Final Report: Integration of Waste Treatment with Algal Cultivation for Sustainable Aquaculture Feed and Renewable Biofuel Production

EPA Grant Number: SU835318
Title: Integration of Waste Treatment with Algal Cultivation for Sustainable Aquaculture Feed and Renewable Biofuel Production
Investigators: Bouwer, Edward J. , Betenbaugh, Michael J. , Bohutskyi, Pavlo , Byers, Natalie , Fung Shek, Coral J , Khaled Nasr, Laila , Liu, Kexin , Rosenberg, Julian
Institution: The Johns Hopkins University
EPA Project Officer: Lank, Gregory
Phase: I
Project Period: August 15, 2012 through August 14, 2013
Project Amount: $15,000
RFA: P3 Awards: A National Student Design Competition for Sustainability Focusing on People, Prosperity and the Planet (2012) RFA Text |  Recipients Lists
Research Category: Pollution Prevention/Sustainable Development , P3 Challenge Area - Agriculture , P3 Challenge Area - Energy , P3 Awards , Sustainability


There is a growing awareness about the global impact of waste accumulation together with the need to develop energy alternatives to fossil fuels. Another major concern is the impact of carbon, nitrogen (N), and phosphorous (P) emissions on our environment. Our project addresses these multiple themes of sustainability through holistic methodologies involving bioconversion. Specifically, this project implements innovative technologies that utilize microalgae to recycle waste carbon and other nutrients, including N and P, for renewable biofuel and potentially animal/aquaculture feed. Algae have multiple advantages as a feedstock for renewable biofuel and feed production including: 1) high growth rates; 2) minimal competition with traditional food crops; 3) cultivation on non-arable lands; 4) use of low quality water resources and solar energy to produce fuels, chemicals and food; and 5) CO2 sequestration and recycle. Unfortunately, algal technologies are not yet economically viable at large scales due to the costs associated with expensive nutrients including N and P fertilizers. However, if these nutrients could instead be obtained from inexpensive sources such as wastewater, we have the opportunity to both revolutionize algal processing and obtain a linked sustainable solution for waste treatment and energy and potentially aquaculture. This project coordinates students from different backgrounds and departments at Johns Hopkins University (JHU), business partners at Clean Green Chesapeake (CGC) and GlycoPure, Inc., and local government workers at Baltimore’s Back River Wastewater Treatment Plant (BRWWTP) owned and operated by the City of Baltimore into a team to advance a sustainable, environmentally friendly process for integrating waste treatment with energy generation and algal aquaculture development. The proposed research addresses a number of vital affecting people, planet, and prosperity through innovative synergies of water resources management, pollution control, renewable energy generation, and food production. Most importantly, this research provides sustainable solutions for our environment by channeling the capabilities of microalgae and other microbes for the conversion of waste streams into useful industrial products. Finally, this project provides a framework for educating the next generation of undergraduate and high school students in preparation for lifelong careers devoted to sustainable innovation.

Objective.The objective of our P3 Phase I proposal was to develop, test, and optimize a robust and sustainable process to convert agricultural and domestic organic waste, carbon dioxide, and sunlight into energy (in the form of biodiesel and biomethane) and/or algal-meal for animal/fish feed. Specifically, the project included investigations of algal cultivation in anaerobic digestion (AD) effluent and comparison of three alternative technologies for algal biomass utilization: (1) biological conversion to biogas through AD; (2) preliminary lipid extraction and AD of defatted residues to biogas; (3) utilization of algal biomass for fish farming. Contribution to Pollution Prevention or Control. The majority of technologies used to process livestock manure as well as industrial and domestic wastewater generate large amounts of potentially hazardous sludge containing organic waste materials, anthropogenic pollutants, pathogenic bacteria, viruses, and protozoa. Disposal of untreated sludge is a potential threat to surface and underground water sources, human, animals, plants, aquatic life and emission sources of greenhouse and odor gases. Application of AD as treatment technology reduces the number of pathogenic microorganisms emerging from waste sludge. Moreover, AD processes are a source of non-fossil fuel – biogas that can be used as a local source of electricity and heat generation or to power motor vehicles. Substitution of oil and coal with biogas reduces emission of greenhouse gases (CO2, CH4, and N2O), air pollution with acids, salts and particulates. Implementation of the proposed technology will not only induce expansion of AD for noxious sludge treatment, but also utilize AD effluent with high nutrient content either for biofuel or food production through microalgal cultivation. Indeed, nutrient-runoff presents a worldwide concern as manifested by “dead zones” in the coastal waters near most major cities. Excessive nutrients result in unbalanced aquatic ecosystems and are detrimental to aquaculture. One approach to capturing the organic wastes that are fouling our waterways and oceans is development of integrated bioprocessing approaches combining microalgae and AD in order to meet rapidly increasing demands for improved wastewater treatment and novel energy and food sources. Description. The technical milestones of this proposal provide innovations in coupling AD effluent and photosynthetic algae cultivation. The overall project goals are to:

