Chemical and Biological Characterization of Wastewater Generated from Hydrothermal Liquefaction of Spirulina Mai Pham, † Lance Schideman, †, * John Scott, ‡ Nandakishore Rajagopalan, ‡ and Michael J. Plewa †, * † University of Illinois at Urbana-Champaign, College of Agricultural, Consumer and Environmental Sciences, Urbana, IL, United States ‡ Illinois Sustainable Technology Center (ISTC), a division of the Prairie Research Institute at the University of Illinois at Urbana-Champaign. * S Supporting Information ABSTRACT: Hydrothermal liquefaction (HTL) is an attrac- tive method for converting wet biomass into petroleum-like biocrude oil that can be refined to make petroleum products. This approach is advantageous for conversion of low-lipid algae, which are promising feedstocks for sustainable large-scale biofuel production. As with natural petroleum formation, the water in contact with the produced oil contains toxic compounds. The objectives of this research were to: (1) identify nitrogenous organic compounds (NOCs) in waste- water from HTL conversion of Spirulina; (2) characterize mammalian cell cytotoxicity of specific NOCs, NOC mixture, and the complete HTL wastewater (HTL-WW) matrix; and (3) investigate mitigation measures to reduce toxicity in HTL-WW. Liquid-liquid extraction and nitrogen-phosphorus detection was used in conjunction with gas chromatography-mass spectrometry (GC-MS), which detected hundreds of NOCs in HTL- WW. Reference materials for nine of the most prevalent NOCs were used to identify and quantify their concentrations in HTL- WW. Mammalian cell cytotoxicity of the nine NOCs was quantified using a Chinese hamster ovary (CHO) cell assay, and the descending rank order for cytotoxicity was 3-dimethylamino-phenol > 2,2,6,6-tetramethyl-4-piperidone > 2,6-dimethyl-3- pyridinol > 2-picoline > pyridine > 1-methyl-2-pyrrolidinone > σ-valerolactam > 2-pyrrolidinone > ε-caprolactam. The organic mixture extracted from HTL-WW expressed potent CHO cell cytotoxic activity, with a LC 50 at 7.5% of HTL-WW. Although the toxicity of HTL-WW was substantial, 30% of the toxicity was removed biologically by recycling HTL-WW back into algal cultivation. The remaining toxicity of HTL-WW was mostly eliminated by subsequent treatment with granular activated carbon. ■ INTRODUCTION Hydrothermal liquefaction (HTL) is a thermochemical conversion process that transforms wet biosolids into valuable, self-separating coproducts (bio-oil and biochar). 1-7 This process produces a gaseous product (mostly CO 2 ) and wastewater with high dissolved organic content. HTL releases oxygen and nitrogen from biomass into the gaseous and aqueous fractions. Thus, the biocrude oil fraction has a higher energy density than the original biomass, which can approach that of petroleum. HTL can resolve several practical limitations associated with current large-scale algal biofuel production. It resolves problems associated with contamination of target high- oil algal species because HTL can convert low-oil algae, bacteria, and other grazing micro-organisms into oil. Second, the parasitic energy demand for dewatering/drying is minimized because HTL can use wet feedstocks, and the resulting bio-oil is self-separating from the aqueous fraction. Finally, nutrients and CO 2 released to the aqueous and gaseous fractions can be recycled back into algal cultivation lowering the input costs for algal cultivation. These factors combine to make the production of algal biofuels via HTL conversion a promising alternative for sustainable and cost-effective bio- energy. However, it is important to characterize the coproducts of this process for potential adverse impacts to the environment and the public health. HTL conversion of algae and biowaste feedstocks into bio-oil was demonstrated with conversion efficiencies of 30-75% and a net positive energy yield of 3-10 times input heat energy. 8 The chemical properties of bio-oil from HTL conversion of different feedstocks such as Spirulina, swine manure, and anaerobically digested sewage sludge were characterized by a variety of analytical techniques. 1,3,7,9 Limited information is available from past studies on HTL-WW residuals. One study reported that wastewater from HTL conversion of swine manure contained very high concentrations of biological oxygen demand (0.42-5.9 g/L), high concentrations of ammonia (1.86-7.07 g/L), and other characteristics that make HTL-WW unsuitable for surface water discharge. 10 A wide variety of organic compounds were found in HTL-WW Received: November 6, 2012 Revised: January 6, 2013 Accepted: January 10, 2013 Published: January 10, 2013 Article pubs.acs.org/est © 2013 American Chemical Society 2131 dx.doi.org/10.1021/es304532c | Environ. Sci. Technol. 2013, 47, 2131-2138