Thin stillage sampling and collection
Pretreatment, and fungal saccharification followed by yeast fermentation, was used to generate ethanol at the BP Advanced Biofuel Demonstration Plant (1.4 million gallon per year/40 ton per day capacity) located in Jennings, Louisiana. Three 55-gallon drums of energy cane thin stillage and three of sugar cane thin stillage were obtained that were representative of the whole single production runs from either energy cane or sugar cane according to the plant manager. After the drums were obtained, they were transported under refrigeration to the Department of Animal Sciences, UIUC, aliquoted in 5-gallon portions in around 35 sealed buckets (Homer bucket, Home Depot, USA) of energy cane stillage as well as of sugar cane stillage and stored at 4 °C.
Liquid chromatography of thin stillage
Anions and organic acids were analyzed by hydroxide-selective anion exchange chromatography. Samples were injected onto a 250 × 4 mm AS11-HC column (Thermo Scientific) equipped with a 50 × 4 mm AG11-HC IonPac guard column kept at 30 °C. The instrument used was a Thermo Scientific IC-5000+ with cooled autosampler, isocratic pump, eluent generator, thermostatted column compartment, and suppressed conductivity detector. Compounds were eluted at a flow rate of 2 mL/min and a hydroxide gradient of 0.2 mM isocratic for 6 min, then over 5 min to 5 mM, then over 16 min to 40 mM. Detection was performed by suppressed conductivity.
Monosaccharides were analyzed by anion exchange chromatography using a Thermo Scientific IC-5000+ instrument as described above except that a pulsed amperometric detector was used. The column used was a 150 × 3 mm CarboPac PA20 (Thermo Scientific) equipped with a 30 × 3 mm CarboPac PA20 guard column (Thermo Scientific). Compounds were eluted isocratically with 2 mM hydroxide at a flow rate of 0.4 mL/min and detected by pulsed amperometric detection.
Glycerol, ethanol, 5-HMF, and furfural were analyzed using a 300 × 7.8 mm HPX-87H column (BioRad, Richmond, CA, USA) equipped with a 30 × 4.6 mm Micro-Guard Cation H cartridge (BioRad). Compounds were eluted isocratically with 5 mM sulfuric acid at a flow rate of 0.6 mL/min and detected by refractive index detection (glycerol, ethanol) or UV (5-HMF, furfural).
All compounds were quantified by external calibration using mixtures of standards (purity ≥98 %) or using the combined seven anion standard (Thermo Scientific, San Jose, CA, USA).
Elemental analysis of thin stillage
Elemental analysis was performed using a Varian Vista Pro CCD simultaneous inductively coupled plasma optical emission spectrometer (radial torch configuration) with an SPS 3 autosampler (Varian, Palo Alto, CA, USA). Samples were nebulized for transport into the radio frequency ICP, where each of the elements emits a specific spectrum. Wavelength intensities were measured by the photosensitive CCD microchip, and data were computed and stored with the ICP Expert computer software (Varian). Particles in thin stillage were analyzed with a Costech 4010 elemental analyzer (Costech, Valencia, CA, USA). Acetanilide and apple leaves were used as standards. A combustion process using chromium oxide as a catalyst with masses separated using an internal GC column was used.
Experimental setup and operation of hybrid reactors
Six 1.25-L laboratory scale hybrid bioreactors, based on a previous design , were used in this study. High-rate upflow recirculation of 300 mL/min in the UASB compartment is combined with an internal bioreactor filtration support carrying a packed bed of around 80 biofiltration rings (Siporax, Pentair Aquatic Eco-Systems, Inc, Apopka, FL, USA) to further improve anaerobic digestion. The warm water flow in the water jacket was set to 325 mL/min. The temperature of the water bath connected to the water jacket was set to 55 °C for MHR and to 70 °C for THR resulting in internal reactor temperatures of 40 and 55 °C, respectively. Reactors were fed continuously with thin stillage using a flow of 0.06, 0.36, 0.54 or 0.9 mL/min. This gave hydraulic retention times of 15, 2, 1.5, and 1 day(s), respectively. Chemical oxygen demand (COD) measures the oxidation of organic matter in a substrate and is an indirect measure of the amount of organic compounds present. The tCOD (total chemical oxygen demand in g/L) of the stillage was used to calculate the OLR (organic loading rate in g COD/L/d). Different OLRs were chosen to test bioreactor performance of hybrid reactors treating thin stillages derived from the bioethanol production with energy cane and sugar cane as source biomass. Reactors were run using individual independent schedules. Only periods in which the reactors were running with each OLR for a minimum of three turnovers were chosen, with the assumption that the three turnovers allowed a pseudo steady state in the reactor at that particular operating condition. This was also evaluated from the observation that the percentage of methane produced in biogas was within a 10 % range deviation. Only data obtained after the three turnovers were used for further analysis of bioreactor performance.
Inoculum for the bioreactors
MHR and THR were seeded with sludge from different sources ensuring presence of mesophilic and thermophilic microbial communities capable of anaerobic digestion of thin stillage under the respective conditions. MHR1 and MHR2 were inoculated with material from stable mesophilic methane-producing communities from cattle manure (Sieber et al., manuscript in preparation) and MHR3 from a mixture of sludge derived from MHR1 and MHR2. THR1 was inoculated from a mixture of samples from five different thermophilic anaerobic digesters from the temperature-phased anaerobic digestion system of wastewater treatment plants, and THR2 and THR3 were seeded with a mixture from the same samples that THR1 was inoculated with plus supplemental sludge from THR1. Initially, the bioreactors were filled with thin stillage that was four times diluted (¼ stillage COD) and adjusted to pH 7, after at least three turnovers, this was changed to two times diluted stillage (½ stillage COD) and subsequently to full strength stillage.
pH determination of bioreactor effluent
Effluent of the HR was used for analysis using an Accumet AB15 pH meter (Fisher Scientific, Pittsburgh, PA, USA) on a daily basis.
