In this paper, replacing complete or partly of growth mediums with secondarily wastewater was studied. Lipid content of Neochloris oleoabundans grown in a 0.3 X SE medium and autoclaved secondarily treated wastewater mixture was 22.27 % (w/w). The maximum biomass concentration of N. oleoabundans grown in wastewater with no additional nutrients was 0.636 g/L with 33% (w/w) glucose. Two culture lines, MA, and NA were isolated within our laboratory and could grow in secondarily treated wastewater with no additional nutrients. The maximum biomass concentration of MA in batch culture was 0.860 g/L and the sum of glucose and xylose was 40% (w/w). The maximum biomass concentration of NA was 1.562 g/l and the sum of glucose and xylose was 33.8% (w/w). The maximum specific growth rates of NA and MA were determined to be 0.0566 and 0.0337 per hour.

Algae extracts are used as cosmetic products and used as additives in specific foods because of their antioxidant activity. Reactive oxygen species (ROS) such as hydrogen peroxide and superoxide radicals are toxic on the skin and can be scavenged by antioxidants, which are abundant in microalgae. Antioxidant substances protect the skin against external factors such as ultraviolet light (UV). Many creams that are concerned with treating the skin have antioxidant molecules. Researchers are performing many studies to achieve natural and non-chemical skin maintenance for the skin. Conducting in vivo studies to analyze the antioxidant potential of extracts on skin needs ethical permission to recruit a specific number of people. In this study, pig ear skin was used with the help of a skin-membrane-covered oxygen electrode (SCOE) to analyze the antioxidant effect of an extract from the microalgae Chlamydomonas reinhardtii. A study proved that this method was effective in measuring other antioxidants. Hydroquinone was used as a control to see if the system worked correctly. Hydroquinone showed that it could penetrate the skin and give antioxidant activity. When the algae extract was used, the same effect as for hydroquinone could not be detected. With a 2,2-diphenylpicrylhydrazyl (DPPH) assay, the algae extract was analyzed for its antioxidant capacity, and the assay revealed a positive antioxidant effect of the extract. The spectrophotometric measurement of the amount of bioactive antioxidant molecules in the extract in different solutions suggested that the one resuspended in ethanol presented a higher amount of carotenoids and chlorophylls than the extract resuspended in an aqueous buffer. Additional research will be needed to characterize the antioxidant potential of the extract from the microalgae Chlamydomonas reinhardtii.

