Plastic products are important commodities on a global scale and have been used extensively in almost every aspect of human life. The massive production and use of conventional plastics have created major concerns, including the unsustainable use of fossil carbon feedstocks, the emissions of greenhouse gases in the manufacturing processes, and the disposal of most of the plastic waste and consequent impacts on the natural environment at the end of the lifecycle. As a response to these problems, there has been a global trend towards alternative bioplastic products that are more environmentally friendly than traditional ones. As one popular alternative, polyhydrolxyalkanoates (PHA) are a family of naturally occurring biodegradable plastic materials used as a replacement for the conventional plastics in various applications. Since PHA has similar thermal and mechanical properties to a broad spectrum of different types of conventional plastics, it has been widely used in many applications including packaging, food service, agriculture, medical supplies, consumer products, chemicals, and environmental services. PHA has an established market in recent decades, however, the current bottleneck for the market growth is its high production costs, which are commonly three to four times higher than other bioplastic and traditional plastic products of similar properties and use in the market. There is an urgent need to develop a bioprocessing system to produce PHA with high quality at low cost. Therefore, the goal of this study was to develop an integrated PHA production system utilizing inexpensive feedstocks including food waste and cheese byproducts, which provides high production efficiency and product quality, and low production cost.

Previous studies showed that Haloferax mediterranei, a wild-type extreme halophilic archaeon, can be a superior candidate as a microbial PHA producer than freshwater microbes, with regards to the utilization of waste or byproduct feedstocks for PHA production. It can provide robust production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a type of high-value PHA, from various renewable feedstocks. Meanwhile, its high salt tolerance enables natural resistance to contamination introduced by microbes in waste-derived streams, which can reduce the costs from sterilization or pasteurization normally incurred in commercial scale productions. In addition, it offers an easier approach for polymer extraction by salt-free (fresh) water, which consumes less energy and chemicals than current PHA production systems. Food waste and food processing byproducts (such as lactose) have good potentials as feedstocks for PHBV production by H. mediterranei because they are rich in sugars and short-chain carboxylates that can be metabolized to PHBV. Pre-treatment processes including anaerobic fermentation, enzymatic hydrolysis, and membrane filtration are required prior to release and to recover those nutrients from the complex macronutrients contained in the feedstocks. The culture of this strain essentially consists of saline medium, carbon source, nitrogen source, phosphorous source, and trace elements. Besides nutrient conditions, a successful cell cultivation process also requires suitable growth conditions including temperature, salinity, pH, and aeration rate.

With the aim of utilizing food waste as low-cost feedstock to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by Haloferax mediterranei, the effects of acetate (Ac), propionate (Pr), butyrate (Bu), and the short-chain carboxylates derived from food waste were examined on the microbial growth and PHBV production. Results showed that a mixture of carboxylates yielded a 55% more PHBV than glucose. The food-waste-derived nutrients achieved yields of 0.41 to 0.54 g PHBV/g Ac from initial loadings of 450 mg/L to 1800 mg/L Ac of total carboxylates. The consumption of individual carboxylate varied among the different compositions of the carbon source.

Based on previous findings, this study further tested the feasibility of using real-world feedstocks which contain different sugars and VFAs as the main carbon sources for PHBV production by the microbes. Four different types of feedstocks including food waste hydrolysate, food waste fermentate, whey sugar (lactose), and delactosed permeate were collected, pretreated with different processes, and utilized as the sole substrate to culture H. mediterranei. Before microbial cultivation, food waste hydrolysate and fermentate were processed through microfiltration for the recovery of nutrients and removal of suspended particles. The two cheese processing byproduct streams were subjected to enzymatic hydrolysis to convert lactose into monosaccharides. After pretreatment, each substrate was loaded with two sCOD loadings of 22.5 g/L and 45 g/L to culture H. mediterranei and the effects of substrate type and loading on PHBV production were studied. The results show that with the same substrate loading, the substrate derived from food waste fermentate gave higher final cell mass and PHBV concentrations than food waste hydrolysate; and 45 g/L sCOD resulted in the highest PHBV production (3.92 ± 0.08 g/L) within the same culturing time. Comparing substrates derived from whey sugar and delactosed permeate, with 45 g/L sCOD loading, cells grew much faster in whey sugar hydrolysate. Food waste fermentate resulted in higher HV contents than food waste hydrolysate; and substrate loading influenced the HV content of PHBV derived from delactosed permeate, which may be due to the prolonged growth time.