Measurement of methane percentage in total gas and specific methane production of bioreactors
Analysis of methane concentration in the biogas was performed on a daily basis and by direct injection of 0.5 mL gas produced by the bioreactors in a gas chromatograph (Series 580 Thermal Conductivity Gas Chromatograph, Gow-Mac Instrument Co., Bethlehem, PA). The GC column was 183 cm × 6.4 mm o.d. packed with Porapak Q and the temperatures for the injection port, detector, and column were 80, 80, and 75 °C, respectively. Biogas production was monitored on a daily basis and measured by a Milli Gas Counter (MGC-10, Ritter, Bochum, Germany). Methane production per day was calculated from the total volume of gas produced (in mL), and the methane percentage was determined analytically. Subsequently, specific methane production was calculated using the soluble chemical oxygen demand (sCOD) that was used per day.
COD analysis of thin stillage and bioreactor effluent
The organic strength of thin stillage and bioreactor effluent samples was determined using the COD2 Mercury-free reagent (Hach, Loveland, CO, USA) according to the manufacturers’ instructions. COD removal was based on the difference in COD of the feed and effluent.
Differences of the concentration of thin stillage compounds were statistically tested using the students t test. Bioreactor performance results were used for statistical analysis with the stats package in the R environment using three-way ANOVA with the lm command .
Pyrosequencing analysis of bacteria and archaea in the bioreactors
Effluent and biofilm (rings) samples from mesophilic and thermophilic hybrid reactors performing anaerobic digestion of energy cane or sugar cane stillage were used for DNA isolation (FastDNA SPIN kit for soil, MP Biomedicals, Irvine, CA) and following the manufacturer’s instructions. Both no input and clean ring samples were used as negative controls for the DNA isolation procedure. DNA integrity was checked on 1 % agarose gel, and DNA concentration was determined using NanoDrop (ND 2000, Thermo Fisher Scientific, Waltham, MA). DNA was subjected to PCR targeting the 16S rRNA gene and using 515F (GTGTGCCAGCMGCCGCGGT ) and 806R (GGACTACVSGGGTATCTAAT ) sequences to construct the primers generating a ~300 bp amplicon. The primers were assembled as follows: general forward primer = 454 Titanium Lib-L primer B/Library Key (TCAG)/515F, sample specific reverse primer = 454 Titanium Lib-L primer A/Library Key (TCAG)/12-base Multiplex Identifier/806R. PCR reactions were carried out using 1 × Phusion High Fidelity PCR Master Mix with HF buffer (New England Biolabs, Ipswich, MA), 0.4 µM of the forward and the reverse primers, 5 % DMSO, and 20 ng template DNA per reaction and reactions. The PCR program consisted of initial denaturation at 98 °C for 30 s, followed by 35 cycles of denaturation at 98 °C for 8 s, annealing at 60 °C for 25 s, and extension at 72 °C for 2 min with a final extension at 72 °C for 10 min after the last cycle. For PCR, reactions with no DNA template were used as negative control, and in these reactions, no visible PCR product was produced. DNA from a soil microbial community, which should have no or low archaeal relative abundance , was used as a control, and both the PCR negative reaction and DNA isolation negative samples were used as negative controls. PCR reactions were performed in triplicate, and each set of triplicates was combined and purified using the Zymo DNA clean and concentrator kit (Zymo Research, Irvine, CA) and quantified using the Qubit dsDNA BR assay kit (Life Technologies, Carlsbad, CA). These quantified samples were combined in equimolar ratios. Sample pools were quantified (Qubit dsDNA BR assay kit) and further processed at the Keck Center (University of Illinois at Urbana-Champaign, Urbana, IL). Sample pools were subjected to quality control including qPCR and quality check on a High Sensitivity DNA chip (Agilent, Santa Clara, CA). Subsequently, the pools were used for emulsion PCR using the Roche emPCR method (Roche Group, Basel, Switzerland). 454 pyrosequencing was performed using Roche GS FLX + system, v2.9, flow pattern A and analyzed through amplicon signal processing using Roche software version 2.9 (Roche Group, Basel, Switzerland).
Analysis of the pyrosequencing data
The pyrosequencing data obtained, were analyzed using the QIIME pipeline . We excluded reads with lengths below 200 bp and quality scores less than 25. No mismatches were allowed in the forward primer. The sequences were denoised and binned into operational taxonomic units (OTU) at a cut-off of 97 % similarity using uclust . Cluster seed was used as representative sequence. Chimeric sequences were detected with Chimera Slayer and excluded . Subsequently, the sequences were aligned with PyNAST using the Greengenes core set alignment as Ref. [17, 18]. Taxonomy was assigned by comparing to the database of the Ribosomal Database Project . An OTU table was prepared, and phylogeny was constructed using RAxML . Taxonomy results were plotted using Microsoft Excel. Further processing of the data involved between-sample diversity analysis, which compared the abundance and presence of microorganisms between the different samples. Based on the collated data, an unweighted UniFrac distance matrix was determined to compare the extent of similarities among samples, and the matrix was utilized for principal coordinate analysis on 3D biplots generated in Emperor .