Multipurpose Use of Microalgae to Treat Municipal Wastewater and Produce Biofuels

Chapter 16

Multipurpose Use of Microalgae to Treat Municipal Wastewater and Produce Biofuels Jason L. Selwitz1, Hossein Ahmadzadeh2, Stephen Lyon3 and Majid Hosseini4 1

Department of Energy Systems Technology, Faculty of Workforce Education, Walla Walla Community College, Walla Walla, WA, United States, 2Department of Chemistry, Ferdowsi University of Mashhad, Mashhad, Iran, 3Alga Xperts, LLC, Madison, WI, United States, 4 Manufacturing and Industrial Engineering Department, The University of Texas Rio Grande Valley, Edinburg, TX, United States

16.1 INTRODUCTION Fundamental to regenerative design is the principle of looking to nature to help inform process and product development. To produce a regenerative system, we go beyond mere sustainability and contribute to the restoration and rejuvenation of ecological systems while concurrently integrating the needs of human communities [1]. For instance, freshwater microalgae produce oxygen which stimulates the growth of bacteria. In turn, bacteria of the genus Nitrosomonas and Nitrobacter improve water quality by performing nitrification, the conversion of ammonium to nitrite and nitrate, which turns nitrogen into a form readily available for use by plants [2]. With knowledge of these interactions as a guide, scientists and engineers have researched the effectiveness of using various microalgae species to improve wastewater quality to the extent the resulting effluent can be used for irrigation and/or aquifer recharge. Microalgae are a source of fatty acids (oils) that can potentially replace current levels of petroleum diesel use for transportation fuels [3 5]. Certain types of algae produce the equivalent of vegetable oil within their biomass. Scientists speculate that algae generate oil to regulate their position in the water column, depending on light intensity, to maximize photosynthesis. In contrast to petroleum, algae oils have the potential to be grown locally and, when burned, produce cleaner emissions. Algae are attractive due to high Advanced Bioprocessing for Alternative Fuels, Biobased Chemicals, and Bioproducts. DOI: https://doi.org/10.1016/B978-0-12-817941-3.00016-4 © 2019 Elsevier Inc. All rights reserved.

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reported yields per acre, potential for regular daily harvests and extraction of oil, and the concept of developing algae production facilities on nonarable land next to free sources of carbon dioxide (CO2) such as power plants [5 9]. Renewable fuels from algae grown to treat wastewater could achieve greater than 80% emission reductions compared to petroleum diesel [10]. As of 2007, oil recovered from microalgae produced in low-cost photobioreactors was estimated to cost $10.60 per gallon, whereas palm oil (the cheapest oil to produce) was sold for $1.97 per gallon in 2006 [11]. Assuming that algae oil has about 80% of the energy content of crude petroleum, for algae oil to be able to displace crude oil, the selling price of algae oil should not exceed $1.55 per gallon if the prevailing price of crude oil is $60 per barrel [11]. As of 2012, the reported production costs for microalgae oils were $7 per gallon [10]. To bring costs down, refinement of harvesting and extraction techniques and genetic engineering should be explored in addition to using the algae biomass to produce power to run wastewater treatment plant operations [12,13]. Based on life cycle assessments, microalgae-based renewable fuels will not be plentiful or cheap in the near future, despite steady yields of algae oil per hour. This analysis is based on land, water, and CO2 costs, the capital costs and operational costs of operating an algae wastewater biorefinery, ideal sunlight (i.e., number of annual average daily solar irradiation hours), and temperature considerations [10]. Although wastewater is attractive as a nutrient source for algae because of high levels of phosphates and nitrates, algae cannot grow off of wastewater alone and need additional CO2 which can be sourced from the biogas produced from a wastewater treatment plant’s anaerobic digester. This approach could save power and reduce capital costs of wastewater treatment if high rate algae ponds are used with added CO2 to perform secondary and tertiary stage wastewater treatment [12,14]. A study using Chlorella sp. determined that when growing algae on wastewater for biofuel production, the ammonium concentrations of the wastewater influent could be a limiting factor in algae biomass and lipid production [15]. Several public private partnerships have sought to further research and commercialize the treatment of wastewater with algae coupled to production of renewable energy from the resulting biomass [12,16]. In January 2012, the City of San Luis Obispo (California, USA) commissioned a pilot algae wastewater treatment facility with nine raceways each with 30 m2 of area at depths of 0.3 m. The system was expected to treat 20 27 m3 per day (5283 7132 gal/day) and the research was to be conducted to measure the efficiency of removing nitrogen and phosphorus from wastewater [17]. In a study involving the treatment of municipal wastewater and diluted dairy wastewater, CO2 was added to accelerate growth of a mixed species of algae. Lipid production in the resulting biomass ranged from 4.9% to 29% and the highest productivity was 2.8 g/m2/day, which was higher than reported yields for oilseed crops [18].