Since food waste fermentate was proven to be one of the better candidates among the four real-world inexpensive feedstocks, more research was conducted to develop an integrated PHBV production system utilizing fermented food waste from commercial anaerobic digesters as feedstock. The effects of feedstock type, substrate loading, and aeration rate were studied and the optimum levels (FWP 1, 40 g/L sCOD, and 2.5 vvm) was determined to yield the highest productions of cell mass and PHBV. A 6-L benchtop bioreactor system has been designed, constructed, and operated to demonstrate the integrated PHA production system from food waste. Through dynamic monitoring of this bioreactor system, the dissolved oxygen level of cell broth was found to drop dramatically to below 20% sat. during the exponential growth phase with a fixed aeration rate. A strategy of maintaining a higher DO level above 50% sat. resulted in faster cell growth but led to lower final cell densities, final PHBV concentration and yield. A novel approach was developed to recycle and reuse the spent salts within the system, should reduce the costs of purchasing raw materials and wastewater treatment and disposal. The spent saline medium (SSM), after the treatment by H2O2 and concentrated to a brine solution through rotary evaporation, was successfully reused through four consecutive batches, where both 80% and 90% SSM recycling strategies yielded comparable cell growth and PHA production to the original batch. The PHA production system developed in this study has the potential for practical applications in the current bioplastic industry. The additional revenue from PHA production can help promote the circular economy for the current food system, increase the market share of bioplastics, and create incentives for better practice of organic waste management in general.

In addition to food waste, the study has also developed an integrated PHA production system with utilizing whey sugar from commercial cheese making facilities as feedstock. Through the dynamic monitoring in a 6-L benchtop bioreactor system, different aeration strategies led to similar results as food waste that with a higher DO level above 50% saturation resulting in faster cell growth, and higher consumption of galactose, but lower final cell densities and PHBV production. The direct reuse of spent saline medium (SSM) was also successfully achieved through four consecutive batches with various substrate loadings, where both 80% and 90% SSM recycling strategies yielded comparable cell growth, PHBV production and PHBV profile to the original batch. This novel PHA production system has the potential of practical applications in the current bioplastic industry; meanwhile, through utilizing whey byproducts and waste streams, an additional revenue can be provided to the dairy industry.

Based on the system design developed here, economic feasibility assessments were conducted to project integrated industrial scale processing. Byproduct streams from a local cheese plant, with an input of 168.7 metric ton/day (MT/day) lactose (186 US ton/day), were used as the feedstock. SuperPro® Designer (Intelligen Inc., USA) was used as the software for the process design and economic modeling. Three scenarios with different processes for the treatments of used enzyme and spent medium were investigated and the major factors that influence the overall economics were identified. The simulated system produces 9700 MT/year PHBV with an assumed PHA yield of 0.2 g PHBV/g lactose and an overall process efficiency of 87%. The breakeven price was found to be more sensitive to the lactose price than enzyme price. The scenario with enzyme reuse and spent medium recycling achieved the lowest breakeven price among the scenarios, which can be less than 4 $/kg PHA based on a delactosed permeate (DLP) unit price of 0.1 $/kg. The study suggests utilizing dairy derived feedstocks has the potential to make PHA competitive in the bioplastic market, which could be beneficial to both the dairy and bioplastic industries.

