Bioresource Technology 161 (2014) 423–430

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Efficient anaerobic digestion of whole microalgae and lipid-extracted microalgae residues for methane energy production Baisuo Zhao a,c, Jingwei Ma a, Quanbao Zhao a, Lieve Laurens b, Eric Jarvis b, Shulin Chen a, Craig Frear a,⇑ a

Biological Systems Engineering, Washington State University, Pullman, WA 99164, USA National Renewable Energy Laboratory, Golden, CO 80401-3305, USA c Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, China b

h i g h l i g h t s  SMP for whole microalgae were better than lipid-extracted microalgae residues.  The control of I/S ratio during BMP could overcome pH drop and LCFA inhibition.  No signs of VFA or ammonia inhibition occurred when digested at I/S P 1.0 ratio.  Higher methane production was not related to species but to lipid content of biomass.

a r t i c l e

i n f o

Article history: Received 8 February 2014 Received in revised form 11 March 2014 Accepted 14 March 2014 Available online 26 March 2014 Keywords: Anaerobic digestion Microalgae Lipid-extracted microalgae residues Methane production

a b s t r a c t The primary aim of this study was to completely investigate extensive biological methane potential (BMP) on both whole microalgae and its lipid-extracted biomass residues with various degrees of biomass pretreatment. Specific methane productivities (SMP) under batch conditions for non-lipid extracted biomass were better than lipid-extracted biomass residues and exhibited no signs of ammonia or carbon/ nitrogen (C/N) ratio inhibition when digested at high I/S ratio (I/S ratio P 1.0). SMP for suitably extracted biomass ranged from 0.30 to 0.38 L CH4/g VS (volatile solids). For both whole and lipid-extracted biomass, overall organic conversion ranged from 59.33 to 78.50 as a measure of %VS reduction with greater percentage biodegradability in general found within the lipid-extracted biomass. Higher production levels correlated to lipid content with a linear relationship between SMP and ash-free lipid content being developed at a R2 of 0.814. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae are currently considered a renewable source of liquid and gaseous biofuels and a practical technology for the capture of carbon dioxide. Microalgae represent one of the most sustainable and promising of biofuel feedstock, demonstrating particularly high growth rates (Scott et al., 2010) as well as an ability to thrive in harsh environments such as seawater (salinity 35 g L 1), alkaline lakes (pH P8.5), non-potable industrial wastewater, and arid and barren land areas—both which potentially allow for production while not directly competing with fresh water and arable land. Microalgae and their entrained lipids, can offer several different types of biofuel and bioenergy production options including trans-esterified biodiesel, fermented bioethanol, photo-biological hydrogen, hydrocarbon biofuels for drop-in ⇑ Corresponding author. Tel.: +1 509 335 0194; fax: +1 509 335 2722. E-mail address: [email protected] (C. Frear). http://dx.doi.org/10.1016/j.biortech.2014.03.079 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

replacements of gasoline, diesel, and jet fuel, and anaerobically generated methane (Sialve et al., 2009; Scott et al., 2010). While the biofuel potential of microalgae is more focused, at both research and commercial levels, on production of drop-in fuels, the production of methane from either whole cell or algal residue has been regarded with general interest. From a whole-cell perspective, microalgae blooms cultivated in open ponds or waterways, often for wastewater treatment and environmental protection purposes, are typically low in lipid content, warranting a lower-value use and simpler processing approach representative of anaerobic digestion (AD) (Sialve et al., 2009). From a residue perspective, algal bio-refineries are anticipated to produce algal biomass composed of >30% by dry weight (Scott et al., 2010). As a result, as much as 35–73% of the original energy (Lardon et al., 2009) of all cultivated solids will be residue, requiring disposal, particularly if value-added approaches for the use of residual proteins, polysaccharides, and other chemicals cannot find cost effective separation technologies as well as suitably large and viable

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Nomenclature BMP C1 C2 C/N DW FAME GH HRT ID I/S LC LCFA N1 N2 NF1 NF2 NREL NS1

biochemical methane potential un-extractedChlorella vulgaris UTEX 395 lipid-extracted Chlorella vulgaris UTEX 395 carbon to nitrogen ratio dry weight fatty acid methyl ester greenhouse hydraulic retention time In diameter inoculum/substrate lipid concent long chain fatty acid un-extracted Nannochloropsis sp. lipid-extracted Nannochloropsis sp. un-extracted Nanofrustulum sp. lipid-extracted Nanofrustulum sp. National Renewable Energy Laboratory un-extracted Nannochloropsis salina