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The original research referenced in this chapter investigated whether certain strains of algae could improve the quality of preliminary treated human wastewater while generating biomass-containing biodiesel quality oil. The ability of the algae to treat human wastewater which already had the bulk of its floatable and heavy solids removed (i.e., preliminary treated) was examined. After the 5-day residence time for the algae in the wastewater, algae lipid yields were determined using gas chromatography mass spectrometry (GC/MS).

16.2 TYPICAL WASTEWATER TREATMENT Wastewater is any water that has come in contact with humans or animals through domestic, commercial, industrial, and/or agricultural use. Human wastewater contains feces, urine, and/or other bodily fluids. Sources of wastewater, or “blackwater,” include flush toilets, kitchen sinks, and/or a concentrated animal feeding operation. In contrast, “graywater” is generally deemed to be wastewater from a bathroom sink, shower, and/or washing machine. Domestic human wastewater is 99.9% liquid and 0.1% solids, of which the solids comprise 70% organic material (fats, carbohydrates, and proteins) and 30% inorganic particles such as grit, salts, and metals [19]. Solids are traditionally separated into two distinctions: the “screenable solids” (paper, sanitary napkins, condoms, and fish) and the “nonscreenable solids” which are suspended and create the cloudy appearance (bacteria, fecal particles, food particles, fats, oils and greases, detergents and soaps, and sediment). The dissolved materials give color, but not cloudiness, to the liquid fraction and include organic matter (proteins, urine, carbohydrates, and fatty acids), ions of ammonia, chloride, nitrogen, phosphate, sulfur, oxygen, minerals, metals, and trace elements [20]. The actual treatment of wastewater in a typical system involves four steps. “Preliminary treatment” removes large, floatable, and heavy solids by means of physical screening and settling. The “primary” stage slows the flow of effluent in a chamber to allow gravity time to pull organic solids out of suspension. During this stage, fats, oils, and greases rise to the top due to buoyancy and are skimmed away. Biochemical oxygen demand (BOD) and total suspended solids (TSS) are lowered by 30% 50%. After primary treatment, wastewaters are nutrient rich containing a diverse mixture of microorganisms and organic nutrients. “Secondary,” or biological treatment, involves the infusion of oxygenrich air bubbled into the treatment basin. The steady flow of air feeds aerobic bacteria which breaks down organic matter. The material in the water settles out to form a sludge layer and is harvested and used to inoculate the bacteria that can work to breakdown the settling solids. Nitrification occurs as the aerobic bacteria convert NH3 (ammonia) to NO32 (nitrate) in the later stages

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of secondary. Some treatment plants discharge “enhanced” wastewater flows following only a secondary level of treatment. The US Environmental Protection Agency’s (EPA’s) wastewater discharge limits are 30 mg/L for TSS, 50% reduction of BOD, and 80% reduction in ammonia [21]. “Tertiary” is a combination of treatments that can be coupled to secondary treatment and work to remove the remaining BOD, TSS, nitrate, ammonia, and phosphorus; strip the water of pathogens; and remove salts before final discharge. This can be accomplished through treatment with chlorine, ultraviolet light (to remove pathogens), constructed wetlands, reverse osmosis (to remove salts), sand filtration (to eliminate suspended solids), and activated charcoal [20,22].

16.3 MUTUALISM In algae-based wastewater treatment systems, water quality improvement occurs through symbiosis between the algae and bacteria (mutualism). As microalgae release dissolved oxygen (DO), bacteria grow to break down organic matter and complex nutrients into forms utilized by the algae to generate biomass [23]. This mutualism is seen in plant species where mycorrhizae (fungi) take needed sugars (carbohydrates) from the plants and in return collect essential minerals, such as phosphorus, for plant’s uptake. Mutualism also occurs when rhizobia (bacteria), living on plant roots, fix needed nitrogen from the atmosphere for plant uptake (e.g., legumes) and as compensation are provided sustaining carbohydrates [2]. Mutualism also occurs quite effectively in constructed wetlands as oxygen is released via root hairs to enrich bacteria that breakdown nutrients found in surface waters. As a result of this relationship, plants can uptake needed nutrients and water quality is improved. Microalgae have been found to adequately perform water treatment by working with bacteria to transform nutrients into a salvageable biomass [24].

