Browsing by Author "Kuhn, David D."

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    Developing Serum-Free Media Via Bioprocessing For Cultivated Seafood Products
    Batish, Inayat (Virginia Tech, 2022-09-08)
    Global food production management has become a challenge with an anticipated population of 10 billion people by 2050 and the ongoing COVID-19 epidemic. Seafood is a vital food source due to its widespread consumption, excellent nutrient profile, and low feed conversion ratio, rendering its sustainable production quintessential. Cellular agriculture or cultured meat can increase seafood production; however, the conventional use of Fetal Bovine Serum (FBS) in culture media restricts its utilization at an industrial level. FBS is effective but has many limitations: unethical animal extraction, high demand and low supply, poorly defined ingredients, variable performance, and high cost that impedes the feasibility and commercial viability of cellular agriculture. Thus, employing serum-free media becomes a quintessential need for cellular agriculture. This project aims to replace or reduce the typical 10% serum usage in Zebrafish embryonic stem cell (ESC) production media with protein hydrolysates derived from low-cost natural sources with high protein content. Enzymatic hydrolysis was performed on nine sources: insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine invertebrates (oyster, mussel, and lugworm). The resulting hydrolysates were evaluated for serum replacement in zebrafish ESCs. All hydrolysates were used at five different concentrations (10, 1 0.1, 0.01 and 0.001 mg/mL) in serum concentrations of 10%, 5%, and 0% with four biological replicates. The best hydrolysate sources and concentrations were selected for further testing at 2.5% and 1% serum concentrations. All hydrolysates, except for cricket, could restore or significantly increase cell growth with 50% less serum at a concentration of 0.1-0.001mg/mL. Protein hydrolysate concentration of 10 and 1mg/mL was toxic for cells. Additionally, the eight hydrolysates could reduce serum concentrations up to 75–90%. However, no protein hydrolysate could completely replace serum, as cells using only protein hydrolysates exhibited morphological aberrations and decreased growth. Replacing serum with protein hydrolysates lowers cellular agriculture's overall cost, thus enabling the commercialization of cultured meat and the development of a sustainable food system. In the future, blending various protein hydrolysate sources with or without the addition of conventional growth factors could be done to create the ideal serum-free media.
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    Development of Omega-3-Fatty Acid Enriched Finishing Feed and Value Added Tilapia Product
    Stoneham, Tyler R. (Virginia Tech, 2016-06-29)
    Despite being a low fat fish and consequently a low omega-3 fish, tilapia have widespread consumer acceptability due to its mild taste, cheap price and low mercury content. However some sources claim that farmed tilapia can be detrimental to human health due to high omega-6:3 ratios and low omega-3 content specifically eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. The objective of this study was to create an omega-3 enriching feed that would increase omega-3 content in tilapia and subsequently decrease the omega-6:3 ratio. An 8 week feeding trial was conducted. Tilapia were cultured in a recirculating aquaculture system on one of eight diets (control, commercial, 1, 3, 5% fish oil or 1.75, 5.26, 8.77% ALL-G-Rich (algae). Water quality, selected fish biometrics and growth performance were recorded. Fillet and rib meat tissues were collected at weeks 4 and 8, and liver and mesenteric fat tissues were collected at week 8. Fat was extracted, trans-methylated and identified as fatty acid methyl esters using gas chromatography-mass spectrometry. Docosahexaenoic acid increased in concentration in all tissues as percent fish oil and ALL-G-Rich increased in the diets with 8.77% ALL-G-Rich resulting in significantly (P<0.0001) greater concentrations in the fillet and mesenteric fat compared to all other diets after 8 weeks. The 8.77% ALL-G-Rich diet resulted in significantly (P=0.003) greater cumulative accumulation of EPA, DPA and DHA on a mg/4oz fillet basis after 4 weeks compared to control. The results of this study suggest that an ALL-G-Rich finishing feed could be produced that would result in a value added farmed tilapia fillet.
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    Effects of water chemistry and panning on flavor volatiles and catechins in teas (Camellia sinensis)
    Sheibani, Ershad (Virginia Tech, 2014-12-03)
    In the first experiment, effects of brewing time, chlorine, chloramine, iron, copper, pH and water hardness were investigated for their effects on extraction of epigallocatechine gallate (EGCG) and caffeine in green tea and oolong tea aqueous infusions. The extraction of EGCG and caffeine were lower when green tea was brewed in hard water compared to distilled water. Brewing green tea and Oolong tea in tap water resulted in higher extraction of caffeine but had no effect on EGCG compared to distilled water. The extraction of EGCG and caffeine were significantly increased (P<0.05) when green tea and Oolong tea were brewed in the chlorinated water at 4.0 mg free chlorine per liter. The purpose of the second experiment was to optimize SDE conditions (solvent and time) and to compare SDE with SPME for the isolation of flavor compounds in Jin Xuan oolong tea using Gas Chromatography- Mass Spectrometry (GC-MS) and Gas Chromatography- Olfactrometry (GC-O). The concentration of volatile compounds isolated with diethyl ether was higher (P<0.05) than for dichloromethane and concentration was higher at 40 min (P<0.05) than 20 or 60 minutes. For SDE, 128 volatiles were identified using GC-MS and 45 aroma active compounds using GC-O. The number of volatiles identified using GC-MS was lower in SPME than SDE. For SPME, 59 volatiles and 41 aroma active compounds were identified. The composition of the volatiles isolated by the two methods differed considerably but provided complementary information. The goal of the third experiment was to determine effects of panning on flavor volatile compositions of oolong using GC-MS and GC-O. Simultaneous Distillation and Extraction (SDE) and Solid Phase Microextraction (SPME) techniques were applied for extraction of volatiles in panned and unpanned teas. A total of 190 volatiles were identified from SDE and SPME extractions using GC-MS and GC-O. Trans-nerolidol, 2- hexenal, benzaldehyde, indole, gernaiol, and benzenacetaldehyde contents were significantly decreased (P<0.05) by panning; however, panning increased (P<0.05) contents of linalool oxide, cis jasmone, methyl salicylate in oolong tea. Overall, panning significantly changes the volatile compositions of the tea and created new aroma active compounds.