ABSTRACT IMPROVED MICROALGAL BIOMASS HARVESTING USING OPTIMIZED ENVIRONMENTAL CONDITIONS AND BACTERIAL BIOFLOCCULANTS DEREK CONTE MANHEIM The cost and energy balance of microalgae biofuel production is sensitive to the algae harvesting method, among many other factors. Bioflocculation and settling of suspended microalgae cultures is a harvesting method with potentially low cost and energy input. However, bioflocculation (the spontaneous flocculation of algal cells without chemical addition) has not been a reliable process with cultures grown in ponds. To provide insights to help improve algae settling, factors affecting the settling of algae were investigated in the laboratory using pure cultures of two common microalgae species: Scenedesmus sp. and Chlorella vulgaris. Bioflocculation of these algae was studied with and without the addition of bioflocculants produced by the bacterium, Burkholderia cepacia, to improve settling efficiencies. The bioflocculant produced by this bacterium was used in two different forms: a cell suspension including capsular and dissolved extracellular polymeric substance (EPS) components of B. cepacia, and dialyzed filtrate of the bacterial culture (only dissolved EPS fraction). The effects of algal growth phase, mixing time, bioflocculant dose, and environmental conditions such as pH and nutrient deprivation of bacterial bioflocculant cultures on settling of the algae species were studied. Settling characteristics were different for the two algae cultures, and their settling was affected differently by the many factors studied. Scenedesmus settling was best in later growth stages, while Chlorella settled much better in early growth phases. Addition of B. cepacia cells as a bioflocculant improved settling of Scenedesmus, with the greatest effect during mid to late exponential growth of the Scenedesmus. In contrast, addition of B. cepacia filtrate as a bioflocculant best improved Chlorella settling during stationary growth of Chlorella. Longer mixing times (contact time between the algae cells and bacterial bioflocculant) improved the settling of Scenedesmus, while Chlorella settled better with a shorter mixing time. Reducing the pH to 3 (a typical isoelectric point for microalgae) improved the settling of both algae cultures, with and without bioflocculant addition. Increasing the pH to 11 autoflocculated Scenedesmus cultures, but not Chlorella cultures, at early growth stages. EPS produced by the algae, bacteria, and wastewater organisms was quantified using dialysis separation followed by total organic carbon (TOC) analysis. Wastewater organisms were included because wastewater is a potential growth medium for biofuel algae. Improved settling of both species of algae depended on both the quantity and type of EPS (dissolved or capsular) produced by both the bacterial bioflocculant, and the algae themselves. Scenedesmus settled the best during late growth phases while its own EPS production was high, and combined EPS (capsular and dissolved) from B. cepacia improved settling at a higher dosage of bacterial cells to algae (1:2 B. cepacia cells to algae cells). Since Chlorella settling was not improved at later growth stages when its own EPS production was greatest, it appears that Chlorella’s settling rate was less affected by the production of its own EPS. For Chlorella, B. cepacia EPS addition (capsular and dissolved) was effective only in low doses (1:6 B. cepacia cells to algae cells). Settling results with the addition of bacterial bioflocculants with the pure algae cultures were compared to settling results of lab experiments with algae pondwater sampled from high-rate algae ponds (HRAPs). These algae samples were used to test the addition of return activated sludge (RAS) to improve settling. RAS addition improved the settling of Chlorella, which was the dominant algae species in the HRAP during the time of this study, at two different doses (a ratio of RAS to algae pond water of 1:3 and 1:6). Nutrient deprivation of B. cepacia cells before use as a bioflucculant was found to improve settling for Scenedesmus, especially during early phases of growth when EPS production of Scenedesmus was low. The EPS produced by the starved bacterial cells was about 30% greater than that produced by cultures which were not nutrient-limited. For the bacterial cultures, EPS production peaked at mid stationary phase for non-starved cultures and during early stationary phase for starved cultures. Chlorella settling improved in early growth with starved bacterial cell addition and in later growth with non-starved bacterial cell addition. These results suggest that the settling of microalgae can vary dramatically by species and that the settling of different species is affected differently by growth phase and environmental conditions. In addition, species of algae respond differently to addition of bacterial bioflocculants. Given the dramatically different settling behavior of the two species of algae used in this research, more research should be directed to studying settling of other microalgal species. Based on this research, the use of bacterial bioflocculants is promising for improving algae settling and may contribute to the development of a reliable, low cost harvesting process for commercial biofuel production from microalgae.

This research sought to optimize anaerobic co-digestion of microalgae biomass harvested from a wastewater treatment pond facility with locally-available wastes. The goal was to produce high methane yields and stable digestion without the need for supplemental alkalinity addition. A key research question was if algae digestion could be improved via the synergistic effects of co-digestion. Cell disruption to increase digestibility was not pursued due to its relatively high mechanical complexity and high energy use. For the wastewater treatment ponds studied, the most practical co-substrates identified were municipal wastewater sludge and food waste (sorted organic municipal waste). Although wastewater sludge does not have a particularly high carbon:nitrogen (C:N) ratio, it readily and stably digests and is available in large quantities at wastewater treatment plants. This research investigated the methane productivity of algae co-digestion with municipal wastewater sludge and food waste in semi-continuous bench-scale anaerobic digesters at 37.5˚C. Digesters fed pure algae biomass loaded at a rate of 4 g Volatile Solids (VS)/L-day with a 20-day residence time exhibited stable digestion and yielded an average of 0.23 L CH4/g VS Introduced. For digesters that contained algae biomass in the feed, the greatest methane yield of 0.40 mL CH4/g VSin was observed in a digester containing 50% algae co-digested with both sorted organic municipal waste (40%), and municipal wastewater sludge (10%) at a loading rate of 2 g VS/L-day with a 20-day residence time. While adding co-substrates increased yields in all digesters, prevention of ammonia toxicity did not appear to be the mechanism. Instead, the co-substrates simply increased the concentration of readily-digestible organic carbon, leading to increased methane yields and productivities. For algae biomass, total ammonia nitrogen concentrations of 3370 mg/L did not appear to inhibit methane yield. Digesters with the same feed contents and residence time loaded at 2 and 4 g VS/L-d had similar yields but total ammonia nitrogen concentrations of 1740 and 3370 mg/L respectively. From the data from these laboratory studies, descriptive models were developed for ammonia nitrogen, alkalinity, volatile fatty acids, yield, biogas quality, and volatile solids destruction. The variables from the descriptive models with p-values above 0.05 were then used to create a compact model.

