Project: Cultivation Of Filamentous Green Algae For High Value Industrial Products

The goal of this project is to develop a scalable method of cultivating freshwater filamentous green algae (FFGA) as a source of industrially useful cellulose fibers. The project is on-going under USDA’s Small Business Innovation Research program and involves designing, constructing and operating a pilot plant at a municipal wastewater treatment plant to generate operating and process parameters for cultivating FFGA at commercial scale. FFGA are a group of cosmopolitan algae that to date have not been considered for commercial purposes. They are known to grow prolifically in eutrophic waters (e.g., wastewater) and have morphological characteristics that facilitate harvesting and processing. FFGA grow under various environmental conditions and are resistant to losses such as predation that affect cultivation of microalgae. Their cell composition is typically high in carbohydrates (starch and cellulose) and low in lipids and proteins. Most algae lack lignin which facilitates extraction of cellulose. This implies that cellulose from algae may be substantially lower in cost to produce compared to cellulose from woody and plant biomass. In addition to the economic value of cellulose fibers, the inherent value associated with algae removal of nutrients make the proposed technology innovative and sustainable.

Project: Algal Bioremediation Of Wastewater Inputs To Great Lakes Ecosystems

AlgaXperts was part of a team led by the University of Wisconsin-Milwaukee to determine the feasibility of algal bioremediation at regional wastewater treatment plants to reduce nutrients content in effluent discharged into the Great Lakes. The goal of the project was to test the overall hypothesis that integration of algal cultivation into existing municipal wastewater treatment processes could provide a cost effective way to reduce N and P loads in treated effluent, reduce energy required for nutrient removal, and provide valuable biomass for production of biofuels and other value-added products. To address the overall goal, specific project objectives were addressed in a 2 year pilot plant at the Milwaukee Metropolitan Sewerage District’s wastewater treatment plant. AlgaXperts provided input for assessing options for open tanks, holding ponds or other facilities at the WWTP, for suitability for cultivation of Cladophora algal assemblages. Locations where filamentous algae already grow in effluent were examined. Other key criteria considered included depth, hydraulic motion, light availability, options for aeration and/or stirring and substratum surface. AlgaXperts conducted an economic feasibility study of algal bioremediation in WWTP using data collected over the two years of nutrient bioremediation performance, algal biomass production rates, and biofuels compounds production. Cost estimates were based on a 5.0 million gallons per day municipal WWTP, and a system designed to meet projected nutrient limits of 3.0 mg L-1 total N and 0.1 mg L-1 total P in final effluent.

Project: Amplification Of Existing Algae To Remove Nutrients

The purpose of this project was to determine if algal growth could be established on rotating (moving) mesh screen and function as a biofilter to uptake nutrients in treated secondary effluent. A device consisting of four leaves of 8 inch x 20 inch (½ inch x ½ inch) mesh screens affixed to motorized rotating arms was placed in a rectangular tank so as to rotate through the tank contents. The hydraulic retention time was approximately 2.5 hrs. Within three to four days after start-up, algal growth was apparent on the mesh screen. This algal growth was due to filamentous algae which had sloughed-off from attached growth upstream of the rotating device (e.g., final settling tank effluent collection channels) and had been “screened” by the mesh. The screen assembly was designed to be about 40% submerged. The rotation was intended to raise the algal growth on the screen above the water surface to expose the algae to higher light while maintaining the biomass wet and exposed to dissolved nutrients in the wastewater. The circulating screens also contributed mixing to the tank contents. The rpm of the assembly was adjustable to create variable hydrodynamic stress on the attached algal growth and eliminate formation of boundary layer via hydraulic shear. Despite the mesh screens supporting dense growth of filamentous green algae, the device did not demonstrate consistent uptake of nutrients. Analysis of algae cells indicated that the cells were saturated with nutrients suggesting that algal biomass would be necessary for such a system to work effectively. The community of algae that grew on the mesh screen was taxonomically evaluated and found to include primarily filamentous species. The dense growth of filamentous green algae persisted through the winter months. This experiment clearly showed that existing algae in municipal wastewater treatment plants could be amplified (increased area of growth substrata and regular biomass removal) to remove nutrients.