markets. Both of these materials can be viewed as attractive feedstock for the production of energy and/or compressed fuels as well as the recovery of associated nutrients, mainly nitrogen and phosphorous. Therefore, the potential of AD using low-lipid whole cells or lipid-extracted residual biomass can offer significant benefits for reducing an algal bio-refinery’s cost and environmental impacts (Sialve et al., 2009). A review of microalgae AD research shows a wide range of production values, on the range of 0.09–0.54 L CH4 g 1 volatile solids (VS) (Sialve et al., 2009). Such a wide range is non-preferred for modeling of costs and benefits, which is essential to algal bio-refinery development. Species dependency is considered important due to macro-molecular content, which allows for variation in inherent energy potential as well as cellular wall/membrane form and subsequent recalcitrance or access to biological degradation of the cellular material (Mussgnug et al., 2010). It has also been proposed that the range, and particularly low values in some studies, is due in part to concerns related to biological inaccessibility to whole cells with intact walls/membranes (Sialve et al., 2009) as well as inhibitory conditions and agents experienced during digestion. Inhibitory conditions include low C/N ratios (Yen and Brune, 2007) and high salinity (Lakaniemi et al., 2011) while inhibitory agents include product inhibitors such as ammonia (Sialve et al., 2009) and long-chain fatty acids (LCFA) (Frigon et al., 2013). Independent of species is the possibility that the wide range in gas production is due to use of a corresponding wide range of experimental methodologies with no consistent control of important anaerobic digestion parameters such as inoculum concentration, substrate concentration, organic loading rate (OLR), etc. (Ehimen et al., 2009; Alzate et al., 2012; González-Fernández et al., 2013). While recent publications have reported on methane production from microalgae biomass (Ehimen et al., 2009; Park and Li, 2012), questions still remain regarding the previously reported wide range of results and the possible biological or methodological explanation for those results. In addition, relatively little data is available to date to fully explore the maximum methane productivity from whole microalgae and lipid-extracted biomass residues and to compare their economic effect. In this study, five potential industrial whole microalgae strains and their residues, i.e. the lipid-extracted microalgae biomass, were characterized for accessing the impact of their conversion to methane. Moreover, parameters consisting of chemical inhibition for extraction and the ratio of inoculum/substrate (I/S) on the Biochemical Methane Potential

NS2 ODW PBR P1 P2 SD SMP TAN TMP TMPE TMPT TS VFA VS WSU WWTP

lipid-extracted Nannochloropsis salina oven dry weight photobioreactor Phaeodactylum tricornutum CCMP 632 lipid-extracted Phaeodactylum tricornutum CCMP 632 standard deviation specific methane productivity total ammonia nitrogen total methane productivity total methane potential from experimental calculation from VS destruction percentage total methane potential from theoretical calculation total solids volatile fatty acid volatile solids Washington State University wastewater treatment plant sludge

(BMP) assay were performed for obtaining maximum methane productivity. 2. Methods 2.1. Whole cell microalgae and lipid-extracted microalgae residue Five model microalgae strains, similar to those generated in pilot and commercial algal fuel production facilities, were selected for their span of diversity as well as specific tolerable growth conditions, lipid and protein contents, and other distinct physiological features. Each strain was delivered and studied in both whole cell and extracted residue form, generating ten specific algal biomass treatments (Table 1) that were studied throughout the project. Growth and extraction methods varied by strain and source, amongst them, Chlorella vulgaris UTEX 395 (C) and Phaeodactylum tricornutum CCMP 632 (P) were cultivated by the National Renewable Energy Laboratory (NREL); Nannochloropsis sp. (N), Nannochloropsis salina (NS), and Nanofrustulum sp. (NF) were kindly provided by Seambiotic, Solix Biosystems, and Cellana, respectively. N and NS belong to Eustigmatophyceae, P and NF to Bacillariophyceae, and C to Chlorophyceae. Replete and nitrogen deplete conditions were used to grow C using a suspended polyethylene bag system while P was grown in a 250-liter raceway pond with 5% carbon dioxide sparge and natural daylight (Laurens et al., 2012a) also under replete conditions. Outdoor production systems were used to grow N1 (raceway), NS1 (photo-bioreactor), and NF1 (raceway), with limited information on growth conditions available due to proprietary relations. All microalgae biomass were harvested and centrifuged at room temperature. Subsequently, the biomass were dried and frozen at 20 °C prior to lipid extraction or AD treatment. 2.2. Extraction methods and evaluation While original experimental designs for production and evaluation of all lipid-extracted species were to utilize Soxhlet reactors via Bligh–Dyer extraction chemistry involving a 2:1 (v/v) chloroform/methanol solvent ratio (Lee et al., 1998), subsequent BMP studies showed extensive biological inhibition, apparently as a result of this methodology. To evaluate this hypothesis, five different solvent systems (chloroform/methanol (2:1, v/v) (control), hexane/isopropanol (3:2, v/v), dichloroethane/methanol (1:1, v/v),