16.4 WASTEWATER TREATMENT WITH ALGAE The overabundance of nutrients in water from animal and human waste streams occurs throughout the world. As these eutrophication events unfold, there remains a definite need to apply ingenuity to mitigation efforts. Examples from the tropics and subtropics have demonstrated that algae can be used successfully to improve the quality of waterways surrounding pig and fish farms [25]. Within the field of phycology, research efforts using algae to perform wastewater treatment began in earnest more than 60 years ago with William J. Oswald’s efforts at the University of California, Berkeley [26]. Oswald’s research culminated with the development of advanced integrated wastewater pond systems (AIWPS). Many researchers have confirmed that algae do hold the ability to cost effectively treat a

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variety of wastewaters. However, the freshwater microalgae have been for the most part unicellular phytoplankton and filamentous cyanobacteria [22,24,25,27,28]. Effective nutrient removal from wastewater is defined by determining the optimal conditions for algae growth and productivity based on the climate and season, where the specific system is operated. It has been noted that light intensity plays a larger role in algae productivity than temperature [29]. Cost-effective harvesting of algae biomass is one of the determining factors of economic viability, and the use of select long strain algae monocultures rather than local small cell strains is advocated. Multicultures of Arthrospira platensis with Rhodobacter sphaeroides and Chlorella sorokiniana may be effective in removing all pollutants [29]. It has been recommended that research be directed toward technical and economic assessments that establish the viability of using specific algae strains within certain environs. Along these lines, important research should continue to look at isolating individual strains of algae to enhance their treatment effectiveness [30]. Using algae to improve the quality of industrial wastewaters, such as from pulp and paper mills, has been accomplished and holds potential for further study [31]. In total, the research emphasizes that algae-based wastewater treatment systems should be cost effective, remove nutrients efficiently, have low-cost harvesting strategies, and use algae strains with a biomass that can serve purposes in addition to wastewater treatment [29]. Green et al. [22] detailed an AIWPS that uses four integrated ponds to mimic the conventional wastewater treatment system. Primary treatment and removal of floatables was achieved via an “advanced facultative pond” (AFP). Well-designed AFPs remove 60% 80% BOD and nearly all TSS. The second pond in the AIWPS series is a shallow, elongated oval shaped “high rate ponds” (HRPs), one of the most widely used and effective designs for outdoor algae cultures [32]. Here, algae work to improve water quality in a relatively short retention time. In HRPs, through the use of a simple paddlewheel (15 20 cm/s) in a shallow, oblong tank, water and algae are circulated around a central dividing line [21,22,33]. The appearance of an HRP is much like that of a raceway. Water treatment in HRPs occurs primarily through mutualism as microalgae release the DO required by bacteria to break down organic matter nutrients into forms utilized by the algae to generate biomass [23]. HRPs are able to provide levels comparable or exceeding secondary treatment (aeration/biological treatment phase) in conventional plants for TSS, BOD, nitrogen, phosphorus, and coliform bacteria [20,34]. In HRPs, competition from locally occurring microalgae strains and the establishment of grazers (such as zooplankton) are likely to be the factors for the growth of the cultivated strains. Under optimal conditions, 34 90 kg of algae biomass (ash free dry weight) can be generated. As algae have an influence over pH during sunlight hours, pH can reach levels approaching

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9.2. This alkaline level is sufficient to disinfect the water of harmful bacteria and other pathogens [22]. Effluent discharged from an AIWPS may be used for irrigation on agricultural lands and/or landscaping [22]. Constructed wetlands, followed by treatment with ultraviolet light, may serve as residence time decreasing substitutes the additional ponds in the AIWPS system. Using algae to treat wastewater in a HRP system compares favorably to conventional wastewater treatment systems (Table 16.1). Two strains of freshwater green algae [Chlorella protothecoides (Cp) and Botryococcus braunii (Bb)] and a locally grown mixed culture (consisting of alga from the genera: Gomphosphaeria, Hildenbrandia, Palmella, and Aphanotheca) were studied at the John T. Lyle Center for Regenerative Studies at the California State Polytechnic University, Pomona. The Cp, Bb, and the local mix of “pond 6” (P6) algae were each discretely assigned a circular vessel to treat 1 L of primary treated wastewater over a 5-day period. After the residence time, the water quality of each vessel was tested and compared with the quality of the original preliminary treated wastewater sample. In addition, after separating the algae from the wastewater posttreatment, an analysis of oil content and quality was conducted using GC/MS. Related Chlorella sp., strains have been used in water treatment and the production of nutritional supplements. The C. protothecoides strain has been TABLE 16.1 Comparison Between Conventional and Advanced Integrated Wastewater Pond Systems (AIWPS) Wastewater Treatment Systems Conventional Wastewater Treatment G G