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    Enhancing Seafood Quality and Safety by Reducing Reliance on Antibiotics: Applying a Novel Antibody in Tilapia
    Garry, Jordan Nicole (Virginia Tech, 2018-06-14)
    Disease outbreaks have overwhelmed the aquaculture industry as a whole and have been catastrophic for many single operations. To minimize disease outbreaks, efforts are underway to enhance animal health and disease resistance to pathogens without the use of antibiotics. The overall purpose of this study was to explore a potential prophylactic, a novel antibody diet, for the bacterial pathogen, Aeromonas hydrophila, in tilapia. The tilapia were on an anti-interleukin-10 antibody diet as a neutralization of interleukin-10 (IL-10), an anti-inflammatory cytokine. The anti-inflammatory function of IL-10 has been shown to allow persistence of gastrointestinal pathogens. Tilapia were fed the novel diet and were challenged via bath immersion or oral gavage with A. hydrophila. Four trials of challenge studies were conducted. Clinical signs of the disease and survival were monitored post-challenge of the bacteria. Out of the 4 trials, one bath immersion trial showed significantly lower survival in the group fed the novel antibody diet (p=0.044) compared to the control fed group, after challenged with A. hydrophila. The other trials tested showed no significant differences in survival between diets. Among the survival percentages collected as a whole, it cannot be determined from in vivo results whether this anti-IL-10 diet is effective in preventing mortality from A. hydrophila in tilapia. Therefore, an in vitro study using an enzyme-linked immunosorbent assay (ELISA) was used to determine the neutralization capability of anti-IL-10 on IL-10 using tilapia splenocytes. Interferon-γ, a pro-inflammatory cytokine, was quantified in order to find a trend in expression of IL-10 in vitro in various tilapia cell treatments. The protocol for the ELISA study is under development being that the use of this antibody is novel and has never before been done in fish.
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    Establishment and Utilization of Tools for Enhancing Foodfish Health
    Galagarza, Oscar Andres (Virginia Tech, 2018-01-29)
    Aquacultured products assist the human demands for seafood so that foodfish supplies can remain sustainable and consistent. Although the fish-farming industry has seen dramatic growth and intensification in recent years, the latter has led to an increase in bacterial diseases and fish health management problems, resulting in major economic losses around the world. In addition to the lack of understanding of fish physiology, these complications are exacerbated by the inappropriate and controversial use of antibiotics. This work addressed these issues in striped catfish (Pangasius hypophthalmus) and Nile tilapia (Oreochromis niloticus), two economically important foodfish, by investigating alternative, more cost-effective options to promote fish health. The first two studies established reference intervals for immunology, hematology and plasma chemistry analytes in striped catfish in a recirculating aquaculture system (RAS). In a third study, the immunomodulatory effects after directly feeding probiotic strains of Bacillus subtilis NZ86 and O14VRQ in Nile tilapia were ascertained. This last study revealed that supplementation with both of the probiotic strains for 51 days stimulated several local and systemic innate immune responses of tilapia. When these transient probiotic bacteria were present in the gut, a pro-inflammatory environment was developed as evidenced by the localized higher expression of the cytokines tumor necrosis factor (TNF) – α and interleukin (IL) – 1β. Significant increases (p < 0.05) were noted differentially by both probiotic strains throughout the trial in plasma lysozyme content, alternative complement activity, and in the peripheral blood leukocyte profiles. Additionally, there were trends for increased levels of phagocytosis and respiratory burst in leukocytes of the anterior kidney and spleen at the end of the trial, suggesting the potential use of these probiotic strains for improved immune-competence. These findings help to understand and clarify the potential mechanism of action associated with the increased disease resistance recorded in preliminary studies with the same probiotic strains. Implementation of the tools established and validated in this work could be useful in evaluating fish welfare situations involving striped catfish grown in RAS conditions, and also show promise for a healthier foodfish supply where antibiotic applications practices could be minimized.