P. tricornutum CCMP 632 Eustigmatophyte Marine Hexane GH Pond 17.00 83.00 38.62 5.70 28.07 6.80 1.28 32.50 6.12 16.14 n.d. 28.24 5.68 Nanofrustulum sp. Diatom algae Marine Whole cell Raceway 44.82 45.18 27.45 4.23 29.31 2.90 0.96 12.52 12.95 8.97 6.42 20.74 9.47 n.d.—Not determined. All data are means of duplicate determination.

P1 NF2

Nanofrustulum sp. Diatom algae Marine Pentane Raceway 51.15 48.85 20.19 2.93 33.96 2.93 0.76 8.70 2.55 11.01 8.12 26.59 6.89

NF1 NS2

Nannochloropsis salina Green algae Marine Hexane PBR 10.31 89.69 47.80 6.90 31.80 5.65 0.77 26.72 11.82 17.04 11.68 34.10 8.46 Nannochloropsis salina Green algae Marine Whole cell PBR 7.04 92.96 56.20 8.76 25.63 3.78 0.55 17.21 37.16 11.52 7.95 27.07 14.87

NS1

2.3. BMP assay and performance monitoring

Nannochloropsis sp. Green algae Marine Hexane Raceway 18.08 81.92 43.47 5.13 34.00 6.84 0.80 32.70 3.03 9.56 n.d. 36.63 6.36

N2 N1

Nannochloropsis sp. Green algae Marine Whole cell Raceway 11.71 88.29 52.84 6.00 23.41 7.00 0.73 34.03 10.65 7.64 4.15 35.97 7.55 C. vulgaris UTEX 395 Green algae Estuarine Hexane Suspended 7.70 92.30 44.90 5.03 27.82 8.15 0.79 38.96 2.83 12.15 n.d. 38.36 5.51 C. vulgaris UTEX 395 Green algae Estuarine Whole cell Suspended 11.23 88.77 52.81 6.13 29.33 7.77 0.72 35.13 9.81 16.84 5.29 28.04 6.80 Algal species Morphology Saline Extraction Source Ash VS/TS Carbon Hydrogen Oxygen Nitrogen Sulfur Protein N Lipid-FAME Total carbs Glucose Unkown C/N ratio

C2 C1 % DW

Table 1 Characteristics of non-extracted and lipid-extracted algal biomass.

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dichloroethane/ethanol (1:1, v/v), and acetone/dichloromethane (1:1, v/v)), all widely used in industry (Lee et al., 1998) were evaluated for possible BMP inhibition using whole cell N1 biomass. N1 were immersed in sealed tubes for 24 h into the above organic solvents (i.e. 1.5 g microalgae biomass/10 ml solvent), respectively. Then, those solvents were evaporated in the hood at room temperature and dried at 105 °C until constant weight was achieved, upon which BMP trials were then conducted. Subsequent recognition of a chloroform/methanol inhibition and no similar inhibition involving a 3:2 (v/v) mixture of hexane/ isopropanol at 70 °C and pressure of 1500 psi (Laurens et al., 2012b) led the authors to utilize this extraction methodology for lipid-extracted C, N and P biomass residues, with the exception being the NF2 grown by Cellana, which utilized a methyl pentane solvent that showed no inhibitory effects. The authors noted that even with multiple runs using the hexane/isopropanol solvent system extraction in Soxhlet reactors, fatty acids did remain in the residue due to incomplete extraction as discussed in Laurens et al. (2012b).

P. tricornutum CCMP 632 Eustigmatophyte Marine Whole cell GH Pond 20.92 79.08 44.12 5.14 28.30 6.43 1.29 26.53 7.61 18.95 2.92 25.99 6.86