There are over 1.1 million tons of almond produced in California resulting in over two million tons of almond hulls generated each year. Currently, those hulls are mostly used for dairy feed and bedding at low value. However, the almond industry is growing faster than the dairy industry, producing large amounts of excess hulls. Finding alternative uses for almond hulls while increasing their value is essential for the almond industry to achieve its zero-waste goal and improve sustainability. Due to their high sugar and fiber contents, almond hulls have the potential to be utilized for producing edible filamentous fungal biomass for food applications. Fungal biomass can serve as an important source of protein and other nutrients for feeding the increasing population. The overall goal of this research was to develop an efficient system for producing edible fungal biomass from almond hulls by integrating sugar extraction, enzymatic hydrolysis, and submerged cultivation.In the first study, almond hulls of three varieties collected from almond processors were characterized for chemical and physical properties, such as total solids, fiber, protein and fat contents. The three varieties were Independence, Nonpareil, and Monterey. Sugars were extracted from hulls and used for the production of edible fungi. Two different grinding methods, continuous and successive grinding, were compared to get desired particle size for sugar extraction using water as an extraction medium. A kinetic model was developed, based on Fick’s law, for sugar extraction from Independence almond hulls using three different liquid-solid ratios. The model was validated with data collected from extraction experiments conducted at different temperatures. Results showed that successive grinding was chosen to prepare almond hulls with particle sizes in the range of 2.36 to 3.38 mm. The established model was applied to predict sugar extraction from Nonpareil and Monterey almond hulls. The model provided a theoretical framework to understand the extraction process, which is helpful in designing sugar extraction processes for different purposes. Experiments were also conducted to optimize sugar extraction from Independence almond hulls. Response Surface Methodology (RSM) based on Box-Behnken Design was utilized to determine the optimal combination of time, temperature, and liquid-solid ratio for achieving the maximum total reducing sugar yield. The optimum extraction conditions were determined to be as follows: extraction time of 86.6 minutes, temperature of 77.4°C, and a liquid-solid ratio of 14. Under these conditions, the experimental sugar yield was 38.1%, which was well matched with the predicted yield 39.3%. The almond hull extract was used for fungal biomass production of Aspergillus awamori (A. awamori). In 250 mL glass bottles or flasks, A. awamori’s growth in pellet form, was investigated under different conditions including inoculum level, aeration, nitrogen source and carbon to nitrogen ratio (C/N ratio). Subsequently, the fungal cultivation was scaled up to a 2 L bioreactor, where configurations that could favor pellet formation were studied. The structure and morphology of fungal pellets at various growth stages were observed and compared using an environmental scanning electron microscope. Results indicated that yeast extract and NH4Cl worked better than peptone and NaNO3, respectively, as supplemental nitrogen sources. To reduce the cost of raw materials, a combination of yeast extract and NH4Cl was chosen to adjust the initial C/N ratio. The highest concentration and yield of A. awamori biomass were observed when the fungus was cultivated at a C/N ratio of 15, compared to ratios of 30 and 45. At a C/N ratio of 15, the crude protein content in pellets was at its highest at 18.10% (dry basis, d.b.). While the fat content was at its lowest at 2.28% (d.b.). The effects of pH control and agitation on A. awamori growth were investigated in the bioreactor. Ultimately, the optimal conditions for A. awamori growth in almond hull extract were determined to be a C/N ratio of 15, an inoculum level of 103 spores/mL, without pH control, and an agitation rate of 150 rpm over a period of five days in a bioreactor with aeration at 1 air volume per working volume per minute to control dissolved oxygen above 25% of the saturation. Under these conditions, the biomass yield was around 0.85 g VSS/g sugar, with an average pellet size of 3.75 mm. In addition to using the water-soluble extracts from almond hulls for fungal biomass cultivation, the potential of the residual almond hull solids (RASH) after sugar extraction for fungal cultivation was studied by using enzymatical hydrolysis. The effect of three different enzymes (Cellic CTec2, Viscozyme L and Pectinex Ultra SPL) and their combinations on hydrolysis performance were investigated and compared. The optimum loadings of enzymes were determined. The ability