014).
In a summary, our results showed that 70% replacement of fishmeal protein with PP protein was not appropriate in shrimp feed, as well as 50% of replacement of fishmeal protein with PP protein was able to maintain the growth (SGR), feed utilization efficiency (FCR and PER), and survival of shrimp.
3. Shrimp whole-body proximate composition
There was no significant difference in the dry matter content of the shrimp whole body (23.85 ± 0.16%) among dietary groups. The protein contents of the shrimp whole body increased with incremental additions of PP in diets. The shrimp in diet group PP70 had a higher crude protein content compared with the shrimp fed control diet (PP0), but the difference is < 2%. PP was produced by the wet fractionation process, where BSG was subjected to the enzymatic hydrolysis to break the peptide bonds of proteins (He et al., 2019; Niemi et al., 2013; Yu et al., 2019). It was reported that the ingestion of protein hydrolysates with small molecular size led to the increase of skeletal muscle protein synthesis (Koopman et al., 2009). On the other hand, the fat content of shrimp decreased successively with the incremental addition of PP in diets, the crude fat content of the shrimp body decreased from 6.42% to 5.46%. This result is in agreement with previous studies on fishmeal replacement with plant protein (Kissil et al., 2000; Robaina et al., 1998). Robaina et al. (1998) found that the total lipid content in sea bream decreased when 30% of soy protein was incorporated in diets. Kissil et al. (2000) also reported that a decrease in fat content in whole-body fish when fishmeal in diet was replaced by soy and rapeseed protein concentrates in an 8-week feeding trial. Our results together with previous studies indicate that the inclusion of some types of plant proteins in diets could affect lipid digestion and accumulation in fish/shrimp. This could be due to several factors, such as the presence of embedded compounds (e.g. fibers) that reduce lipid digestion and the physicochemical properties of lipids themselves in plant protein ingredients. Some lipids are easily digested by the lipases in shrimp digestive systems, while some are not (Gunasekera et al., 2002).
Regarding the mineral contents, high levels of PP in diets resulted in sodium accumulation in shrimp bodies. The shrimp fed PP50 and PP70 diets had significantly higher contents of sodium, magnesium, zinc, copper, and manganese compared with the shrimp fed the control diet. However, no difference in the contents of calcium and iron was observed among the dietary groups. Based on previous studies (Ikram et al., 2017; Mussatto et al, 2006), phosphorus, calcium, and magnesium are the most abundant minerals in raw BSG, whereas, in PP, sodium was the predominant mineral, probably due to the addition of sodium hydroxide for adjusting pH to 8.0 for enzymatic hydrolysis during the PP production from BSG. The sodium in PP was almost double that of fishmeal. Thus, the sodium content in the shrimp body increased in response to the increasing level of PP in diets. However, Litopenaeus vannamei are euryhaline, tolerating salinities ranging from 1 g/L to over 40 g/L (Bray et al., 1994; Van Wyk et al., 1999). Shrimp cultured in lower salinity water will benefit physiologically from receiving higher dietary sodium. There were no significant differences in the threonine, isoleucine or methionine levels between the shrimp fed the PP diets and the shrimp fed the control diet. Furthermore, the amounts of these three essential amino acids in the shrimp fed PP50 were slightly lower than those in the shrimp fed other diets. However, we did not observe any difference in the remaining essential amino acids or non-essential amino acids between any of the dietary groups.
4. Techno-economic analysis of the production of high-protein feed
Based on the annual operating costs, the minimum selling price of PP (MSPP) was calculated at 1,043.5 USD/t. The MSPP of the designed process includes a variable cost (raw materials, utility cost, and co-product credit), fixed cost (labor and overheads), capital depreciation, average income tax, and an average return on investment. Among these costs, raw material cost contributed most to the MSPP; within the materials cost, the enzyme and raw BSG costs accounted for the greatest portion. Especially, the enzyme and raw BSG costs contributed to 492.4 USD/t and 362.9 USD/t of the MSPP, respectively. Although a low enzyme dosage (5 µL/g BSG) was used based on the lab experiments, the unit price of the enzyme (food-grade Alcalase, 34 USD/kg, a bulk price quoted from Novozymes Inc.) still makes it the highest cost for PP production. This high contribution of the enzyme to MSPP highlights the importance of exploring a low-cost but effective enzyme. On-site crude enzyme production using fungi could also be a possible option as several studies have reported that on-site enzyme production could greatly reduce the enzyme cost in the biofuel industry (Olofsson et al., 2017). After the cost of the enzyme, the cost of wet BSG was the second-largest contributor to the overall operating cost. In this study, a cost of 31.4 USD/t wet BSG was used based on a market survey (Buffingt, 2014). To be noted, in reality, the cost could be zero as some breweries are willing to give away their BSG as a means to get rid of the waste materials (McHugh et al., 2020). Thus, the PP production cost could potentially be reduced if a low-cost BSG source can be secured. Besides the cost of the raw materials, utility costs, including the costs of electricity, natural gas, and steam, accounted for 224.9 USD/t (22%) of the MSPP. Among the utility costs, the steam cost constitutes the highest portion (64%), which was attributed to the need to remove a large amount of water from PP slurry during evaporation and spray drying to produce dried PP, and to the fact that the heating source for both evaporation and drying is steam. The fiber product (FP) annual production was 29,825 t, and the selling price of FP was assumed at 120 USD/t, which was based on its nutritional composition identical to soybean hulls. Income from the sale of FP was claimed as a coproduct credit and contributed to around a 20% reduction in PP production cost.
Educational & Outreach Activities
1.1 Participate in the development of an online multi-institution course named Emerging Technologies in Food Engineering and presented the project to over 100 students and professors working in the agricultural areas (Spring 2021).
The outcome of this project has affected agricultural sustainability from three aspects.
- Economic. Through the development of the novel fractionation process, we are able to produce high protein powders from the waste material, brewer's spent grain. Based on the comprehensive techno-economic analysis, the production cost of the high protein powders is about $1,000 per metric ton, which is much lower than the currently popular fishmeal protein at $2,000 per metric ton. Thus, the high-protein powders can potentially provide a cheaper protein source for Aquaculture farmers.
- Environmental. Although most brewer's spent grain is used as low-value cattle feed, some of them are sent to landfills. Brewer's spent grain spoils quickly and has high nutrient (such as nitrogen [N] and phosphorus [P]) content, which, if not handled properly, presents the potential of polluting and damaging our environment. Conversion of brewer's spent grain into high-value protein powders greatly mitigates the environmental burden created by the disposal of brewer's spent grain.
- Social. The rising demand for seafood, together with the overexploitation of marine resources, has made aquaculture the fastest-growing segment in global food production. The rapid expansion of aquaculture has led to a corresponding increase in demand for fishmeal protein, the primary ingredient in aquaculture feeds. This has led to an increase in fishmeal prices from $400 to $2000/metric ton in the last 15 years. The outcome of this research will provide better protein accessibility to aquaculture farmers in the U.S., and thus improving the economic benefits of aquaculture farmers.
- Gained knowledge in the separation, characterization, and application of plant-based proteins from agricultural processing byproducts.
- Gained knowledge in the evaluation of plant-based protein as an alternative protein source to feed shrimp.
- Gained skills in animal feeding trials to test the efficacy of new feed formulations.
- Increased awareness of the importance of sustainable agriculture through the circular economy approach.
It is a great program to support the next generation of agricultural scientists. With the support, the student successfully conducted the Ph.D. research and obtained her doctoral degree.