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    Identification of Inhibitory Compounds in Medicinal Mushrooms against L. monocytogenes and Z. bailii
    Chu, Hyun Sik Stephano (Virginia Tech, 2014-01-06)
    Extracts from medicinal mushrooms were prepared and tested for anti-microbial activity against food pathogens and food spoilage microorganisms. The inhibitory activity was measured using a disk diffusion assay and with optical density (OD). For OD, 7 fractions were collected using HPLC for 4 (A. blazei Murrill, G. lucidum, G. frondosa, I. obiquus) medicinal mushrooms and 6 fractions from L. edodes and 8 fractions from P. linteus. The results from disk diffusion assay showed that most mushrooms displayed significant inhibition compared to the ethanol. The exceptions were: A. blazei Murrill, I. obliquus, and L. edodes against E. coli O157:H7; I. obliquus against L. monocytogenes V7; I. obliquus against S. cerevisiae Y99; L. edodes against Z. bailii Y03; and I. obliquus against Z. bailii/bisporus Y108. Inhibition was more effective in yeasts than bacteria. The result from Bioscreen C showed that against L. monocytogenes V7, fraction 7 in A. blazei Murrill; fraction 1, 4 and 5 in G. lucidum; fraction 4 in G. frondosa; and fraction 4 and 5 in I. obliquus significantly inhibited the growth compared to ethanol. Against Z. bailii Y03, fraction 7 in A. blazei Murrill; all fractions from G. lucidum, G. frondosa, and P. linteus; fraction 1, 2, 3, and 6 from I. obliquus; and fraction 4 and 6 from L. edodes significantly inhibited growth compare to ethanol. The results indicated that there is significant antimicrobial activity against food pathogens and spoilage organisms in the medicinal mushrooms studied.
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    Identification of soil bacteria capable of utilizing a corn ethanol fermentation byproduct
    Packard, Holly; Taylor, Zachary W.; Williams, Stephanie L.; Guimarães, Pedro Ivo; Toth, Jackson; Jensen, Roderick V.; Senger, Ryan S.; Kuhn, David D.; Stevens, Ann M. (PLoS, 2019-03-08)
    A commercial corn ethanol production byproduct (syrup) was used as a bacterial growth medium with the long-term aim to repurpose the resulting microbial biomass as a protein supplement in aquaculture feeds. Anaerobic batch reactors were used to enrich for soil bacteria metabolizing the syrup as the sole nutrient source over an eight-day period with the goal of obtaining pure cultures of facultative organisms from the reactors. Amplification of the V4 variable region of the 16S rRNA gene was performed using barcoded primers to track the succession of microbes enriched for during growth on the syrup. The resulting PCR products were sequenced using Illumina MiSeq protocols, analyzed via the program QIIME, and the alpha-diversity was calculated. Seven bacterial families were the most prevalent in the bioreactor community after eight days of enrichment: Clostridiaceae, Alicyclobacillaceae, Ruminococcaceae, Burkholderiaceae, Bacillaceae, Veillonellaceae, and Enterobacteriaceae. Pure culture isolates obtained from the reactors, and additional laboratory stock strains, capable of facultative growth, were grown aerobically in microtiter plates with the syrup substrate to monitor growth yield. Reactor isolates of interest were identified at a species level using the full 16S rRNA gene and other biomarkers. Bacillus species, commonly used as probiotics in aquaculture, showed the highest biomass yield of the monocultures examined. Binary combinations of monocultures yielded no apparent synergism between organisms, suggesting competition for nutrients instead of cooperative metabolite conversion. © 2019 Packard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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    Impact of Brewing Industry Byproducts Used as Feed Additives for Aquaculture-Raised Fish: Studies of the Host-Microbe Relationship
    Layton, Anna Rayne (Virginia Tech, 2024-04-15)
    Aquaculture, the cultivation of aquatic organisms in a controlled environment, offers both economic and nutritional benefits to human society. As there is an increased demand to feed a growing human population, many wild-caught fisheries have struggled due to the overexploitation of resources. Currently, production relies heavily on wild-caught fish to produce fishmeal to feed farm-raised fish. The demand for alternative materials in fish feeds has grown rapidly as fishmeal resources have become limited. Antibiotic resistance emergence in aquaculture systems is another area of concern. Reducing antibiotic use via alternate prophylactic measures to increase host health is an essential area of research; modulation of the host intestinal bacterial community via prebiotics is one possibility. Prebiotics refer to non-digestible food ingredients that are thought to stimulate the growth of beneficial bacteria, consequently benefiting host health by indirectly reducing the possibility of bacterial pathogen proliferation. This occurs through various measures such as competition for space and resources. The intestinal bacterial community has a significant impact on a variety of host factors that include host development, physiology, immunity, and nutrient acquisition. In turn, there are multiple factors impacting the bacterial community, including the presence of pathogens and/or antibiotics, environmental conditions, host genetics, and the diet consumed. To promote environmental sustainability and improve production and animal health in aquaculture, a collaboration was created with Anheuser-Busch of the brewing industry and Maltento, a functional ingredient company. With breweries around the globe, Anheuser-Busch produces consistent, food grade byproducts that are safe for human consumption. Two of the most prevalent brewery byproducts are brewer's spent yeast (BSY) and brewer's spent grain (BSG). BSY contains a variety of beneficial nutrients such as proteins, essential amino acids, and carbohydrates. BSG is high in fiber but low in protein; however, black soldier fly larvae can be cultured on BSG to convert the low-value product into insect biomass to be used in fish feed, as insects themselves are full of beneficial lipids and proteins. The objective of the work presented in this thesis was to evaluate the efficacy of using low-value brewery waste products, converted into high-value feed additives, for aquaculture practices. Specifically, the effects of dietary feed additives on the production, health, and intestinal bacterial community of aquaculture-raised rainbow trout were examined. Inadvertently, benefits of the feed additives on fish subjected to chronic and acute thermal stress were also assessed. Overall, the results of the study found that the feed additives did not significantly change the production efficiency of the rainbow trout, though some increase in growth was observed. When subjected to chronic thermal stress conditions, fish fed the experimental diets outperformed those fed the control diet regarding growth parameters. The intestinal bacterial community of the fish was significantly altered from the beginning of the trial compared to the end of the trial, though differences were not attributed to the feed additives. Instead, the resulting intestinal dysbiosis is believed to have stemmed from the physiological response of the fish to thermal stress conditions. When the fish underwent an acute thermal stress event, causing mortality, fish fed three of the five experimental diets were found to have higher survival rates compared to the control. Ultimately, results of this project suggest that the BSY and BSG-fed insect feed additives may have increased the health and robustness of the fish during a period of thermal stress. However, further research under controlled conditions is needed to evaluate if the observed host health benefits can directly be attributed to the feed additives.