Project: Identification And Enumeration Of Algae In Water Treatment Plant

AlgaXperts was engaged by a water utility that is operationally impacted by seasonal presence of algae in the utility’s source water and within the treatment plant. The utility provides an average of 65 million gallons per day of drinking water to half million customers. Conventional treatment technologies are used including lime softening, recarbonation, coagulation/flocculation, filtration and disinfection. Filtration consists of three parallel systems; sand media, dual media, and membrane ultrafiltration. Chemicals used in treatment include lime, alum, carbon dioxide, powdered activated carbon (PAC), potassium permanganate, ferric chloride, ammonia, fluoride, are chlorine. AlgaXperts conducted algae identification and enumeration (ID&E) program consisting of weekly sampling over three months. Samples were taken at strategic points within the treatment process and collected by utility’s operating staff and shipped to AlgaXperts for analysis. The resulting ID&E data yielded taxonomic diversity of algae as well as population and how species and population changed over time and by process. AlgaXperts integrated ID&E data with understanding of treatment process to establish how and why algae were impacting the utility’s water treatment operation. Additional sampling for ID&E is planned to furnish data beyond the three months in order to confirm findings of first phase and to develop an operating strategy for controlling and mitigating the negative impacts of algae on water treatment.

Project: Feasibility Study To Integrate Algae Cultivation In Municipal WWTP

This project analyzed the feasibility of integrating algae cultivation into existing municipal wastewater treatment plants that use heterotrophic bacteria technology such as activated sludge and oxidation ditch. The paddle-wheel-mixed open raceway technology similar to High Rate Algal Pond (HRAP) was assumed to maintain cell density of 0.75 g L-1 at growth rate of 0.5 d-1 or hydraulic retention time of 2 days. The algae are assumed to be a mix of phytoplankton and periphyton of unicellular, colonial and filamentous body types. Treatment train consisted of fine screens, filters, and dewatering in addition to raceway ponds. Process variables such as nutrients, carbon, light, and mixing are controlled at or near optimum on sustained basis.

Operating records for the existing 32 mgd activated sludge plant were analyzed and used to conceptualize an integrated algae cultivation system. The conceptualized system consisted of 8 paddle-wheel-mixed open raceway ponds treating 23% of the influent flow including the sludge processing return (plant side-streams). The raceway pond dimensional data were as follows: width 60 ft, length 600 ft, liquid depth 5.15 ft, side slope 2.5 to 1.0, total surface area 16.5 acres, and liquid volume 60 million liters. A center wall divides the pond to form a continuous raceway and two paddle wheels are located on opposite ends. This configuration at the assumed growth rate and harvesting efficiency produces dewatered algal biomass of 20 dry tons per day.

The feasibility study indicated that integrating algae cultivation system at an existing municipal wastewater treatment plant provides the potential for significant cost savings. The annualized cost for nutrient removal at a 32 mgd wastewater treatment plant using: a) conventional technologies (5-stage Bardenpho) was $6.0 million; b) conventional technologies supplemented with integrated algae cultivation (5-stage Bardenpho 27.1 mgd plus algae cultivation 7.9 mgd) was $3.78 million. Cost saving was $2.3 million per year.

Project: Freshwater Diatoms As A Source Of Lipids For Biofuels

The purpose of this project was to determine whether it was possible to produce lipid rich freshwater diatoms in hypereutrophic media optimized for silicon content. Until recently, biodiesel production has been derived from terrestrial plants such as soybean and canola, leading to competition between biodiesel production and agricultural production for source materials. Microalgae have the potential to synthesize 30 times more oil per hect- are than terrestrial plants without competing for agricultural land. We examined four genera (Cyclotella, Aulacoseira, Fragilaria, Synedra) of common freshwater diatoms (Bacillariophyceae) for growth and lipid content in defined medium (sD11) that replicates hypereutrophic conditions in lakes and wastewater treatment plant eZuents and optimized the medium for silicon content. Cyclotella and Aulacoseira produced the highest levels of total lipids, 60 and 43 µg total lipids/ml, respectively. Both diatoms are rich in fatty acids C14, C16, C16:1, C16:2,7,10, and C22:5n3. Of the diatoms examined, Cyclotella reached the highest population density (>2.5 £ 106 cells/ml) in stationary phase when many of the cells appeared to be filled entirely with oil. Silicon enrichment studies indicated that for optimal utilization of phosphorus and nitrogen by diatoms growing in wastewater effluent, the amount of silicon present or added to the effluent should be 17.5 times the mass of phosphorus in the effluent. With high growth rates, high lipid contents, and rapid settling rates, Cyclotella and Aulacoseira are candidates for biodiesel production.

Publication: James M. Graham JM, Graham LE, Zulkifly SB, Pleger BF, Hoover SW, and Yoshitani J. Freshwater diatoms as a source of lipids for biofuels, Journal of Industrial Microbiology and Biotechnology (2012) 39:419–428.