of A. awamori to grow on the resulting hydrolysate was evaluated. The most effective hydrolysis in terms of liquefaction and sugar yield was achieved through the combination of Cellic CTec2 and Viscozyme L. Using 200 uL/g RASH of Cellic CTec2 and 60 uL/g RASH of Viscozyme L resulted in a total sugar yield, total fiber conversion, and liquefaction efficiency of 41.36%, 86.01%, and 51.61%, respectively. Applying these optimal conditions at a larger scale resulted in improved liquefaction efficiency, reaching 72.53%. While the sugar yield was similar to small scale hydrolysis. After cultivating A. awamori in hydrolysate for five days, uniform yellow fungal pellets were observed, resulting in a biomass yield of 0.8 g VSS/g sugar. This study suggests that RASH could be a promising source for fungal-based food production, thus optimizing almond hull utilization and increasing the yield of fungal biomass from almond hulls. A techno-economic analysis was conducted for an industrial-scale system for the production of A. awamori fungal biomass. Almond hulls were utilized as the feedstocks, with an input of 50 MT/batch (wet basis, w.b.). SuperPro Designer software was used for process simulation and economic assessment. Two different scenarios were evaluated: the first system was using sugar extract alone as a source of nutrients needed for fungi cultivation and the second was similar to the first system plus the nutrients produced from the enzymatically hydrolyzed residual almond hull solids after sugar extraction. A sensitivity analysis was carried out for studying different parameters, affecting the breakeven price of fungal biomass, such as almond hull price and facility processing capacity. Using the same amount of hulls, the simulated systems produced approximately 2,971 MT/year fungal biomass using the sugars extracted from the hulls using hot water; and around 4,653 MT/year fungal biomass using the sugars extracted from the hulls using hot water plus the sugars produced from the enzymatic hydrolysis of the hulls with the same amount of almond hulls. The breakeven price of the fungal biomass from both systems ranged from $6 to $7 per kg of fungal biomass (d.b.). In both scenarios, the breakeven price of fungal biomass is more sensitive to the capacity at small scale (lower than 50 MT/batch) and more sensitive to almond hull price at larger scale. The results of this study indicate that almond hulls are very good feedstock for the production of myco-foods that can be competitive in the markets of proteins, probiotics, and other food categories, benefiting both the almond and food industries. Finally, the potential of using A. awamori pellets for developing different products (myco-foods) was explored. The color and characteristic of the pellets were studied using both artificial food dyes and natural colored media derived from agricultural and industrial byproducts. Pellets’ colors obtained from natural colored media were more stable after one-month storage. A method to create multilayered color pellets was developed successfully by deactivating pre-cultured pellets and then growing new spores on them. The texture of pellets was improved by coating with potato dextrose agar (PDA) and re-growing to form condensed mycelium inside fungal pellets. The potential for developing A. awamori biomass into dried products was also evaluated. A mice study was conducted to determine the health benefits of the developed products. It was found that the biomass could potentially be processed into protein/fiber powder-based products through freeze drying or transformed into crispy, snack-like myco-chips through hot air drying. Lighter color was obtained after freeze drying, while hot air drying led to darker color of the fungal biomass. No mycotoxins were detected in the A. awamori-based foods, and the foods showed significant health benefits in the mice study. The spent media collected after pellet production contained enzymes, suggesting the potential for its utilization in the development of beverages. The findings from this study provided important information of the potential of developing A. awamori biomass into novel functional food. In summary, based on the findings from this study, California’s two million tons of almond hulls can be potentially transformed into 0.6 million tons of fungal biomass. This conversion could increase the revenue generated from almond hulls, from a previous 0.4 billion dollars (when sold as dairy feed) to 4.2 billion dollars in fungal products. This research demonstrated converting almond hulls into fungi-based food could significantly add economic value to the almond hulls. Further investigations can be undertaken to scale up the production of fungal biomass from almond hulls to large scale. Additionally, exploring the viability of utilizing almond hulls for cultivating various fungal strains could be a promising way forward for future research.