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    Impact of environmental complexity and stocking density on affective states of rainbow trout (Oncorhynchus mykiss)
    Anderson, Mallory G.; Campbell, Andrew M.; Kuhn, David D.; Smith, Stephen A.; Jacobs, Leonie (Springer, 2022-04-16)
    Environmental condition, such as environmental complexity or stocking density, can directly or indirectly influence animal emotion and ultimately, affective state. Affective states of animals can be assessed through judgement bias tests, evaluating responses to ambiguous situations. In this study, we aimed to determine whether environmental complexity and stocking density impacted rainbow trout affective state. Rainbow trout (n = 108) were housed in recirculating aquaculture systems under commercial conditions while trained at tank-level to discriminate between a positively reinforced chamber (feed) in one location and a negative chamber (positive punishment; chase by net for 1 s) in the opposing location. Fish from successful tanks (two out of five tanks) were then housed in treatment tanks of either high- or low- environmental complexity at either high (165 fish/m3) or low (69 fish/m3) stocking density. Trained fish were tested for latencies to approach three intermediate, ambiguous chambers. Fish housed in high-density tanks were faster to enter all chambers than those housed in low-density tanks (8.5 s vs. 15.2 s; P = 0.001), with faster entries into the positive (7.4 s vs. 15.2 s; P = 0.02) and near-negative chambers (10.2 s vs. 17.4 s; P = 0.006), suggesting that these fish were more optimistic to receive a feed reward. Tank complexity did not affect test outcomes. No differences between treatments were observed between body weight, length, and plasma cortisol. Overall, rainbow trout are capable of discriminating between cues during a judgement bias test and fish housed in high-density environments respond more optimistically in ambiguous situations compared to fish in low-density environments.
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    The Impact of Marine Resource-Free Diets on Quality Attributes of Atlantic Salmon
    McLean, Ewen; Campbell, Kelly B.; Kuhn, David D.; Tlusty, Michael F.; Barrows, Frederick T. (MDPI, 2024-01-17)
    The influence of feeding Atlantic salmon for 90 days on diets that excluded fishmeal (FM) and fish oil (FO) was examined for influence on various quality traits. In addition, the effect of adding krill meal (KM; 0%, 2.5%, and 5%), as a putative feed palatant was also examined. Total replacement of FM/FO had a limited effect on production characteristics, affecting percentage yields of headed and gutted control fish and their standard length (p < 0.05). Variances between dietary groups were observed for pigmentation, and plant protein-based KM-free-fed fish returned deeper hues across their belly, NQC (Norwegian Quality Cut), and back portions (p < 0.03). No differences were measured for relative fin condition. δ13C and δ15N concentrations were lower and higher, respectively (p < 0.05) for fish fed the FM/FO-based diet. δ13C:δ15N likewise differed between treatments with FM/FO-fed salmon expressing higher ratios. Fillet mechanical characteristics varied with fish fed on animal protein-based diets, without KM expressing higher springiness and resilience (p < 0.05). Fish fed plant-based diets were generally preferred by younger taste testers. The results from this trial illustrate that FM/FO can be completely removed from salmon diets without problematic effects on quality and palatability attributes.