G

G

G

Cleans water thoroughly Removes burden of waste treatment from individuals and communities Costs of aeration and disposing of accumulated sludge deposits High energy consumption 5 fossil fuel emissions 5 relates to air quality (uses 1 kW h per 1 kg sludge produced) Centralized—highly mechanized; operated by technicians

Wastewater Treatment With AIWPS G

G

G

G G

G

G

G

Primary (AFP) removes solids and produces methane for electricity generation Secondary (HRP) removes nutrients and produces usable algal biomass Tertiary removes algae and pathogens Smaller footprint required Energy demand for paddlewheels and harvesting (uses 10 kW h per 1 kg algae produced) Reduced emissions 5 improved air quality Can be built, operated, and maintained by local people on farms Up to 30-day residence times

Conventional wastewater treatment uses more energy and is more expensive than an AIWPS which has increased residence times. AFP, Advanced facultative pond; HRP, high rate pond.

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harvested to make biodiesel [33,35]. B. braunii has been shown to improve quality of diluted secondary treated wastewaters and its biomass can contain lipids in upwards of 20% 60% [23,36,37]. While plants convert about 1% of the solar energy that strikes their leaves, microalgae have been found to convert solar energy at a rate of 2% 3% because they have a higher portion of biomass devoted to photosynthesis (i.e., no roots, leaves, or woody material) [21,38]. One result of microalgae biomass development is the production of lipids (i.e., oil or fatty acids) [3,8,21]. Although not definitive, some researchers speculate that algae oil production is related to buoyancy. Certain strains may produce oil to control their position in the water column relative to photo-intensity to optimize photosynthesis [23,39,40]. Furthermore, the research points to limiting nitrogen quantity in growth media as a factor in high oil content [21]. This relates directly to the need to harvest algae from their water source at a certain point during low nitrogen periods before they lose affinity to form colonies. Once colony separation occurs, any potential beneficial use of algae biomass and oil is greatly negated by the difficulty of harvest. Since several strains are effective factories of solar energy and oil production, further research has been suggested on using microalgae to produce renewable energy, animal feed, and fertilizer [21,23,34]. In order to model nature, regenerative algae systems should clean polluted water sources and provide useful resources, such as energy, to produce income streams and reduce emissions from petroleum.

16.5 BIODIESEL FROM ALGAE Through the burning of fossil fuels, in just a few centuries, quantities of CO2 have been released which comparatively took hundreds of millions of years to be bound up [21]. As petroleum reserves diminish, the effects of climate change increase. As a result, developing supplies of clean, renewable energy has become increasingly important [41]. Biodiesel is a highly biodegradable, nontoxic, nonpetroleum-based fuel that has been commercially used in Europe since the late 1980s. Throughout many countries, diesel engines can be found in ships, trains, semitrailer trucks, heavy construction equipment, small fishing vessels, rice harvesting equipment, and generators. In order to improve the emissions of diesel engines, without causing engine replacement or major modification, biodiesel is applicable [42]. At present, the most common source of biodiesel is canola, or rapeseed, at about 84%, producing about 125 gal/acre/year [43]. Soybeans yield roughly 45 gal/acre/year [43]. The US National Renewable Energy Lab (NREL) estimates that biodiesel can be produced from microalgae at a rate of approximately 3500 gal/acre/year which is 28 times higher than canola [43]. While providing partial reductions in greenhouse gas emissions, biodiesel fuel may serve as an intermediary to bridge the existing gap between

