Article pubs.acs.org/EF
Hydrothermal Liquefaction of Bacteria and Yeast Monocultures Peter J. Valdez, Michael C. Nelson, Julia L. Faeth, Henry Y. Wang, Xiaoxia Nina Lin, and Phillip E. Savage* Department of Chemical Engineering, University of Michigan, 3074 H.H. Dow Building, 2300 Hayward Street, Ann Arbor, Michigan 48109-2136, United States S Supporting Information *
ABSTRACT: We hydrothermally treated monocultures of Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Saccharomyces cerevisiae at isothermal (350 °C for 60 min) and fast (rapid heating for 1 min) liquefaction conditions. Fast hydrothermal liquefaction (HTL) of P. putida and S. cerevisiae produced the highest biocrude yields of 47 ± 13 and 48 ± 9 wt %, respectively. Biocrudes generated via fast HTL were always richer in O and N and had a higher yield of hexane-insoluble products. Isothermal HTL of all microorganisms always produced an aqueous phase richer in NH3 than the aqueous phase from fast HTL. Up to 62 ± 9% of the chemical energy in the biomass could be recovered in the biocrude product fraction. These results demonstrate the feasibility of applying HTL to produce high yields of biocrude from bacteria and yeast that are high in protein [>80 wt %, dry and ash-free basis (daf)] and low in lipids (99% purity, from Fisher Scientific. We measured approximately 20 mg of dried biomass into a glass test tube and then added 2 mL of a 5% (v/v) solution of acetyl chloride in methanol and a magnetic stir bar. We vigorously stirred the reaction mixture (>100 rpm) for 90 min at 100 °C using a magnetic stir plate and temperature-controlled heating block. After the holding period, we quenched the reaction by adding 1 mL of room-temperature deionized water. After the solution cooled for 10 min, we added 4 mL of n-heptane and agitated each tube for 10 min on a vortexer set to 1000 rpm. We centrifuged the mixture for 3 min at 1500 rcf to separate and then collect the heptane layer for gas chromatographic analysis. We injected 1 μL of sample, with a 2:1 split ratio, into an Agilent 7890 gas chromatograph equipped with an Agilent DB-FFAP column (30 m × 320 μm × 0.25 μm). Helium at a column flow of 1 mL/min was the carrier gas. The injector temperature was 250 °C. The oven temperature was maintained at 60 °C until the injection and then increased to 200 °C at a rate of 20 °C/min and then to 240 °C at a rate of 5 °C/min. The final temperature was held for 3 min. We generated calibration curves using a RESTEK Marine Oil mixture of 20 fatty acid methyl esters as an external standard. We estimated the protein content (wt %) of the biomass by multiplying its N content (wt %) by 6.25.27,28 We calculated the carbohydrate content as the difference between 100 wt % and the sum of the lipid, protein, and ash contents. HTL. We constructed Swagelok reactors using 3/8 in. port connectors fitted with a cap on one end and a 3/8 to 1/8 in. union on the other end. The union allowed for the attachment of a 15 000 psi-rated High Pressure Equipment Co. valve, with grafoil packing, for sampling the gas products. The valve was attached via 8.5 in. of 1/8 in. outer diameter tubing. The nominal volume added by the valve was 68
dx.doi.org/10.1021/ef401506u | Energy Fuels 2014, 28, 67−75
Energy & Fuels
Article
Table 1. Elemental and Biochemical Compositions (wt %) and Higher Heating Value (MJ/kg) of the Biomass E. coli TB E. coli MM P. putida B. subtilis S. cerevisiae
C
H
N
S
O
ash
lipid
protein
carbohydrate
HHV
46.54 47.32 46.58 42.65 46.47
6.69 6.88 7.08 6.56 7.31
13.70 13.17 13.23 11.45 12.04
0.67 0.58 0.55 0.43 0.47
25.58 27.15 21.48 25.91 29.03
6.82 ± 0.02 4.9 ± 0.1 11 13.0 ± 0.4 4.68 ± 0.01
0.57 ± 0.36 2.6 ± 0.1 2.7 ± 0.7 0.55 ± 0.03 2.7 ± 0.6
86 82 83 72 75
7 10 4 15 17
22 23 23 21 22