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    Improving Disease Resistance for Shrimp Through Application of Probiotics in Feed
    Choi, Moonyoung (Virginia Tech, 2015-06-12)
    Diseases affecting shrimp contribute to billions of dollars of economic loss yearly to the aquaculture industry. Recently, one of the primary causative agents of disease has been Vibrio parahaemolyticus; in 2009, a new strain causing Early Mortality Syndrome (EMS) or acute hepatopancreatic necrosis disease (AHPND) in shrimp emerged. Shrimp losses attributed to pathogens can be greatly reduced through probiotic use, which are known to act as natural immune enhancers and promote pathogen resistance. However, research on probiotic treatment against EMS disease is lacking. The overall project goal was to improve intensive shrimp production through direct application of probiotics in aquaculture feeds. The value of probiotics for the shrimp industry was evaluated by (1) reducing severity or mortality of V. parahaemolyticus disease in shrimp, (2) qPCR confirmation of Bacillus spore germination in shrimp gut, and (3) probiotic effectiveness evaluation for improving disease resistance. The virulence of several Vibrio spp. strains was examined and it was concluded the V. parahaemolyticus strain identified as the causative agent of EMS was the most lethal; EMS-infected shrimp exhibited 100% mortality within 36-hours of feed inoculation. The number of bacterial cells added to feed directly correlated with pathogenicity and only cells, not filtrate, was capable of causing death. Probiotic strains were evaluated and it was concluded that probiotic strains A, A/B blend, and B were the best candidates for promoting disease resistance against EMS. This research will provide the shrimp farming industry with information vital to developing a means for reducing economic loss from Vibrio-infected shrimp.
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    The Influence of Prebiotics, Probiotics, and Exposure to an Opportunistic Pathogen on the Intestinal Microbiome of White Shrimp (Litopenaeus vannamei)
    Kesselring, Julia Jiang Hao (Virginia Tech, 2022-06-13)
    Prebiotics and probiotics, proposed alternatives to antibiotics in shrimp aquaculture, are reported to improve growth parameters, promote disease resistance, and influence the gut microbial community. This study aimed to investigate the influence of prebiotic- or probiotic-coated feed and/or exposure to the Early Mortality Syndrome-causing strain of Vibrio parahaemolyticus ( VP-EMS) on the mid and hindgut microbiome of Pacific white shrimp (Litopenaeus vannamei). A monoculture probiotic strain of Bacillus subtilis spores: O14VRQ, and a prebiotic product of cultured Saccharomyces cerevisiae cell walls: MOS, were administered to shrimp as feed additives for 14 days, before a pathogen challenge to VP-EMS. Based on previous efforts, animals in this study were fed experimental diets for 14 days to allow ample amount of time for the prebiotic to be metabolized by health-promoting bacteria and for the probiotic spores to germinate. The pathogen challenge consisted of negative disease control (no VP-EMS exposure, commercial feed), positive disease control (VP-EMS exposure, commercial feed) and two treatment groups, probiotic (VP-EMS exposure) and prebiotic (VP-EMS exposure). DNA extraction, 16S rRNA gene amplicon sequencing, polymerase chain reaction (PCR), and sequencing were utilized to create an overview of the mid and hindgut microbial composition. No significant differences in survival were shown between experimental diets following exposure to sublethal levels of VP-EMS. Bioinformatic analyses revealed no distinct shifts in the mid and hindgut microbiome of shrimp across experimental diets and time points. Results of this data revealed that dominant members of the intestinal microbiome, Proteobacteria, Actinobacteriota, Bacteroidota, Verrucomicrobiota, Flavobacteriaceae, Demequinaceae, Vibrionaceae, Shewanellaceae, Rhodobacteriaceae, and Rubritaleaceae were relatively stable across treatments and time points. Sequencing methods such as metagenomics or metatranscriptomics should be utilized for a higher microbiome resolution. Furthermore, the use of quantitative polymerase chain reaction to quantify ingested probiotic spores, prebiotic-associated bacteria, and VP-EMS is recommended.
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    Integrated processing of brewer's spent grain into value-added protein feedstuff and cellulose adsorbent
    He, Yanhong (Virginia Tech, 2021-09-16)
    Brewer's spent grain (BSG) is the major byproduct generated by the brewing industry, which contains 14–30% protein and 50–70% of fiber. Currently, BSG is predominantly used as low-value cattle feed or buried in landfills, which is a considerable loss of valuable resources, leading to economic loss and environmental problems. Although research has been done on BSG valorization, the studies are limited to producing a single product (e.g., polyphenols, ethanol, or active carbon) and without further utilization of the produced products. Besides, the economic information available about the production of value-added products from BSG is insufficient. The overall goal of this research is to develop an integrated process to convert BSG into value-added protein-rich feedstuff and cellulose absorbent. The objectives of the research detailed here were to 1) develop a process to simultaneously produce protein-rich (PP) and fiber-rich products (FP) from BSG, 2) assess the replacement of fishmeal with PP in shrimp feed, 3) evaluate the economics of the overall process of PP production at a commercial scale, and 4) explore the potential use of cellulose adsorbent obtained from the FP in removing heavy metals from contaminated water. To attain these objectives, BSG was first subjected to a wet fractionation process to produce PP and FP using different chemical/biological treatments, where the effects of sodium hydroxide, sodium bisulfite, and a protease (Alcalase) at different concentrations were investigated. Under the optimized conditions, the produced PP contained 46% protein and less than 1% fiber. The effectiveness of using PP to replace fishmeal at increasing levels (10–70%) was then evaluated by performing shrimp feeding trials. The results showed that up to 50% of fishmeal in shrimp feed can be replaced by PP without affecting shrimp growth and feed utilization. Moving forward, a techno-economic analysis was conducted to evaluate the economic feasibility of the production of PP. The experimental conditions and results were input into the process simulation model for determining the mass and energy flows. For a processing plant with a capacity of 590 t wet BSG per day, the minimum selling price of PP to achieve a 5% return was determined to be $1044/t, lower than the price of fishmeal, indicating that the use of PP to replace fishmeal in shrimp feed could potentially reduce shrimp farming cost. The utilization of FP will further improve the economic feasibility of the fractionation process. FP was sequentially treated by dilute acid, alkali, and bleach to produce purified cellulose fibers, which were then modified by 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) oxidation to produce a cellulose adsorbent. The feasibility of the adsorbent in removing heavy metals (especially lead and manganese) from contaminated water was then investigated. Based on the results, the produced cellulose adsorbent showed high adsorption capacities for lead (272.5 mg/g) and manganese (52.9 mg/g). Overall, this study demonstrated that BSG can be upcycled into multiple value-added products via an integrated process. The outcomes of this study not only provide a low-cost and sustainable protein source to the aquaculture industry, and provide a novel adsorbent for the water treatment industry, but also offer alternative ways for the brewing industry to manage BSG.