Project: Cultivation Of Cladophora In Municipal WW Secondary Effluent

The objective of this project was to investigate the potential for cultivating the native nuisance algae, Cladophora glomerata, in municipal wastewater to be used as biomass feedstock for conversion into biofuel. Unchlorinated secondary effluent obtained from the Madison Sewerage District’s Nine Springs Plant, Madison, WI was used as growth media, and found to be an adequate substitute for synthetic laboratory prepared growth media. The addition of gravity belt thickener filtrate did not enhance Cladophora growth, as expected but appeared to enhance growth of heterotrophic bacteria. Both “wild” and laboratory cultivated Cladophora were successfully maintained in the secondary effluent for 10 months confirming previous findings that wastewater effluent could be used as media for long-term dedicated cultivation of Cladophora in municipal wastewater effluent. Relatively fast depletion of bioavailable phosphorus during daily growth experiments indicated that Cladophora has the ability to uptake enough phosphorus for several divisions. This suggests that if Cladophora could be grown to be sufficiently dense, the biomass could be used as a nutrients biofilter for secondary effluent. “Wild” Cladophora and previously lab-cultivated Cladophora appeared to grow similarly well when grown in wastewater effluent. Growth occurred in floating and attached state. Attached growth occurred best under photoperiod cycle of 8 hours light and 16 hours dark. The results of this investigation suggest that Cladophora could be used for nutrient removal and production of biomass as feedstock for conversion to biofuel. Observation of “wild” Cladophora suggests high level of resistance to herbivory.

Project: Bacterial Production Of Free Fatty Acids From Freshwater Macroalgal Cellulose

The objective of this project was to demonstrate that cellulose extracted from Cladophora-dominated periphyton could be used as feedstock to make biofuel. The predominant strategy for using algae to produce biofuels relies on the overproduction of lipids in microalgae with subsequent conversion to biodiesel (methyl-esters) or green diesel (alkanes). Conditions that both optimize algal growth and lipid accumulation rarely overlap, and differences in growth rates can lead to wild species outcompeting the desired lipid-rich strains. Here, we demonstrate an alternative strategy in which cellulose contained in the cell walls of multicellular algae is used   as a feedstock for cultivating biofuel-producing micro- organisms. Cellulose was extracted from an environmental sample of Cladophora glomerata-dominated periphyton that was collected from Lake Mendota, WI, USA. The resulting cellulose cake was hydrolyzed by commercial enzymes to release fermentable glucose. The hydrolysis mixture was used to formulate an undefined medium that was able to support the growth, without supplementation, of a free fatty acid (FFA)-overproducing strain of Escherichia coli (Lennen et. al 2010). To maximize free fatty acid production from glucose, an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible vector was constructed to express the Umbellularia californica acyl–acyl carrier protein (ACP) thioesterase. Thioesterase expression was optimized by inducing cul- tures with 50 μM IPTG. Cell density and FFA titers from cultures grown on algae-based media reached 50% of those (∼90 μg/mL FFA) cultures grown on rich Luria– Bertani broth supplemented with 0.2% glucose. In comparison, cultures grown in two media based on AFEX-pretreated corn stover generated tenfold less FFA than cultures grown in algae-based media. This study demonstrates that macroalgal cellulose is a potential carbon source for the production of biofuels or other microbially synthesized compounds.

Publication: Hoover SW, Marner WD, Brownson AK, Lennen RM, Wittkopp TM, Yoshitani J, Zulkifly S, Graham LE, Chaston SD, McMahon KD and Pfleger BF, Bacterial production of free fatty acids from freshwater macroalgal cellulose, Microbiol Biotechnol (2011) 91:435–446

Project: Cultivation Of Periphyton Using Submerged Leds

The primary goal of this project was to demonstrate the feasibility of producing periphyton algal biomass using a light source consisting of submerged light emitting diodes (LEDs). The secondary goal was to quantify differences in algal biomass production due to use of various configuration of white LEDs and blue/red LEDs. Waterproof LEDs mounted on bars were used as light source for each reactor. Irradiance was measured by a cosine corrected light meter. Algae to be cultivated were obtained in samples taken from Lake Mendota in Madison WI, isolated and cultured. Secondary wastewater effluent from Madison Sewerage District’s Nine Springs Plant, Madison, WI was used as cultivation media. Microscopy of algal samples indicated a mixed community of filamentous green algae Cladophora and Spirogyra and diatoms. Testing results did not indicate significantly different algal biomass production due to different light wavelengths. Ancillary test parameters of interest included nutrient uptake rate, pH, temperature, and growth rate. A thick growth of filamentous algae, mostly Cladophora, developed in each bioreactor indicating that Cladophora can be grown in free floating state. Attached growth was also observed. Biofilm accumulation on submerged components did not occur as was expected. The investigation suggests that LEDs can be effectively employed in the cultivation of algal biomass.