Abstract

Plastic products have become indispensable in various industrial sectors and everyday life worldwide. However, conventional plastics present significant sustainability challenges due to their reliance on unsustainable petroleum sources, contribution to greenhouse gas emissions, and their resistance to biodegradation, which leads to environmental waste accumulation. In response to these issues and the need to decrease carbon emissions, there has been a growing interest in bioplastics. One promising family of bioplastics is polyhydroxyalkanoate (PHA), a group of natural polyesters that can serve as a sustainable alternative to traditional plastics in numerous applications. PHA stands out among bioplastics for several reasons, including production from organic waste substrates. The polyester can be produced from various organic waste materials, reducing the demand for costly virgin resources and providing an environmentally friendly solution for waste management. PHA additionally possesses high biodegradability compared to other bioplastics on the market, allowing it to break down more efficiently in the environment and minimize long-term pollution. PHA can replace several families of petroleum-based plastics, making it a versatile option for a wide range of products and applications. However, despite these promising attributes, the high production cost of PHA remains a significant barrier to its widespread adoption. The expensive carbon sources utilized in microbial fermentation are the primary factor contributing to this cost, limiting the growth of the PHA market. There is a critical need to develop an efficient bioprocessing system that can produce high-quality PHA at a significantly lower cost. To address this imperative, the primary goal of this study was to create an integrated PHA production system that utilizes inexpensive cheese byproducts feedstock, making PHA production more economically viable while maximizing resource efficiency and minimizing waste. Whey and whey byproducts generated from cheese production offer a promising source for bioplastic production. Researchers estimate that approximately half of this whey produced from cheese making is processed into useful forms for human or animal consumption, with the rest ending up as waste material, contributing to high carbon emissions. While some cheese manufacturers can convert whey byproducts into valuable food ingredients, like whey protein concentrates and lactose powder, additional byproducts continue to be generated. Whey permeate is produced after concentrating the liquid whey with membrane filtration, containing most of the lactose and minerals. The whey permeate can be further processed into lactose powder with evaporation and drying systems creating byproduct delactosed permeate (DLP). Crystallization limitations prevent the complete recovery of lactose for food products. As a result, approximately one-third of the lactose remains as delactosed permeate (DLP), which is either sold as low-value animal feed or disposed of as waste. DLP is the main cheese byproduct without a widespread commercial application, which presents an opportunity for biomaterial production. Therefore, this study aimed to convert low-value cheese byproducts DLP into high-value bioplastic PHA. The PHA producer Haloferax mediterranei (H. mediterranei) appears to be a suitable candidate for utilizing cheese byproducts as feedstock. This fit is due to the archaea’s unique ability to thrive in extreme halophilic conditions, tolerating salinity levels of up to 20%. The presence of high salt-containing cheese byproducts provides a compatible source of nutrients for H. mediterranei, compared to other PHA producers and common contaminant organisms that can become inhibited from such high salt concentrations. The high salt environment removes the need for an expensive sterility unit operation, such as high-temperature exposure or antibiotics, reducing production costs. Moreover, the intracellular PHA extraction from H. mediterranei can be achieved through a straightforward process of water addition, causing osmotic shock and cell lysis. High salt-containing cheese byproducts provide Another advantage of H. mediterranei is its ability to produce a higher-value PHA polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). This PHA polyester exhibits enhanced properties compared to the more commonly produced polyhydroxybutyrate (PHB). The presence of 3-hydroxyvalerate units in PHBV enhances its flexibility, making it suitable for various applications, especially in the food industry. The potential applications of PHBV in food packaging, such as films, bags, containers, or cutlery, align with the circular economy principles. By utilizing cheese byproducts and producing high-value PHBV, the cheese industry can take significant steps toward sustainability and waste reduction. A pretreatment method of the DLP was developed and optimized utilizing centrifugation and enzymatic lactase hydrolysis. The centrifugation pretreatment was shown to increase lactase hydrolysis efficiency and H. mediterranei cell growth. The optimal lactase enzyme conditions with the centrifuged DLP to achieve a lactose hydrolysis efficiency of 92.5% were determined to be a loading of 0.025 g lactase/g lactose and shaking incubation at 40 ℃ for 12 hours. The effects of hydrolyzed DLP on H. mediterranei cell growth and PHA production were studied. The hydrolyzed DLP achieved high PHA yields of 0.33 ± 0.01 g VS/g total sugar that surpassed yields obtained with glucose feedstock. However, lower galactose consumption by H. mediterranei was observed to limit PHA production with this substrate. After demonstrating that hydrolyzed DLP could result in effective H. mediterranei growth and PHA