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    Long Chain n-3 PUFA and Oleic Acid Modification Strategies to Enhance Fillet Quality in Tilapia, Oreochromis species
    Chu, Hyun Sik Stephano (Virginia Tech, 2017-05-26)
    Tilapia are freshwater fish that have become important in aquaculture and as a stable global source of seafood due to their ability to thrive in different environments. However, tilapia are sometimes considered nutritionally undesirable due to their high n-6 to n-3 fatty acid ratios. A market study was conducted first to determine fatty acid compositions in tilapia fillets in different US markets. Then a research was conducted to enhance nutritional value of tilapia by improving the n-3 and oleic acid contents in fish fillets without compromising fish growth or feed conversion ratios. Feeds were formulated with combinations of high and low n-6, n-3, and oleic acid levels using soybean oil, fish oil, algae oil, and high-oleic sunflower oil. Then 12 diets, including a commercial diet, were assigned to 24 tanks, each with 25 tilapia per tank. A Recirculating Aquaculture System (RAS) was used to grow the fish for 8 weeks. Fatty acid compositions of tilapia fillets were determined and samples were vacuum packed and stored at -10oC and -20oC to test oxidative degradation and fatty acid compositional changes. The market survey data showed that there were significant differences in fatty acid composition, lipid content, and n-6:n-3 fatty acid ratios depending on the country of origin. Samples from USA had ideal n-6:n-3 ratios (1.3 ±0.85) while samples from Southeast Asia had higher n-6:n-3 fatty acids ratio (6.6 ±0.54). Algae oil incorporation significantly increased DHA level while fish oil incorporation significantly increased both EPA and DPA. High-oleic sunflower oil based diets improved oleic acid levels and reduced linoleic acid compared to the soybean oil based diets. Sensory evaluation indicated that lipid source did not significantly impact preference or overall fillet quality, including texture. Interestingly, a survey showed people were interested in value-added tilapia, and would pay up to 30% more for nutritionally enhanced fish compared to the $5.00/lb fresh fillet price currently available in supermarkets. There was no observable oxidation during long term frozen storage. The oxidation study proved that value-addition would not be compromised during the long term storage conditions, even under temperature abuse. It is possible to improve tilapia nutritional quality through diet to provide consumers with value-added products that maintain quality during frozen storage.
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    Microbiology at the Nexus of Food, Energy, Water and Health
    Badgley, Brian D.; Boyer, Renee R.; Dufour, Monique; He, Zhen (Jason); Hungerford, Laura L.; Kiechle, Melanie A.; Kuhn, David D.; Lawrence, Christopher B.; Marr, Linsey C.; Melville, Stephen B.; Pierson, F. William; Popham, David L.; Senger, Ryan S.; Sumner, Susan S.; Vinatzer, Boris A.; Schmale, David G. III; Stevens, Ann M. (Virginia Tech, 2017-05-15)
    Microorganisms are absolutely critical to myriad aspects of the human existence. As a field of study, microbiology could and should serve a greater role on our campus, as it has key connections with many of the Destination and Strategic Growth Areas. We propose the development of a broad concept area in microbiology that will serve as a nexus, as it is applied to solve critical global challenges related to food, energy, water and health, by bridging across multiple disciplines at Virginia Tech (VT). There is increasing recognition of microbes as a driving force in natural and managed environments, biological processes, and ecological structure. Conversely, the importance of culture and individual behavior in affecting microbial communities has also become apparent....