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current diesel engine technology and a possible zero emission future. Furthermore, biodiesel produced from the algae used to treat wastewater could offer a palliative solution to fuel needs in the transportation sector. To date, studies have shown that compared to conventional diesel engines, fuels containing 100% biodiesel (i.e., B100) can reduce emissions as follows: 70% for particulate matter, 50% for carbon monoxide, 40% for total hydrocarbons, and 100% for sulfur [42]. As for the major smog component nitrogen oxide (NOx) emissions, a number of studies on diesel engines, with adjusted engine timing, using soy based B20 (20% biodiesel) show reduced net NOx emissions compared to 1% 12% increases that had previously been reported [44]. The prospects for biodiesel become economically favorable as petroleum prices rise and incentives, such as tax shifting and subsidies, are adopted. Three main considerations exist with the production of biodiesel produced from nonpetroleum-based oils: increasing land costs, the prevailing priority to maximize the extent to which agricultural lands are utilized to grow food not fuel, and the reported net energy loss associated with vegetable-based oil sources [45]. After decades of studies, the NREL’s “aquatic species program” recommended research into the use of microalgae to not only treat wastewater, but to concurrently produce biodiesel [21]. From 1978 to 1996, research was done to develop renewable transportation fuels from algae. Algae were gathered from all over the world and it was discovered that nutrient deficiency (i.e., limiting nitrogen) is often a lipid production trigger. Locally occurring algae strains in open ponds produced high levels of oil with nitrogen and silica depletion techniques, although the average production was limited to 10 g/m2/day due to cold nighttime desert temperatures and nutrient disturbances from birds. The highest utilization of delivered CO2 by algae occurred at the Roswell, New Mexico (USA) study site where more often than not, a greater amount of delivered CO2 was lost to the atmosphere [21]. Overall, algae are highly efficient compared to higher plants at taking CO2 and converting it into oil. Natural algae oils are too thick for diesel engines designed to operate on cheap petroleum diesel and thus the process of transesterification (converting triglycerides to less viscous alkyl esters) is the norm to lower viscosity. The issues of high land costs and the need to ensure food production were raised in the final NREL report. Overall, algae are deemed to be efficient converters of sunlight to energy because of their simple cellular structure (no roots, leaves, woody material). In addition, their heightened efficiency is due to their ability to thrive in water where they have direct access to nutrients needed for photosynthesis. The solar energy conversion efficiency of many algae was determined to be 2% or a rate of 15 g/m2/day [21]. Some species were found to be capable of producing 30 times the amount of oil per unit area of land compared to agricultural crops. In the future, if algae biodiesel is able to achieve production on a commercial scale, the fact that the oils can be harvested from a vastly

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smaller area than that of crops such as soybeans or canola will greatly contribute to its importance as a petroleum diesel substitute. Furthermore, the proposal that algae biodiesel production can be wedded to wastewater treatment is appealing because productive domestic agriculture land will not have to be sacrificed in the name of fuel production from crops. As early as 1980, the aquatic species program (ASP) moved away from researching the dual utility of algae to treat wastewater and produce biodiesel, to the dedication of producing algae with high oil yields. However, the dual use of algae for wastewater treatment and oil production was a central recommendation of the report [21]. By 2006, there were a limited number of scientific works on the subject of biodiesel production from the alga C. protothecoides. For example, one article detailed a scientific experiment where C. protothecoides strains were grown heterotrophically, to consume nutrients from media rather than photosynthesis, to create increased biomass with a maximum of 60% oil content [35]. Algae oils were extracted using hexane. The algae oils were then turned into biodiesel using a sulfuric acid catalyst for the process of transesterification.

16.6 USING ALGAE TO TREAT WASTEWATER AND PRODUCE RENEWABLE FUELS Using microalgae to treat primary wastewater is comparable to traditional secondary treatments but may require longer residence times [28]. However, new systems are being developed to significantly reduce treatment times [9,34]. Costs of treatment are reduced when biomass of the microalgae is used to make feedstock or mechanically digested to produce electricity [32,34]. Algae water treatment processes have produced significant decreases in nitrate, phosphate, and bacteria contents of wastewater [28,32,34]. Having reclaimed water available for irrigation and safe discharge into rivers or aquifers reduces overall energy costs, mitigates water pollution, and improves human health [34]. While on the one hand, there is a body of scientific literature using microalgae to treat wastewater, on the other are studies using algae to produce biodiesel. However, there are little research completed combining both processes. The potential cost savings, lowered emissions, reduced land footprint, attempt to meet multiple needs, and use of already in place infrastructures have made an algae-based wastewater treatment biodiesel production system very appealing.