production, nitrogen source optimization was conducted to devise a cost-effective feedstock for H. mediterranei growth on hydrolyzed DLP substrate. Three nitrogen sources were tested at equivalent C/N ratios of 8, yeast extract, ammonium chloride, and an equal nitrogen loading of yeast extract and ammonium chloride. Supplementation of ammonium chloride resulted in the highest PHA yield, reaching 0.34 ± 0.03 g VS/g total sugar. Furthermore, the media supplemented with ammonium chloride nitrogen source gave the lowest PHA production of the feedstocks investigated. These cost savings make ammonium chloride an economically favorable choice for PHA production compared to other nitrogen sources containing yeast extract. The results of these studies demonstrated that cheese byproduct DLP could be used as an effective feedstock for PHA production, helping to create a circular economy for the cheese industry. The hydrolyzed DLP supplemented with ammonium chloride nitrogen source was studied as feedstock for H. mediterranei cultivation with three feeding strategies; batch, fed-batch, and continuous feeding. With batch feeding, four different hydrolyzed DLP and whey permeate loadings of 10, 20, 30, and 40 g total sugar/l were studied. The loading of 10 g total sugar/l was determined to be the maximum loading of hydrolyzed DLP, as higher loadings resulted in considerable inhibition. However, hydrolyzed whey permeate resulted in a higher maximum loading of 30 g total sugar/l. This difference in maximum loading between the two hydrolyzed cheese byproduct feedstocks indicated that inhibitory constituents were present in DLP that were not in whey permeate. High-heat reaction products that form during whey permeate processing to lactose powder and DLP, such as Maillard products that are inhibitory to other microorganisms, could be the source of increased inhibition with DLP. The PHA yields of the hydrolyzed DLP and whey permeate were similar at 0.30 ± 0.03 and 0.27 ± 0.02 g VS/g total sugar, respectively. These yields were on the higher end of those reported for glucose feedstock of 0.07-0.33 g PHA/g sugar, demonstrating that hydrolyzed cheese byproducts are an effective substrate for PHA production. The maximum specific growth rates of H. mediterranei growth with hydrolyzed DLP and whey permeate were estimated with batch feeding and found to be similar for the two byproducts. These results can assist in developing large-scale PHA production facilities utilizing the cheese byproduct substrates. Based on the batch operation findings, a 6 L fed-batch bioreactor with an initial working volume of 3 L was operated to attempt to decrease substrate inhibition with the hydrolyzed DLP. Hydrolyzed DLP and whey permeate feedstocks were fed every four days at a loading of 10 g total sugar/l. A 50% increase in the final PHA concentration of the hydrolyzed DLP was achieved with the fed-batch operation and two feedings than with the single batch loading of 10 g total sugar/l. This increase in PHA production with fed-batch operation was due to the additional loading of 10 g total sugar/l that was able to be fed without inhibition occurring. However, the culture became inhibited and unstable past the third feeding of hydrolyzed DLP. For the whey permeate, the PHA concentration increased with the first three feedings, and a stable culture was maintained after three additional feedings. These results confirmed that increased inhibition occurs with hydrolyzed DLP compared to hydrolyzed whey permeate feedstock. The results indicated that fed-batch bioreactor operation with hydrolyzed DLP and hydrolyzed whey permeate feedstocks can improve PHA production compared to batch cultivation. Since fed-batch feeding only increased PHA production with hydrolyzed DLP marginally, a continuous stirred tank reactor (CSTR) with hydrolyzed DLP feedstock was studied to try to reduce substrate inhibition further and increase PHA production. The CSTR was operated at a hydraulic retention time of 10 days with an organic loading rate (OLR) of 2.5 g total sugar/l/day, and a hydraulic retention time of 20 days with various OLRs of 1.25-2 g total sugar/l/day. The 10-day HRT CSTR resulted in a PHA yield of 0.18 g VS/g total sugar, comparable to that achieved with the fed-batch bioreactors. However, the 10-day CSTR became unstable after 22 days of continuous feeding, potentially due to hydrolyzed DLP substrate inhibition. Although the 20-day HRT CSTR was able to maintain a stable culture for 130 days, considerably lower PHA yields were achieved of < 0.10 g VS/g total sugar. Therefore, continuous feeding at the applied HRTs and OLRs was not recommended for PHA production with hydrolyzed DLP. Lower galactose consumption was observed for the batch and fed-batch studies than for glucose. Therefore, a galactose acclimation study was conducted to determine if growing H. mediterranei on successive batches of galactose could increase consumption and PHA production with the sugar. The archaea was cultivated on galactose for five consecutive batches. The final cell dry mass (CDM) concentration of H. mediterranei significantly increased with three batches, the specific growth rate with four batches, the final PHA concentration with five batches, and the galactose consumption with three batches (total galactose consumption), demonstrating effective galactose acclimation had occurred. The research can potentially lead to improved PHA yields with galactose-containing feedstocks such as hydrolyzed cheese byproducts. Overall, the study demonstrated that cheese byproduct delactosed permeate could successfully be utilized as feedstock for PHA bioplastic production with H. mediterranei. The results of the study should be used to further scale the proposed process leading to commercialization.