  1. augment the productivity of conventional algal growth systems and eliminate fertilizer usage through utilization and recycling of nutrients from sewage sludge and residual algal biomass;

  2. control and manage pathogens in complex algal-bacterial population as well as chemical contaminants that can be present in wastewater and affect algal biomass as aquaculture feed;

  3. optimize the conversion of algal biomass and delipidated residues into biomethane, through thermochemical or enzymatic pretreatment;

  4. analyze nutrient balances, life cycle and perform economic analysis on demonstration facilities by comparing efficiency of different strategies for algal biomass utilization.

Equally important is the opportunity to train students at the undergraduate (through academic research projects) and graduate levels in the critically emerging fields of sustainability, pollution control, and biofuels. Three of the greatest challenges to society lie at the intersection of these global problems. Educating students to address these challenges is essential for the future technological development of long-term solutions to critical global societal issues.

Summary/Accomplishments (Outputs/Outcomes):

For the Phase I study, significant progress was achieved on the following Tasks and resulted in the preparation of 5 publications/presentations.

I. Optimize Algal Growth and Nutrient Assimilation from AD Effluent

  1. A variety of sixteen algae species were tested as candidates for cultivation in wastewater sources. Fortunately, several robust algal species that were able to proliferate in wastewater spiked with AD effluent were identified. Two species – Chlorella sorokiniana (CCTCC M209220) and Scedenesmus acutus f. alternans (UTEX B72) were found to have the highest growth rate and to be particularly suitable for cultivation in non-sterilized wastewater media. In addition, our group isolated a local algal strain with high potential for cultivation in wastewater.

  2. The optimal parameters for algae cultivation in wastewater media were investigated in batch cultures including percentages of AD effluent, light intensity and inoculum concentration. An AD dose of 5-10% was found to be sufficient to provide the essential nutrients and significantly intensify algal growth rate with studies on optimal light and inoculum concentration in progress.

  3. Initial process scale-up was achieved by conducting algal cultivations at the 8-L and 150-L scales. Cell density and nutrient concentrations were measured in order to determine growth rates and nutrient consumption demands for the scaled up process.

Task II: Evaluate bacterial & chemical contamination in algal biomass cultures

  1. Preliminary results demonstrated elimination of 3 and 4 Logs of total coliforms and E. coli, respectively, with a 2 Log removal of Enterococcus and Pseudomonas aeruginosa expected during a 10-day cultivation process.

  2. Advanced studies were performed to determine the level of wastewater contamination in the algal biomass generated. The metals Cr, Ni, Cu, Pb, Cd, Zn were detected but only in trace amounts. These results demonstrated that algal biomass produced in wastewater meets the animal and fish feed quality requirements for Cd and Pb content, but As and Hg need to be examined in subsequent studies.

  3. Finally, our studies showed that algal cultivation can significantly reduce N and P contamination in wastewater. A substantial reduction in these Chesapeake Bay nutrient contaminants was observed over the 10-day algal cultivation period.

Task III. Enhance algal biomass conversion following anaerobic digestion

Several methods of algal biomass hydrolysis pretreatment have been tested to overcome limitations in algal biomass biodegradation.

  1. Thermochemical pretreatment was found to be superior to thermal and thermochemical methods alone for algal biomass hydrolysis. Both biogas and methane production increased by 30-40% compared to untreated samples. In addition, the optimal chemical levels for thermochemical pretreatment were determined such as doses of NaOH in the range of 4-5 g/L.

  2. An evaluation of enzymatic pretreatment methods for algal biomass is in progress. A number of prospective hydrolytic enzymes were characterized on standard materials including cellulose and xylose before experimental trials are to begin on algal biomass.

  3. We also made substantial progress towards stable expression of self-lytic enzymes (as an alternative to enzyme addition) using both nuclear and chloroplast expression systems in the green alga Chlamydomonas reinhardtii.