16.7 REGENERATIVE RESEARCH AT CALIFORNIA STATE POLYTECHNIC UNIVERSITY, POMONA Bench scale research and analysis was conducted focusing on water quality improvements performed by selected strains of microalgae followed by a

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thorough characterization of the oils yielded. Within the applied research, two freshwater microalgae species (Cp and Bb) and a mixed culture of locally occurring microalgae (P6) were studied. Experiments measured the ability of the algae to treat human wastewater which already had the bulk of its floatable and heavy solids removed (i.e., preliminary treated). After the 5-day residence time for the algae in the wastewater, algae lipid yields were determined using GC/MS. Previous phycology studies had recommended completing bench scale tests on algae strains that can offer the potential to both treat wastewater and produce a harvestable biomass for energy generation (e.g., biodiesel). The microalgae were inoculated into 1 L volume of nutrient rich, “preliminary treated” effluent with low levels of DO and high BOD. The primary level was obtained from the Inland Empire Utility Agency’s regional plant 5 (Chino, CA, USA). On days 1 and 5 of each experiment, the quality of the water was tested for DO, pH, turbidity, water temperature, ammonia, nitrite, nitrate, phosphorus, BOD, and TSS (Table 16.2). The results were compared TABLE 16.2 Control Variables Remained Unchanged From Week to Week and Provided Consistency to the Study Control variables (not altered)

G

G G G

Used 1 L quantities of primary treated human wastewater from the IEUA Air/CO2 flow mix 5 103 155 Torr 5-day residence times Light 5 16 h ON, 8 h OFF

Independent variables (adjusted from week to week) G

G

G

Quality of algae used, i.e., unable to keep Chlorella sp. cultures axenic: 1. Cp 2. Bb 3. Local P6 mix Quantity of algae inoculant increased each week Larger suspended solids of the primary were removed via settling and/or separation with coffee filters before weekly testing

Dependent variables (affected by fluctuations in the independent variables) G G G G G G G G G G G G

TSS Turbidity pH Temperature DO BOD5 Ammonia Nitrite Nitrate Phosphorus Fatty acid (lipid) yields: Palmitic, stearic, oleic, linoleic, and linolenic

Outcomes for the dependent variables related to changes in the quantity and quality of the algae tested and the variability of the preliminary treated human wastewater used. IEUA, Inland empire utility agency; TSS, total suspended solids; DO, dissolved oxygen; BOD, biochemical oxygen demand; P6, pond 6; Bb, Botryococcus braunii; Cp, Chlorella protothecoides.

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between days 1 and 5 to determine to what degree the water parameters had changed. Water quality improvement would have been indicated by a decrease in both BOD and nitrogen levels. Water quality tests were replicated in triplicate for each sample to establish means and standard deviations. Experiments were repeated four times: weeks 1, 2, 3, and 4. After each 5-day residence period, the TSS, DO, pH, turbidity, and water temperature readings were taken and the algae were separated from the wastewater using a centrifuge (the algae harvest). Algae oils were extracted from the samples using hexane, transesterified using methanol and potassium hydroxide (creating biodiesel), and both qualitatively and quantitatively analyzed using GC/MS to determine the presence of five fatty acid methyl esters (FAMEs) common to biodiesel. The nitrogen, phosphorus, and BOD5 (i.e., standard BOD testing with a 5-day residence time) parameters of the wastewater were tested after the algae were harvested.

16.8 RESULTS Several wastewater quality outcomes from the research could have been expected: an increase in TSS and turbidity (indicating algae growth), a decrease in BOD followed by an increase in DO (indicating algae productivity), higher temperatures reducing overall net gain in DO (ambient conditions were a limiting factor), and noticeable decreases in nitrogen and phosphorus levels (indicating nutrient utilization by the algae). As for oil production, the harvested biomass of Cp should have had 10% 15% oil content, while the range for Bb should have been at least 25% 35%. However, the observed outcomes did not fully mirror these expectations and it is possible that several variables limited the algae’s ability to treat wastewater (“water”) and produce biodiesel quality fatty acids (lipids). Both Cp and Bb affected waters showed net increases in TSS concentrations once higher volumes of algae were used by week 4. pH levels were maintained at a fairly uniform level during this time as well and may be attributed to the strength of the algae inoculants. In general, when lower pH levels were present, nitrogen existed largely in the form of ammonia (NH3) and when pH levels were elevated, nitrogen existed in the form of nitrate (NO32). The air/CO2 mix provided was consistent throughout the weeks with CO2 supplemented at 103 155 Torr during the morning hours from 7 to 11 a.m. By weeks 3 and 4, the ambient temperatures were comparably warmer and the day 5 results fell within the optimal 21 C 26 C range reported by outdoor-based studies. Temperature and DO concentrations are linked. As temperatures rise, water’s capacity to store oxygen decreases. Throughout each of the weeks, DO levels rose. However, in weeks 3 and 4 when temperatures were elevated, as expected, DO levels did not reach as high an increase in concentration as weeks 1 and 2. The presence, and amount, of algae resulted in more oxygen being provided directly to the