The push for alternatives to petroleum fuels has forced researchers to look for highly productive, renewable, non-food resources. The advantages of using microalgae instead of traditional oil crops for biofuel production include high oil yields, rapid reproductive rates, and versatile growing requirements. In order to reduce the cost of producing microalgae based biofuels, wastewater has been used as a nutrient source instead of specialized plant nutrients. The purpose of this study was to compare the relative effectiveness of different combinations of microalgae strain and dairy wastewater for increasing biomass. The methods for monitoring growth included optical density, cell counting, biomass estimation by chlorophyll-a, and volatile suspended solids. The analyses compared four concentrations of wastewater media as well as four strain treatments: Chlorella vulgaris, Tetraselmis sp., mixed freshwater culture and mixed saltwater culture. Optimum wastewater concentrations for microalgae growth were approximately 0% and 25% for most strain treatments. The results of the wastewater treatments concluded that dairy wastewater could serve as an effective nutrient substitute for plant food at concentrations approximately 25%. Chlorella vulgaris and Tetraselmis sp. prevailed over the mixed cultures for biomass production. Nitrate was the most limiting nutrient and exhibited the greatest reductions, sometimes in excess of 90%. The regression equations derived from the volatile suspended solids data achieved high R2 values and determined that total nitrogen, ammonium, and nitrate were significant in the model. In those equations, increasing either ammonium or nitrate yielded an increase in volatile suspended solids. With regards to comparing biomass quantification methods, the two most useful and reliable biomass quantification methods were optical density and volatile suspended solids.

Research has shown Spirulina to be a viable feedstuff in poultry production. Other species of microalgae are being studied to determine their effects on poultry production. This project compared four microalgaes: Hydrodictyon, Vaucheria, Uronema, and Spirulina and was broken down into three trials. The first experiment investigated the digestibility of an algae species, Spirulina, in broiler chicken diets. Eighty, 25-day-old Ross 708 broiler chicks were sorted into 20 metabolism cages and received one of two diets: Control Diet or Test Diet (75% basal diet + 25% Spirulina). The apparent ileal amino acid digestibility (AIAAD) of lysine, methionine, cysteine, threonine, isoleucine, valine and arginine in Spirulina was 94.5%, 91.3%, 56.1%, 71.4%, 76.8%, 69.8% and 90.4% respectively. Apparent metabolizable energy (AME) of Spirulina was 2279 kcal/kg. The second experiment focused on digestibility of Spirulina and novel algae species in laying hens. Sixty, 80-week-old Lohman LSL-Lite laying hens were randomly assigned to Control Diet, Hydrodictyon Test Diet (87.5% basal diet + 12.5% hydrodictyon), 12.5% or one of three other test diets (75% basal diet + 25% Spirulina, Uronema, and Vaucheria). Spirulina diets contained the highest crude protein (25.5%) and methionine levels (0.68%). Uronema had the highest gross energy content of 3880.5 kcal/kg and lysine levels (1.12%). Hydrodictyon diet was the most nutritionally similar to the control. Crude protein was at 15.3% and 16.6% for control and Hydrodictyon respectively. Gross energy was 3429.6 kcal/g in control versus 3316.1 kcal/kg in Hydrodictyon. Lastly, crude fat was 2.7% compared to Hydrodictyon at 2.4%. The last trial was to determine the effects of the addition of microalgae in the diet on laying hen production. Seventy-eight, 23-month-old laying hens were assigned to 1 of 3 of the following experimental diets: control, control + 1% microalgae, or control + 2% microalgae. Microalgae, regardless of inclusion level, had no significant effect on egg weight in this study. Increasing level of inclusion of Spirulina, Hydrodictyon, and Uronema from 0 to 2 % significantly increased yolk color. Vaucheria influenced yolk color at the 2% level. No significance differences shown from any of the algae species regardless of inclusion level for egg weight, egg mass, shell thickness, shell strength, shell weight, albumen weight, yolk weight, and albumen height. In addition, there was no significant difference in feed conversion ratio, feed intake, body weight, or tibial strength between the treatments. However, as these are novel algaes, further studies are required to determine the true usability in poultry production.