Task IV. Characterization of algae biomass as premium aquaculture feed

  1. Preliminary fish aquaculture studies were performed, but detailed studies were not completed due to a delay in obtaining (animal use committee) approval prior to animal work. Nevertheless, aquaculture techniques were developed and implemented by rearing blue tilapia from fingerlings to adulthood.


Our results from Phase I showed the technological viability and significant potential of combining microalgal cultivation with AD for conversion of liquid waste into biomass for use as biofuel or even aquaculture fish food. Municipal wastewater and sewage sludge AD effluent can be used to support abundant algal growth and sustainable production of feedstocks for bioenergy. Our findings demonstrate that algal productivity in wastewater can be significantly enhanced by selecting robust algal species and optimizing cultivation parameters. Furthermore, microalgae can uptake N and P contaminants in order to reduce environment discharge of this nutrient, leading to contamination of the local watershed. According to preliminary calculations, usage of AD effluent generated on BRWWTP as fertilizer to facilitate algal growth would result in the conversion of about 900 tons of waste N and assimilation about 2,750 tons of CO2 from the atmosphere per year through algal bioconversion processes such as the one proposed here. Especially exciting is the prospect that this technology can be expanded to other sludge and agricultural wastes, including livestock manure and poultry litter. As an example, the poultry litter generated on the DelMarVa Peninsula alone generates about 24,000 and 9,000 tons of N and P, respectively, much of which is deposited in the Chesapeake Bay. This technology will help to prevent release of this toxic pollution and convert it to a valuable energy or food source for improving people, planets, and prosperity locally and around the globe.

Another success achieved during Phase I is that optimized pretreatment processing can increase both the quality and quantity of biodegradable biomass that may be digested by the anaerobic digestor. Such pretreatment can lead to major gains in methane production achieved following AD of the algal biomass. Alternative enzymatic pretreatment studies are in progress with an examination of multiple enzymes for degradation. A number of carbohydrase enzymes were characterized including cellulases, hemicellulases, amylases, and β-glucosidase.

Preliminary fish feeding aquaculture studies are underway, but approval of more complex experiments awaits animal usage studies by IACUC (animal use committee). In an initial experiment, the success of aquaculture was demonstrated through the rearing of blue tilapia from fingerlings. In order for the application of microalgae from this source to be viable, substantial efforts must be undertaken to ensure food safety and to monitor for the presence of potential chemical and biological pathogens. Towards this goal, methods are under development to monitor the biological and chemical safety of harvested algal biomass as fish/animal feed. Importantly, the pathogens’ concentration decreased significantly during microalgae cultivation and only trace amounts of heavy metals were detected in the algal biomass. Nevertheless, the safe application of harvested algae as feed will be studied extensively in the next phase.

Finally, productive collaborative interactions were established both with a commercial partner Clean Green Chesapeake (CGC) and the local government operators of the Back River Wastewater Treatment Plant (BRWWTP) in Baltimore, MD. Intimate industrial, academic, and government interactions will be essential for the successful translation of this technology to industry as well as for the development of a successful Phase II commercial process in large- scale bioreactors. Furthermore, CGC’s laboratory and demonstration-scale algal production facility provides a test bed for research ranging from algae based aquaculture feed to enhanced algal biofuel extraction methodologies and serves as a dynamic environment for graduate and undergraduate student training and engagement in research at a small business.

Journal Articles on this Report : 1 Displayed | Download in RIS Format

Other project views: All 6 publications 1 publications in selected types All 1 journal articles
Type Citation Project Document Sources
Journal Article Bohutskyi P, Betenbaugh MJ, Bouwer EJ. The effect of alternative pretreatment strategies on anaerobic digestion and methane production from different algal strains. Bioresource Technology 2014;155:366-372. SU835318 (Final)
  • Abstract: ScienceDirect-Abstract
  • Supplemental Keywords:

    sustainability, sustainable development, sustainable waste management, wastewater treatment, sewage sludge, animal waste, eutrophication, nutrients, water conservation, resource recovery, closed-loop recycling, waste-to-value, waste-to-energy, carbon sequestration, renewable energy, algal biofuel, biodiesel, biogas, biomethane, anaerobic digestion, algal-meal, aquaculture, integrated biorefinery, enzymatic pretreatment, thermochemical pretreatment

    Relevant Websites:

    AlgaFuture: Sustainable Biofuel, Food and Environment Exit

    Progress and Final Reports:

    Original Abstract