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system during diurnal hours. This can be exemplified by the week 4 BOD data where levels dropped significantly. The distinct reductions in BOD for Cp and Bb in weeks 1, 3, and 4 demonstrate the promise of using algae to improve the quality of primary treated human wastewater. Save for week 2, concentrations of ammonia for the algae inoculated samples rose compared to the level in the wastewater sample. This indicates that algae were not effective at driving nitrification in these experiments. However, in experiments carried out in India, ammonia levels under algae treatment were able to be reduced over 90% [46]. Week 4 ammonia levels may have even been greater, if not for the pH remaining fairly neutral. During this week, due to ample amounts of DO, some ammonia may have been converted to nitrate by the presence of nitrifying bacteria. However, in general, when there is elevated BOD, nitrogen takes the form of ammonia. Nitrifying bacteria need low BOD conditions in order to be in position to flourish over other forms of bacteria [47]. The initial high BOD of the primary treated wastewater may have inhibited the growth of nitrifying bacteria. If this was the case, then future studies should experiment with longer residence times to give the nitrifying bacteria more time to adjust to given DO and BOD levels. In a water treatment study utilizing a combination of aquatic plants and algae, DO levels increased over 70%, while both nitrate and phosphorus levels were reduced by over 80% [46]. In past experiments with gravel-based constructed treatment wetlands, DO levels increased and subsequent reductions in nitrate and phosphorus levels were observed. The US EPA’s maximum contaminant level for nitrate is 30 mg/L (ppm). Looking at just the nitrogen levels and the slight increases in phosphorus, the data suggest that some level of eutrophication was occurring in the water samples. Although, this is not confirmed by the increases in DO and drop in BOD already mentioned. In primary wastewater the major form of phosphorus is organically bound. During the aeration process the organically bound phosphorus was oxidized to yield orthophosphate. Thus, at the point each weekly experiment had concluded, there had not yet been consumption of phosphorus by algae. B. braunii has demonstrated the ability to supply the oxygen levels needed to breakdown pollutants, though often on diluted secondary treated wastewaters [36,37]. Notably, Cp and Bb oil levels in week 3 represented roughly 13% and 16% of biomass, respectively. It is the thought that having richer concentrations of algae at the onset of each experiment would have led to not only a reduction in pollutants but possibly a continued increase in oil production. Specifically, reductions in nitrogen have been found to cause increases in oil production in certain algae strains [48]. Changing the growth media or increasing the concentration of the algae inoculant would have helped the colonies thrive on the wastewater and would have most likely fed oil production.

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As triglycerides are transesterified with methanol in order to complete the GC/MS analyses, the fatty acids present are converted to their constituent FAME forms. Transesterification is the process of making FAMEs (i.e., biodiesel) from plant-based triglycerides (i.e., oils). Common vegetable and animal oils are listed with their relative composition of individual FAMEs (Table 16.3). In addition, the breakdowns per algae strain per week are shown. Palmitic (16:0) and stearic (18:0) acids are most abundant in tallow, as they are in the algae studied [49]. Larger quantities of these saturated fats may produce fuel with reduced emissions. Through GC/MS analysis, the oil produced as a percentage of the total biomass was measured and the percentage of the five FAMEs present in the oil was determined (Fig. 16.1). Further study is needed to understand the correlations between the oil increases and the FAME profile of the algae oils produced. For example, if Cp had been grown heterotrophically in glucose rich media as opposed to autotrophically on the standard media, oil percentages around 50% may have

TABLE 16.3 Percentage of Common Fatty Acids Present in Biodiesel Made From Select Vegetable, Animal, and Algae Oils 16:0