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    Nitrogen Removal from Closed Aquaculture System by Bio-electrochemical System
    Guan, Lu (Virginia Tech, 2018-01-22)
    Removal of nitrogen elements in culture water is one of the major concerns in recirculating aquaculture system (RAS). Maintaining a low concentration of nitrogen compounds is essential for a good quality of aquaculture production. Due to fish is very sensitive to the toxic ammonium/ammonia, nitrification biofiltration tank is often an integrate part of filtration in RAS to remove ammonium via nitrification. However, nitrate accumulation via nitrification in RAS is often observed during the operation, which is usually solved by replacing with the fresh water into the system. With the concern of water consumption, bio-electrochemical system (BES) is introduced in this study to realize simultaneous nitrate removal for the system while generating the electricity through electron transferring. A microbial fuel cell (MFC) with an anion exchange membrane (AEM) was constructed. The removal of nitrate from aquaculture water generated from RAS was achieved by nitrate migration across the AEM and heterotrophic denitrification in the anode chamber. To further investigate the potential application of BES in RAS, the cathode chamber was incubated with biofilm to do the nitrification while the denitrification processing in the anode chamber. The study gave a total inorganic nitrogen removal efficiency of 38.72% ± 4.99, and a COD removal of 86.09% ± 9.83. The average daily electricity generation was 67.98 A m-3 ± 13.91, and nitrate-nitrogen concentration remained at 21.02 ± 2.62 mg L-1 throughout the experiment. These results of treating aquaculture water indicate that BES has a potential to install within RAS for enhanced nitrogen removal.
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    Novel Aquafeed Towards A Sustainable Aquaculture
    Farzad, Razieh (Virginia Tech, 2018-12-19)
    With World Health Organization estimation of population increase to over 9 billion by 2050, malnutrition and food insecurity are predicated to be two of the main challenges facing the world in the future. Fish is a source of protein that can have a significant contribution for solving the global food crisis. Fish for human consumption is provided by both the aquaculture industry and the capture fisheries, however, the flat-lined production of seafood from capture fisheries means aquaculture production should double in number to provide enough protein by 2050. Culturing fish at higher densities is being used as a way to increase the global aquaculture production. Modern aquaculture intensified practices expose the fish to various stressors that can negatively impact their welfare. Good nutrition can improve the health status of aquacultured fish while accumulation of specific nutrient can add value to the fillet. In line with that, the studies described in this dissertation aimed to examine the vital role of fish in human health and evaluate strategies to improve the sustainable aquaculture production of fish using novel aquafeeds. This was completed by conducting a survey of the essential minerals and heavy metals of commercially available catfish, salmon, and tilapia fillets in the United States marketplace and three fish nutritional trials to evaluate the impacts of novel aquafeeds on production and health of fish and the food quality of their respective fillets. Selenium with the established health benefit for human and fish was the trace mineral of interest in this dissertation. This element is the main component of a group of selenium containing proteins called selenoproteins that are antioxidant and maintain oxidative homeostasis of fish and human. Additionally, selenium is an antagonist for many heavy metals such as mercury. The market study revealed that catfish, salmon and tilapia samples available in United States marketplace are good source of selenium, zinc and phosphorous. The concentration of heavy metals in all the samples were lower than safety standards. Nile tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss), which are two economically important aquacultured fish, were used as animal models in the fish nutritional trials. Different forms of selenium supplemented to Nile tilapia diet. The form of Dietary Se form did not have any significant (p>0.05) effect on growth, and plasma and hepatic GPX activity of Nile tilapia. However, organic selenium supplement showed significantly (p<0.05) higher bioavailability and accumulated in the fillets at the significantly higher concentration. In the second fish trial selenoneine, a novel selenium containing compound, was supplemented at various concentrations to rainbow trout diet. Other than growth parameters multiple other health biomarkers (lysozyme, glutathione peroxidase, superoxide dismutase activities) were examined to determine whether selenoneine supplementation had a positive impact on fish health. Selenoneine enriched diet significantly(p<0.05) increased the weight gain, lysozyme and oxidative enzymes activities of rainbow trout. Lastly rainbow trout feed was supplemented with commercially available probiotic (Actisaf®), prebiotic (Safmmann®), and their mixture (synbiotic). These dietary supplements did not have any significant effect on fish production but led to a significant increase in activity of oxidative enzymes in plasma when compared to control diet. These results suggest that feed supplements used in this dissertation can be used to promote the fish health raised in recirculating aquaculture systems and support an economically and environmentally sustainable aquaculture.
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    Post Harvest Transmission of Salmonella enterica to the Roots and Leaves of Butterhead Lettuce Packaged With Intact Roots
    Waitt, Jessie Anne (Virginia Tech, 2013-05-21)
    In the United States, illnesses associated with fresh produce are increasing in frequency.  While contamination risks are present at every aspect of the farm to fork continuum, post-harvest practices holds the potential for cross-contamination of large amounts of product.  Post-harvest contamination risks for hydroponically grown lettuce packaged with intact roots and sold as "living lettuce"" are poorly understood.  In this study, transmission of Salmonella enterica serotype Enteritidis to the roots and leaves of butterhead lettuce was studied when contamination was introduced during typical handling practices.  The effectiveness of random sampling strategies for selection of Salmonella contaminated leaves was assessed by co-inoculating the Salmonella solution with Glo Germ™ and comparing recovery from blacklight selected leaves.  The recovery of Salmonella was improved by only 0.5 log CFU/g when blacklight was used to select Glo Germ™ contaminated leaves (P=0.05). This suggests random leaf selection as described by current FDA protocols is adequate. In addition, this study showed rapid transfer of Salmonella from liquid to the roots and sub-sequentially to the leaves of living lettuce.  Salmonella persisted but did not grow on leaves when stored at 4˚C for 18-days. Storage at 12˚C was associated with 2 log CFU/g increases in Salmonella on roots after 18-days storage (P=0.0002), while 4˚C storage was associated with a decrease of 0.4 log CFU/g Salmonella on roots (P=0.0001). Growth occurred only under temperature abuse conditions.  This reinforces the need for maintaining temperature control and highlights the importance of identifying risks associated with post-harvest handling during hydroponic production and distribution.