This thesis discusses the mechanisms associated with the removal of organic matter, nitrogen and phosphorus in wastewater-fed high rate algae ponds (HRAP) designed to operate as triplicates. Research was conducted at the San Luis Obispo Water Reclamation Facility (SLOWRF) as a pilot-scale study of nine 30-square meter ponds one foot in depth. During period of study, triplicates were operated at hydraulic retention times (HRT) of 4, 3 or 2-days. Main objectives for the study were to determine minimum HRTs required to achieve secondary and tertiary treatment. Experimental conditions such as CO2 supplementation, nighttime aeration and operation of ponds in series were employed to evaluate optimal conditions for efficient nutrient removal. Ponds were continuously fed primary effluent with the following water quality characteristics: 5-day total biochemical oxygen demand (TBOD5) of 124mg/L, 5-day soluble carbonaceous biochemical oxygen demand of 67mg/L (scBOD5), total suspended solids (TSS) of 66mg/L, volatile suspended solids (VSS) of 65mg/L, total ammonia nitrogen (TAN) of 34mg/L-N, oxidized nitrogen of 1.1mg/L-N, total K̇jeldahl nitrogen (TKN) of 42mg/L-N and dissolved reactive phosphorus (DRP) of 3.3mg/L-P. Nutrient removal efficiencies were compared between summer months (April – October) and winter months (November – February). Average pond temperatures during summer and winter were 20.4 °C and 14.9 °C, respectively. Average TAN removal efficiencies of 2-day HRT ponds ranged from 62% in winter to 78% in summer. Operation of ponds at an increased 3-day HRTs resulted in corresponding seasonal increases of TAN removal by 14% and 12%. In 4-day HRT ponds operating in series after a 3-day HRT set, TAN removal efficiency was 98% in winter and 99% in summer. Aeration increased nitrification and nitrate concentrations in 2-day HRT ponds to10mg/L-N ± 4.4mg/L-N. DRP concentrations and BOD removal efficiencies within replicate ponds were similar throughout seasonality. DRP was 1.2mg/L-P ± 0.66mg/L-P at a 4-day HRT operating in series, 2.2mg/L-P ± 0.57mg/L-P at a 3-day HRT and 2.6mg/L-P ± 0.58mg/L-P at a 2-day HRT. Aeration had no measureable effect on BOD removal. BOD removal efficiency was 97% at a 4-day HRT in series with a 3-day HRT and 95% at 3-day and 2-day HRTs.

Quarrying of limestone on the Swedish island of Gotland dates all the way back to the 5th century but saw the rise of its modern form during the early 20th century, with the establishment of Cementa AB at Slite, which since then has developed as an industrial town. Cementa has been progressively mining limestone in three large open pit quarries in Slite. Their factory accounts for approximately three quarters of Sweden’s cement production and is considered to be a vital part of the construction industry. However, the ecological and social damages caused by the extractive procedures far exceed the economic incentive and has triggered numerous debates on whether they should be allowed to continue production.   This project henceforth envisions an alternate post-extraction future for Slite’s mine-scape, where the production of cement will shift from extracting limestone to growing limestone using calcareous microalgae. The leftover quarries will be regenerated, the factory will be repurposed, and the contextual industrial land will be developed, improved, and enhanced for the benefit of the surrounding community and visitors. By shifting to a net-zero carbon method of producing cement, alternate industries will develop in place which will also resolve Slite’s socio-economic dependency on limestone and diversify its mono-cultural economy. A 30-year protocol is planned to transform the urban-industrial fabric of Slite into a microalgae farming field and extreme sports destination - creating an anthroposcenic garden in which production, everyday life and leisure are meant to be compatible. While the quarries will undergo a natural rewilding process, this proposal does not intend to artificially restore the quarries entirely to their original landscapes but rather acknowledge our anthropogenic actions as irreversible and consequential; and hence engage with this damaged landscape to find new uses for it.

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