0.5 mL. The total volume of the reactor and valve is approximately 2.2 mL (depicted in Figure 1). For conventional, isothermal HTL, we loaded 1.35 g of 12 wt % biomass slurry to each Swagelok reactor. At this loading, water would fill 95% of the reactor volume at the reaction conditions. We sealed the reactors and placed them into a Techne fluidized sand bath set at 350 °C. The reactors were submerged in the sand bath and agitated using a Burrell Wrist Action shaker9 for 60 min. For fast HTL reactions, performed with rapid heating, we loaded the reactor with 0.30 g of 12 wt % biomass slurry. This water loading matched previous experiments in our laboratory.16 After sealing the reactors, they were placed in a 600 °C sandbath for 1 min. As previously described by Faeth et al., we used dummy reactors (depicted in Figure 2) fitted with a thermocouple to record the temperature and calculate the heating rate.16 In both cases, after the desired holding time had elapsed, we removed the reactors from the sand bath and quenched them in a room-temperature water bath. Recovery and Analysis of HTL Product Fractions. We followed the procedure published previously to collect and separate the solids, aqueous-phase products, light biocrude (hexane solubles), heavy biocrude (hexane insoluble and dichloromethane soluble), and gas product fractions from each reactor.9,16 We report the gravimetric yield of each product fraction as its mass divided by the mass of biomass loaded into the reactor on a dry basis (wt %). We also report the biocrude yields on a dry and ash-free basis (daf) because the ash content cannot contribute to the yield of biocrude. Solvent-free samples of the light and heavy biocrude were sent to Atlantic Microlab, Inc. for measurement of the weight percent of C, H, N, and S. The O content in the light and heavy biocrudes was calculated by difference. We report elemental distribution as the mass of an element in each of the product fractions per the total mass of that element in the biomass. We diluted the aqueous phase 1:600 with deionized water and measured NH3 in the aqueous phase using methods described previously.9 The hexane-soluble product, also referred to as the light biocrude, was analyzed with an Agilent 6890N gas chromatograph fitted with an Agilent 5973 mass spectrometer. We injected 1 μL into a 300 °C split injection port using a split ratio of 2:1 onto an Agilent HP-5 capillary column (50 m × 200 μm × 0.33 μm). The oven was set to 100 °C, and the temperature increased to 300 °C at a rate of 5 °C/min immediately after injection. The samples exited the column into an electron ionization mass spectrometer. We used matching software to tentatively identify molecular constituents in the biocrude sample based on mass spectra. We calculated the higher heating value of the light and heavy biocrude using the Boie formula29 and the elemental composition data (wt %) on a dry basis.
rpm for 1 h. After mixing, we added 1.2 mL of deionized water, which mimics the water loading in a reactor, and agitated the samples for another 1 h. We then followed the workup procedure described previously to collect and measure the yield of each of the product fractions, except for gas.9 This control experiment provides the yields of each product fraction available from the biomass simply by wet extraction without HTL.
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RESULTS AND DISCUSSION This section first reports the characteristics of the microorganisms that we cultivated and then reports the results of the
Figure 3. Yields of light and heavy biocrude product fractions (wt %, daf) for each biomass and isothermal and fast HTL.
hydrothermal treatment of the biomass. The latter section describes the yield, elemental composition, and selected molecular composition of the product fractions. We also report the heating value and energy recovery of the biocrudes and compare results among the various microorganisms. Feedstock Analysis. Table 1 shows the elemental and biochemical contents of each of the biomass feedstocks. There are but modest variations in the elemental compositions of C, H, N, and S. The C and N weight percents for E. coli are within 6% relative difference of those reported previously.30,31 Although the two E. coli cultures were cultivated using different growth media, their C, H, N, and O contents were within 10% of each other on a relative basis. B. subtilis and P. putida had the highest ash compositions of 13 and 11 wt %, respectively, whereas the other organisms had