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    Production of omega-3 enriched tilapia through the dietary use of algae meal or fish oil: Improved nutrient value of fillet and offal
    Stoneham, Tyler R.; Kuhn, David D.; Taylor, Daniel P.; Neilson, Andrew P.; Smith, Stephen A.; Gatlin, Delbert M.; Chu, Hyun Sik S.; O'Keefe, Sean F. (PLOS, 2018-04-11)
    The goal of this project was to increase the nutrient value of fillets, by-product muscle, and offal of aquacultured tilapia. A diet that includes seafood with a high omega-3 (n-3) fatty acid content, more specifically eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are known to have numerous health benefits for consumers. Improved nutrient value of the offal may also attract new market opportunities for the aquaculture industry. Tilapia were cultured on different experimental feeds that contained various levels of n-3 fatty acids from either fish oil (FO) or algae meal (AM) that were used to replace corn oil. The experimental diets included a control (corn oil 6.3%), FO1%, FO3%, FO5%, AM1.75%, AM5.26%, and AM8.77%. All diets were formulated to be isocaloric, isonitrogenous, and isolipid. Three hundred and fifty tilapia with an initial mean weight of 158±2 g were cultured in a recirculating aquaculture system (seven diets replicated at the tank level, 14 tanks, 25 fish per tank). For all of the production performance data, no differences (P>0.05) were observed between the experimental groups which included survival (overall mean ± standard error, 99.4±0.3%), growth per week (45.4±1.0 g/wk), food conversion ratio (1.32±0.03), fillet yield (44.4±0.2%), hepatosomatic index (1.61±0.02), viscerosomatic index (2.86±0.06), and mesenteric fat index (0.97±0.04). Fillet and rib meat tissues were collected at weeks four and eight, and liver and mesenteric fat tissues were collected at week eight. Fatty acids were extracted, methylated and identified with gas chromatography±mass spectrometry. All tissues had improved fatty acid profiles (higher n-3, lower n-6, n-6:n-3) with increasing levels of FO and AM in the diet. For example, the best diet for significantly (P<0.05) improving the lipid profile in tilapia fillets at week eight was diet AM8.77%. In the fillet, total n-3 was increased (control versus AM8.77%) from 151.2±19.0 to 438.7±14.2 mg per 4 ounce (113 g) serving and n-6: n-3 ratio was improved from 5.19±0.76 to 1.29±0.03.
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    Steelhead trout (Oncorhynchus mykiss) fed probiotic during the earliest developmental stages have enhanced growth rates and intestinal microbiome bacterial diversity
    Hines, Ian S.; Santiago-Morales, Kevin D.; Ferguson, Clay S.; Clarington, Jireh; Thompson, Meaghan; Rauschenbach, Meghann; Levine, Uri; Drahos, David; Aylward, Frank O.; Smith, Stephen A.; Kuhn, David D.; Stevens, Ann M. (Frontiers, 2022-11)
    Sustainable aquaculture practices can help meet the increasing human demand for seafood, while easing pressures on natural fish populations. Studies aimed at increasing fish production in aquaculture have included supplementary dietary probiotics that often promote general health and enhanced growth rates by altering the microbiome of the host. Steelhead trout (Oncorhynchus mykiss) is anadromous, like salmon, and it is a subspecies of rainbow trout capable of rapid growth, making it an attractive fish to the aquaculture industry. In this study, the impact of feeding a Bacillus subtilis probiotic on the bacterial microbiome of steelhead trout was examined temporally across several stages of animal development, from eggs (day -19) through 184 days after hatching, in relation to physiological measures. Diets included: commercial feed only as a control (A), continually-fed probiotic top-coated on commercial feed (B), commercial then switch to probiotic feed (C), or probiotic then switch to commercial feed (D). Validation of probiotic concentrations on feed and in fish tissues was performed using CFU/g and qPCR, respectively. Fish growth was measured and samples for intestinal microbiome analyses were collected at multiple timepoints during fish development. Fish fed diet D yielded higher weights than the other three diets, with little impact on other biometric parameters. However, bacterial microbiome analysis indicated an increasing trend of overall alpha diversity from the egg stage to day 29 for fish fed the various diets with diet D having the highest diversity. Fish fed diets A and D maintained a high alpha diversity beyond day 29 in contrast to a decreased trend for fish still being fed probiotics in diets B and C. The fish fed diets B and C harbored a significantly higher relative abundance of Bacillus sp. in their total microbiomes (feces + mucosa). Interestingly, the mucosal-only microbiome indicated little variation between the four groups of fish. Feeding the probiotic earlier in development, during the hatchery phase, to influence bacterial microbiome composition in the intestine (rather than later after the microbiome has been established) appears to be a more effective aquaculture practice by enhancing microbiome diversity while enabling higher fish yields.