P2

B. Zhao et al. / Bioresource Technology 161 (2014) 423–430

As all whole-cell microalgae or lipid-extracted microalgae residues received from industry and project partners arrived as compacted freeze dried solids, biomass were milled through a 1 mm screen using a direct-drive cyclone sample mill (Cole Parmer, Verner Hills, IL, US) so as to reduce the level of compaction and reduce particle size. Seed inoculum with total solids (TS) of 18.0 gL 1 and VS of 13.4 gL 1 was obtained from Wastewater Treatment Plant (WWTP) (Pullman, WA) primary anaerobic digester treating activated sludge at 35 °C. The inoculums were transferred in sterile N2-sparged bottles, kept at room temperature during transportation, and then stored at 4 °C until use. During BMP assays, biomass treatments were inoculated with seed and placed into 250 ml flasks that served as bioreactors for the BMP studies. No additional external nutrients/trace elements were added to the BMP bottles as it was assumed that basic nutrient requirements for anaerobic microorganisms were provided by the wastewater-based inoculum. The working volume in each BMP reactor was 200 ml with the rest serving as headspace. Cultures were neutralized with acids and bases to pH 6.95–7.05, flushed with N2 gas for 15 min to induce anaerobic condition, and incubated in a 16-cell automated Challenger AER System (Fayetteville AR) maintained at 35 ± 1 °C and mixed continuously with a magnetic stirrer set to 200 rpm. Two sets of samples (triplicates per ratio) were run separately for the total biogas (i.e. carbon dioxide and methane) as well as for total methane via using pellets of sodium hydroxide with color indicator to scrub carbon dioxide. In a primary experiment for determination of optimal seed concentration to ensure effective AD, inoculation of NS1 and NS2, as test biomass, occurred across five-inoculum to substrate (I/S) ratios—0.1, 0.5, 1.0, 1.5, and 2.0. NS1 and NS2 were selected because of its abundance and high lipid content, which can be considered as a model species for the digestion because of its specific features (Table 1). This was achieved by varying the seed inoculum concentration ranging, from 1 to 20 g VS L 1 while maintaining a constant microalgae substrate concentration of 10 g VS L 1 liquid. Subsequent analysis of data from this primary experiment determined that I/S ratio of 1.0 was suitable for effective AD of all biomass treatments—subsequently all future BMP biomass studies utilized this I/S ratio. 2.4. Analytical methods TS and VS were measured using standard analytical methods (APHA et al., 2012). Micro-metal content were analyzed by first

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digesting with a CEM SP-D microwave digestion system (CEM, Buckingham, England) with subsequently diluted samples containing reference standards analyzed with an Agilent 7500cx ICP-MS (Santa Clara, CA, US) equipped with an octopole collision/reaction cell, all according to a method described by Ovissipour et al. (2013). Elemental analysis for carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and sulfur (S) were performed per standard methods as described in Pella (1990) using a Leco CHN-O-S analyzer (St. Joseph, MI, US). Total ammonia nitrogen (TAN) were evaluated using a Tecator 2300 Kjeltec Analyzer (Eden Prairie, MN, US) using standard methods. Lipids were analyzed as fatty acid methyl esters after a one step acid catalyzed in situ trans-esterification reaction using a GC-FID (Agilent 6890N) equipped with an HP-5 ms capillary column (30 m  0.25 mm id  0.25 lm) according to the procedure of Laurens et al. (2012b). Protein were calculated from elemental N content (Lourenço et al., 2004). Carbohydrates were determined by H2SO4 acid hydrolysis followed by HPLC measurement of monosaccharides (Laurens et al., 2012a). Gas composition was determined by a Varian 3800 GC-TCD (Palo Alto, CA, USA) fitted with a Restek (Bellefonte, PA, USA) shincarbon column (2  1/16 inch) using the method by Wen et al. (2007). The concentrations of volatile fatty acids (VFA), including acetate, propionate, butyrate, and valerate in the effluent were determined by a gas chromatograph (Shimadzu Corp., GC-2014, Japan) equipped with a flame ionization detector and a 30 cm length, 0.25 mm in diameter (ID) and 0.25 lm film capillary column (HPINNOWax, Agilent Technologies, Palo Alto, CA, US). The liquor samples were first centrifuged at 12,000 rpm for 5 min. Subsequently, the supernatant liquids were mixed with an equal amount of internal standard solution (4-methyl-n-valeric acid, 1.162 g/L), acidified by formic acid (1% m/v) with the ratio of 1:4 (v/v) between sample and formic acid, and then filtrated through 0.22 lm membrane (Fisher, PA, USA) and finally measured for free acids. The temperatures of the injector and detector were 250 and 300 °C, respectively. The initial temperature of oven was 70 °C for 3 min followed with a ramp of 15 °C/min to final temperature of 230 °C for 3 min. Helium was used as carrier gas with a flow rate of 0.93 ml/min at a split ratio of 40:1. Detector temperature was 300 °C.