California produces 2.55 billion pounds of almond kernels annually, and along with that 4.03 billion pounds of byproduct almond hulls are generated. Almond hulls have high sugar contents and are mainly used as livestock bedding and feed at present. The almond industry is seeking ways to reduce the environmental impact of conventional almond production by reducing dust generation and achieving zero waste. Off-ground harvesting is an emerging harvesting strategy that generates significantly less dust; however, off-ground harvesting produces a stream of wet hulls that can easily spoil if they are not processed properly. Wet almond hulls are an ideal feedstock for fermentation into high quality, probiotic-rich animal feed which can help the almond industry meet its zero-waste goal and increase revenue for almond farmers. Fermented animal feed is an alternative to the use of growth-promoting antibiotics in livestock and has already been recognized as an effective tool to improve gastrointestinal health and improve productivity in swine and poultry. It is possible that similar benefits can be observed in cattle fed fermented feed. Additionally, studies have shown that probiotic supplementation and alternative feeding strategies can reduce ruminant methane generation, the largest source of anthropogenic methane emissions in the US. The goal of this study is to assess the potential of using almond hulls as a feedstock for the production of a fermented cattle feed which can potentially reduce the environmental impact of the almond industry while concurrently increasing the value of almond hulls, improving cattle digestion, and reducing enteric methane emissions. In this study, California almond hulls of Nonpareil, Monterey, Independence, and Fritz varieties from a hulling facility were characterized for their chemical composition. The hulls had high sugar contents ranging from 31.8% to 42.2% by weight on a dry-basis (db) and phenolic compound content ranging from 3.4% to 7.6%, db. On- and off-ground harvested hulls of Independence, Monterey, and Fritz varieties were also characterized and compared. Off-ground harvested hulls had an average moisture content 3.5 times higher than on-ground harvested hulls of the same variety. Hulls were low in protein and fat at an average of 5.7% and 3.7% db, respectively and an average of 24.1% acid detergent fiber and 33.3% neutral detergent fiber. The hulls were characterized for the quantity of contaminants vs. hulls and on average, hull samples contained 88.9% hulls and 10.1% contaminants. Sieving and terminal velocity analysis were conducted to investigate methods for separating hulls from contaminants. The results showed that sieving separation was an effective method for separating hulls from contaminants. Solid-state, inoculated fermentation trials were conducted using Saccharomyces cerevisiae and Lactobacillus plantarum as inoculums. The effect of inoculum type, fermentation duration, inoculum amount, hull variety, particle size, and fermentation temperature were investigated through several fermentation experiments. It was found that fermentations using S. cerevisiae produced higher amounts of ethanol and acetic acid and caused an increase in pH, and fermentations using L. plantarum produced higher amounts of lactic acid and caused a drop in pH. Hull variety had a large impact on the characteristics of the fermented hulls under the conditions tested. 14-day fermentation durations produced similar results as 30-day fermentation durations in the conditions studied. Additionally, in-vitro digestion results indicated that almond hulls fermented with S. cerevisiae for 14 days reduced enteric CH4 production by 96% over 72 hours digestion at a 20% inclusion rate in a cattle diet. The nutritional composition of almond hulls, especially their high sugar content, makes them an ideal feedstock for the creation of fermented cattle feed. Almond hulls fermented with S. cerevisiae and L. Plantarum produce desirable characteristics for fermented feed including a pH below 4.5, high concentrations of lactic acid, and low concentrations of acetic acid. In-vitro digestion tests suggest that fermented feed is potentially an effective strategy to reduce enteric CH4 production.