18:0

18:1

18:2

18:3

Totals

6.4

2.9

13.6

72.9

0

95.8

Rapeseed

3.5

0.9

64.1

22.3

8.2

99

Soybean

13.9

2.1

23.2

56.2

4.3

99.6

Tallow

23.3

19.3

42.4

2.9

0.9

78.8

Cp

16:0

18:0

18:1

18:2

18:3

Totals

Sunflower

Week 1

4.54

10.90

2.16

1.86

0.47

19.93

Week 2

8.44

18.74

0.81

0.97

0.75

29.71

Week 3

0.75

1.86

0.00

0.09

0.00

2.7

18:3

Totals

Week 1

0.74

1.66

0.72

0.15

0.00

3.27

Week 2

3.83

6.70

4.30

0.68

0.93

16.44

Week 3

0.91

1.28

0.00

0.11

0.00

2.3

18:3

Totals

Bb

P6

16:0

16:0

18:0

18:0

18:1

18:1

18:2

18:2

Week 1

0.97

0.16

0.37

0.54

0.66

2.7

Week 2

7.00

19.35

0.50

0.59

0.84

28.28

Week 3

5.43

14.80

0.00

0.00

0.00

20.23

Cp, Chlorella protothecoides; P6, pond 6; Bb, Botryococcus braunii.

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35.00

% of Oil or FAMEs

30.00 25.00 20.00 15.00 10.00 5.00 0.00 % of Oil in sample

% of 5 FAMEs in oil % of Oil in sample 3.33 1.89 12.96

% of 5 FAMEs in oil 19.93 29.71 2.70

Week 1 ~ Botryococcus braunii Week 2 ~ B. braunii Week 3 ~ B. braunii

10.00 5.56 16.36

3.27 16.44 2.30

Week 1 ~ local pond 6 mixture Week 2 ~ local pond 6 mixture Week 3 ~ local pond 6 mixture

3.77 1.92 1.89

2.69 28.28 20.23

Week 1 ~ Chlorella protothecoides Week 2 ~ C. protothecoides Week 3 ~ C. protothecoides

FIGURE 16.1 Percentage of oil in biomass with percentage of FAMEs in oil. FAME, Fatty acid methyl ester.

been observed [50]. In addition, the type of FAMEs produced could be related to buoyancy and the needs of the algae during photosynthesis.

16.9 CONCLUSION Tying algae oil production with wastewater treatment is essential to reduce costs and make most efficient use of available resources. Results from using specific strains of algae to treat wastewater and produce biodiesel quality oil offer the foundation for future research. However, the quality of the oil, indicated by the FAMEs produced, needs to be carefully analyzed and researched. Factors affecting the outcomes of this type of research include: need for standardization of algae stock growing conditions, contamination of axenic cultures by competing strains of algae, variable nature of the

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wastewater received, the amount of algae inoculated into the wastewater, the health of the algae colonies upon inoculation, inadvertent addition of nutrient rich media to the testing vessels, improved techniques to harvest the algae and extract the oils, and a better understanding of algae and bacteria symbiosis to optimize water quality of the effluent. Wastewater treatment was achieved in this work, but not to the extent achieved at conventional wastewater treatment plants. Future experiments should test nitrogen and phosphorus levels of the algae species in the growing vessels prior to inoculation in the wastewater. In addition, though the generated oils were in excess of 10% of the algae biomass, the FAMEs produced were not consistently of biodiesel quality. Algae oils were produced and extracted, although ratios of key FAMEs were lower than anticipated. Due to Week 4 decreases in observed BOD and the leveling off of ammonia levels, an additional week of oil analysis may have produced more favorable results. This suggestion is reinforced by the dramatic increases in oil seen for Week 3 in Cp and Bb. The results of oil quantities produced and the corresponding FAME composition indicate further studies using Cp and Bb are warranted. Future work was indicated to address harvest and extraction of algae, shortening residence times and reductions in nitrogen and phosphorus levels, and conducting an analysis of the suitability of algae oils for biodiesel production.

16.10 FUTURE OUTLOOK Over the next decades, an integrated algae-based water treatment and biofuel/biochemical refinery approach may provide the means of expanding water supplies and generating renewable energy. Ultimately, an integrated wastewater biorefinery approach offers a step toward the goal of operating within a low carbon economy with clean, stable recycled water supplies. Current research and development project that economical commercialization of using algae to treat wastewater and produce renewable fuels and chemicals might still be years or decades in the making. As climate change emerges, the need for expanded reserves of recycled wastewater will continue to coincide with the need to develop domestic sources of low-cost, consistent, and renewable energy that does not compete with agricultural lands used to produce food. More hard work and regenerative design at the nexus of algae, wastewater treatment, agriculture, and the production of renewable energy and chemicals may still hold a key to fulfilling the vision of a low carbon economy.

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