2.5. Statistical analysis All data analyses were performed with SPSS Statistics version 19 for Windows (IBM, New York). The statistical significances were examined using one-way analysis of variances (ANOVA) and the level of significance was defined at P < 0.05. The 95% methane production and maximum methane rate were evaluated using a modified Gompertz equation (Zwietering et al., 1990). First order hydrolysis constant (kh) with various digested substrates was estimated according to the method described by Angelidaki et al. (2009). Total methane potential from theoretical calculation (TMPT) was calculated according to models suggested by Sialve et al. (2009) and Heaven et al. (2011).

3. Results and discussion 3.1. Characteristics of whole microalgae and their biomass residues In order to evaluate the various microalgae for their conversion to methane, a wide range of material and chemical compositions were carefully investigated as shown in Table 1. Across all biomass evaluated, ash content and total metal concentration were between 8–45% and 0.31–1.0%, respectively (Supplementary Table S1). All algal species had considerable levels of sodium and potassium (0.5–3%), due to their culturing conditions. NF species had a higher level of calcium, magnesium and iron compared to others while C species were rich in copper and zinc and P1 had a higher level of manganese. In general, the concentration of the metals within all of the algal biomass was not high enough to inhibit the AD process, while still supplying more than adequate supply of necessary micro-metal nutrients for biomass maintenance and growth. The major non-mineral compositions (Table 1) were C (27.45– 56.20%), H (4.23–8.76%), O (23.41–29.33%), N (2.90–7.77%) and S (0.55–1.29%) for whole-cell microalgae while C (20.19–47.80%), H (2.93–6.90%), O (27.82–34.00%), N (1.93–8.15%) and S (0.76–1.29%) for lipid-extracted microalgae, respectively. C/N ratios for whole cell biomass ranged from 6.8–14.7 with a generalized lowering of this range in lipid-extracted residues, producing a

Table 2 Biogas and methane production from whole microalgae and lipid-extracted microalgal biomass. Algae biomass

C1

C2

N1

N2

NS1

NS2

NF1

NF2

P1

P2

Biogas Production (L biogas/L liquid) CH4 production (L CH4/L liquid) CH4 fraction (%) 95% CH4 production (d) First order hydrolysis constant (kh) Max CH4 rate (L CH4/ L/d) Effluent pH TAN effluent (mg N/L) Total VFA effluent (mg/L) VS reduction (%) SMP (L CH4/g VS fed) TMPE (L CH4/g VS d) TMPT (L CH4/g VS) Biodegradability (% SMP/TMPT)

5.28 ± 0.16

5.31 ± 0.18

5.36 ± 0.20

5.90 ± 0.24

8.36 ± 0.26

5.11 ± 0.13

6.93 ± 0.30

4.37 ± 0.10

5.20 ± 0.15

4.89 ± 0.13

3.37 ± 0.11

3.14 ± 0.18

3.57 ± 0.05

3.99 ± 0.13

5.57 ± 0.05

3.83 ± 0.13

5.07 ± 0.05

3.04 ± 0.05

3.37 ± 0.15

3.39 ± 0.13

63.82 9.75

59.13 12.04

66.60 5.58

67.62 13.94

66.63 12.71

74.95 7.84

73.16 11.36

69.56 9.89

64.81 13.49

69.32 9.70

0.22

0.19

0.41

0.17

0.16

0.24

0.22

0.31

0.16

0.23

0.046

0.037

0.087

0.037

0.074

0.054

0.072

0.056

0.040

0.050

7.17 ± 0.11 401 ± 38 250 ± 40

7.50 ± 0.12 865 ± 29 177 ± 24

7.16 ± 0.19 458 ± 51 n.d.

7.05 ± 0.10 658 ± 44 n.d.

6.94 ± 0.17 228 ± 26 151 ± 12

6.94 ± 0.07 326 ± 24 155 ± 15

7.16 ± 0.04 322 ± 33 n.d.

7.10 ± 0.06 509 ± 23 118 ± 16

7.28 ± 0.10 613 ± 35 190 ± 13

7.45 ± 0.13 690 ± 47 55 ± 6

66.06 ± 3.42 0.337 ± 0.011 0.511 ± 0.012 0.604 55.76

64.21 ± 2.57 0.314 ± 0.018 0.489 ± 0.012 0.552 60.19

65.90 ± 3.19 0.357 ± 0.005 0.542 ± 0.020 0.682 52.36

64.41 ± 1.26 0.399 ± 0.013 0.619 ± 0.013 0.531 75.09

78.50 ± 1.22 0.557 ± 0.005 0.710 ± 0.008 0.749 74.33

73.83 ± 3.40 0.383 ± 0.013 0.519 ± 0.014 0.598 64.06

76.41 ± 3.35 0.507 ± 0.005 0.663 ± 0.023 0.882 57.47

59.33 ± 3.78 0.304 ± 0.005 0.512 ± 0.025 0.457 66.49

70.60 ± 3.04 0.337 ± 0.015 0.477 ± 0.014 0.629 53.54

60.20 ± 3.34 0.339 ± 0.013 0.564 ± 0.013 0.580 58.48

n.d.—Non detected. All data are means ± SD of triplicate determination.