Dairy industry is important for milk production and has substantial contribution to the California’s economy, however, dairy farms also produce manure, which impacts the environment. Dairy manure contains carbon, nitrogen and phosphorus, which are beneficial to soil. Manure also contains many bacteria including human pathogen, such as Salmonella and Escherichia coli (E. coli) which poses risk to public health. In this research, an intensive reconnaissance survey of manure stored in dairy lagoon was conducted to determine the prevalence of E. coli and Salmonella. In addition, an attempt was made to develop the ozone and air-based treatment method to reduce pathogen in liquid manure. First a comprehensive review on microbial pollution of manure, and various control method was performed (Chapter 1). Secondly, a field study was undertaken to collect dairy manure from 20 dairy farms to determine the prevalence of indicator E. coli, Shiga toxin producing E. coli (STEC), and E. coli O157:H7 (Chapter 2). Finally, a lab scale experiment was developed to determine the impact of ozone and air injection on E. coli inactivation in liquid manure (Chapter 3). The first chapter addresses the issues of microbial pathogen in manure and associated risk to human, animal and environment and impacts of various treatment methods on bacteria control. Previous studies showed that anaerobic digestion reduces E. coli and Salmonella under prolonged incubation and high temperature conditions, however, many anaerobic pathogens such as Clostridium survive anaerobic environment. In anaerobic incubation, E. coli level is reduced, but these bacteria are not eliminated completely. In the second chapter, several bacteria testing methods were used to determine the prevalence of E. coli, Salmonella, STEC and E. coli O157:H7 which are common pathogens in dairy manure. About 177 manure samples were collected from 20 dairy farms from primary and secondary lagoons to test the bacteria using agar culture-based methods, polymerase chain reaction (PCR) method for detection of E. coli and Salmonella. In addition, a real- time PCR based method was used to determine the presence of E coli O157:H7. Results showed that the prevalence of Salmonella in manure sample is lower than E. coli. The bacteria detection approach presented here could reduce the number of testing required during downstream confirmation process. The presence of Salmonella was found in 2.26 % of the samples, and both the culture-based and PCR methods yielded comparable outcomes in detecting Salmonella. Moreover, approximately 11.30% of the total samples out of the 177 were identified as positive for STEC by qPCR. None of the lagoon samples were positive to E coli O157:H7 by qPCR. The outcomes of this study hold substantial importance to determine the microbial quality of lagoon manure, and aid in the selection of appropriate methodologies for determining the prevalence of pathogenic organisms in dairy manure. In the third chapter, lab scale experiments were conducted to determine the impacts of ozone injection (Ozonation) and air injection (Aeration) in liquid dairy manure on E. coli reduction. Liquid manure was exposed to ozone and air for multiple durations (0 min, 60 min, 120 min, and 240 min) and E. coli levels were determined. Further, manure samples were analyzed for pH, salt content, potassium (K+), sodium (Na+), nitrate (NO3-), calcium (Ca2+), and electrical conductivity (EC, mS/cm). Results showed that Ozonation can be an effective treatment method to reduce E. coli levels in liquid manure. Further nitrate ions were reduced substantially by Ozonation. The analysis of variance (one way and two way) revealed significance difference in E. coli and nitrate reductions in liquid slurry among ozone and air treatments (p <0.05). The study's results carry significance importance in terms of developing a simple field-scale method for reducing microbial and nutrient pollutions from dairy lagoon manure.