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range from 5.51–8.46. Both these range, and in particular the lipidextracted, are well below optimal C/N ratios of 20–30 preferred for both aerobic and anaerobic treatment of organic wastes. Such low ratios have been implicated as harbingers of product inhibition resulting from the anaerobic conversion of protein N to ammonia N—a known inhibitor to the AD process both in its ionic and, in particular, its free form (Koster and Lettinga, 1984). BMP effluent analysis showed TAN levels ranging from 228–1316 mg N L 1 with the higher range of values arising from lipid-extracted digestion (Table 2), however this entire range is well below threshold TAN inhibition indicators of 1700–2000 mg N L 1 reported by Koster and Lettinga (1984). TAN results produced here are highly accordant with the effluent ammonium concentrations (550–700 mg L 1) from digestion of Scenedesmus biomass containing high levels of proteins (43.5% and 55.1%) (González-Fernández et al., 2013), indicating that the inhibition of methane production via TAN derived from microalgae protein can be ignored during the digestion, at least in this report. Frear et al. (2011) showed similar lack of TAN inhibition during stable digestion of manure at a C/N ratio of 11, even while producing higher effluent TAN concentrations (2600 mg N L 1)—presumably a result of bacterial acclimation to these higher TAN levels (Calli et al., 2005). As will be discussed in succeeding sections, careful control of loading rate and I/S ratio used during AD allowed for effective control of these potential inhibitory actions, yielding stable biogas production curves. Of interesting note is the lipid, protein and carbohydrate compositional analysis of the biomass. Lipid contents in the whole cell microalgae ranged from 7.61% to 37.61%, containing in particular palmitic acid (C16) and palmitoleic acid (C16:1n9) (Supplementary Table S2). This range indicates that despite the commercial interest of these strains as well as the attempts to 1000

3.2. Effect of I/S ratio on biogas and methane production In BMP assay, the ratio of I/S is an essential parameter that is able to affect the production of methane and the accumulation of VFA. As shown in Fig. 1and Table 3, the maximum biogas production, VS reduction, and methane content were reached at the I/S ratio of 1.0 for NS1 while at the I/S ratio of 0.5 for NS2. When above these threshold levels, pH remained stable near neutral (7.10–7.20) while the total VFA in their respective effluent were quite low (146 and 82 mg L 1), both of which are emblematic of effective AD treatment. This pH range is a commonly recognized range during the anaerobic process. When the I/S ratios were increased greater than 1.0, the pH remained near neutral and VFA could not be detected regardless of whole microalgae and lipid-extracted biomass

NS1 I/S 0.1

1000

NS1 I/S 0.5

900

NS1 I/S 1.5

Biogas Production (ml)

600 500 400 300

400 300

100 0 10

15

20

25

NS2 I/S 2.0

500

200

5

NS2 I/S 1.5

600

100 0

NS2 I/S 1.0

700

200

0

NS2 I/S 0.5

800

NS1 I/S 2.0

700

NS2 I/S 0.1

900

NS1 I/S 1.0

800

Biogas production (ml )

accumulate lipids through control of growth conditions, in general, the lipid concentrations were relatively low, with even NS1, at 37.61%, somewhat below industry targets. In addition, attempts at lipid extraction resulted in only moderate removal percentages, ranging from 19.6–80.3% removal when including the P2 outlier and 68.2–80.3% when excluding its presence. The meaning being that a considerable fraction (>20%) of lipids remains within the residual biomass, contributing extensively to AD biogas production as well as potential LCFA inhibition during digestion. While, biomass and extraction processing were not done to scale or truly representative of existing or future industry capabilities, these present numbers offer credence to previous discussions for the role AD can play as a commercial-ready processing technology for whole-cell microalgae to less valued methane (Sialve et al., 2009), not to mention its role in the more discussed option as processor of residual biomass that still contains a large share of the original energy-rich biomass.

30

0

5

10

Time (Day)

15

20

25

30

Time (Day)

a

b Fig. 1. The biogas production from NS1 and NS2 at different I/S ratio.

Table 3 Biogas production from whole microalgae NS1 and its biomass residues NS2 at different I/S ratio. Algae biomassa

NS1

Ratio (I VS/S VS) Biogas production (L Biogas) Effluent pH Total VFA effluent (mg/ L) VS reduction (%) CH4 (%) 5 days 30 days

0.1 0.5 1.0 1.5 2.0 0.1 0.5 1.0 1.5 2.0 0.099 ± 0.015 0.650 ± 0.010 0.836 ± 0.011 0.833 ± 0.011 0.832 ± 0.016 0.173 ± 0.018 0.501 ± 0.016 0.496 ± 0.011 0.496 ± 0.015 0.510 ± 0.025 5.80 ± 0.29 917 ± 52

NS2

6.72 ± 0.24 593 ± 39

6.96 ± 0.17 146 ± 23

19.55 ± 2.18 53.45 ± 3.64 60.10 ± 4.58 4.55 45.71 64.93 40.78 67.32 65.22

7.06 ± 0.13 n.d

7.10 ± 0.11 n.d

6.29 ± 0.17 823 ± 67

6.94 ± 0.13 82 ± 16

60.01 ± 3.63 64.50 64.20

75.38 ± 6.21 64.60 64.05

31.50 ± 2.13 5.46 47.04

59.61 ± 5.32 62.56 ± 4.69 61.88 59.63 63.98 62.33

n.d, not detected by GC. All data are means ± SD of triplicate determination. a All data in this table were from 100 ml solution in the bottles.

7.11 ± 0.15 n.d

7.19 ± 0.12 n.d

7.20 ± 0.17 n.d

62.80 ± 3.23 58.87 62.58

63.41 ± 4.56 59.51 62.94

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residues, although overall biogas production showed at times a slight improvement due to additional VFA conversion. When the I/S ratio was below the threshold, pH dropped to a more acidic zone in the range of 5.80–6.29 while levels of VFA achieved notably higher levels in the digester, both indicating inhibition of methanogens. Thereafter the methane production rate diminished or even stopped. The required higher ratio for NS1 digestion verified the high ratio was capable of converting the produced VFA to biogas at a relatively short time during the anaerobic digestion, and also indicated it was necessary for the degradation of high lipid content algae. The I/S ratio also had some impact on the composition of biogas. For example, the produced methane from NS1, most likely comprising some hydrogen, accounted for 40.78% at the I/S ratio of 0.1 while 65.22% at the I/S ratio of 1.0. Thus while, little improvement in biogas production was seen as the I/S ratio was raised above 1.0, there most likely was a greater conversion to methane as indicated by the reduced VFA concentrations, presumably due to an enriched and more robust methanogenic community, capable of converting both the acetate and hydrogen forms to methane. The threshold I/S ratio of 1.0 is consistent with literature. Hashimoto (1989) determined that a minimum ratio of 0.5 was required for straw digestion at concentrations of 10–40 g VS L 1. Furthermore, Hashimoto showed that maximum methane production rates were achieved when I/S ratios reached 2.0. Owen et al. (1979) as well as Chynoweth et al. (1993) showed similar results and suggested I/S ratios of 1.0 and 2.0, respectively. An explanation for the observed trend in I/S ratio as well as the varying impact on whole cell versus lipid-extracted biomass involves known mechanisms for LCFA inhibition in anaerobic cultures. While lipids are rapidly hydrolyzed to LCFA and glycerol during AD, subsequent further degradation, via beta-oxidation, to acetate and hydrogen is less rapid and often delayed, not allowing for attainment of the true

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biogas potential contained within lipids. Primary reasons for this delay rest on the toxicity of the LCFA intermediate (Palatsi et al., 2009), with studies reporting scum layer production with associated biomass washout (Hwu and Lettinga, 1997) as well as microbial membrane transportation inhibition (Hwu et al., 1998), all due to the tendency for LCFA to form adsorptive layers around the microbial surfaces. Degree to which LCFA is experienced and therefore presumably to which the adsorptive mechanism is elicited depends upon specific surface area, carbon chain length, degree of saturation and most importantly, type of microbe, with methanogens reported to be more susceptible to LCFA inhibition as compared to acidogens (Pereira et al., 2003). The above summary of known LCFA interaction with AD biology fits in nicely with the BMP curves summarized in Fig. 1. As noted, for NS1 a minimum I/S ratio of 1.0 was required to achieve stable digestion, while for NS2, the required I/S ratio was only 0.5. Notably, NS2 has considerably less lipids and therefore LCFA due to the extraction process, thus not exposing the microbes to as high a concentration of surface attaching chemicals. However, according to the gas production in Fig. 1 and the VFA at low I/S ratio in Table 3, pH effect might be a more important factor on microalgal digestion than surface adhesion. If not, the gas production from the NS1 was not that easy to come back after 15 days anaerobic digestion. Conversely, the higher concentrations of LCFA from microalgae presumably induced a combined greater effect on the degree of surface adhesion as well as the pH drop, and therefore microbial inhibition, requiring a greater inoculums concentration to overcome this phenomenon. The conclusion is that in order for effective digestion of whole cell or lipid extracted microalgae biomass to occur without disruption a suitably large inoculums concentration is required, particularly if the remaining lipid concentration is large. This conclusion has implications to future

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scale-up as reactor designs will need to ensure effective biomass accumulation, perhaps requiring biomass retaining reactors such as anaerobic sequencing batch reactors (ASBR), anaerobic sludge bed reactors (ASBR), and hybrid reactors, as opposed to more traditional continuous stirred tank reactors (CSTR). 3.3. Biogas and methane production from the whole and lipidextracted microalgae residues In order to determine the ultimate biogas and methane yield from ten different microalgae used in the present study as well as to evaluate their biodegradability, an array of batch tests were conducted for 25 days. As shown in Table 3 and Fig. 2, overall organic conversion ranged from 59.33–78.50 as a measure of %VS reduction. Notably this range was relatively small, showing that all of the biomass could have a relatively similar percentage of total organic material converted to methane during the 20–30 day digestion, although important variations did exist. For example, each lipid-extracted form resulted in lowered VS reduction as compared to its whole cell companion. This is presumably due to lipidextracted material being composed of a greater fraction of non-biodegradable or recalcitrant organic material such as crystalline cellulose, and chitinous fibril, etc. (Zheng et al., 2011). Solvent extraction methodologies had significant impact on subsequent AD of the residue biomass. The chloroform/methanol solvent mixture resulted in near complete methane production inhibition while all other tested solvent mixtures resulted in similar non-inhibited production. This infers that small amounts of chloroform even after air and oven drying can potentially be retained to a significant inhibitory level during AD. While explanation of how highly volatile chloroform is retained within the biomass, there is merit to chloroform being able to severely inhibit AD. Chloroform severely inhibited both acid fermentation and methane production at high concentrations (Hu and Chen, 2007). However, methanogens were sensitive to chloroform at very low concentration, selectively inhibiting methanogenic activity while not affecting hydrolysis and the fermentation process (Hu and Chen, 2007). Chloroform reportedly inhibited the production of methane from both H2/CO2 and acetate forms of methanogens (Chidthaisong and Conrad, 2000). With respect to this particular microalgae AD observation, Ehimen et al. (2009) reported a low methane production rate from the algal biomass after chloroform/methanol lipid-extraction. It was their assertion that repression of methanogenesis was attributed to the inhibitory effect of chloroform that remained bound to the residual microalgae biomass. Analysis of the biogas production over time as well as the 95% methane production parameter shows that even 30+ day digestion led to little significant increases in appreciable biogas production and therefore organic degradation, with most biomass achieving 95% of realized potential by no later than 15 day. This indicates that as with animal manures, extended digestion time for minimal return on degradation of recalcitrant organics, has diminishing economic returns (Frear et al., 2011). While pre-treatments, not studied in this project could make recalcitrant material more accessible to anaerobic degradation (Sialve et al., 2009; MUssgnug et al., 2010), the already high %VS reductions also show that from a cost perspective this might not be economically rewarded, unless an extremely high price for methane energy off-takes and/or necessary biomass solids reduction is preferred. Unlike literature representing a wide range of maximum methane potentials, this study showed that while important differences did exist, the overall range among all biomass, both whole cell and lipid-extracted was quite small, ranging from 0.304–0.557 L CH4 g VS 1. For comparison purposes, this SMP range is higher than

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that of dairy manure (0.21–0.24 L CH4 g VS 1 (Frear et al., 2011), similar to that of food scraps/green waste now utilized in municipal digesters (0.35–0.55 L CH4 g VS 1 (palatsi et al., 2009), but below that of pure lipids and/or fats, oils, and greases (FOG) (0.90– 1.0 L CH4 g VS 1 (Møller et al., 2004). As might be expected from known high SMP for lipids and FOG, the most important variable to this microalgae SMP spread was lipid content. A linear relationship occurred between ash-free lipid dry weight percentage and the resulting SMP of the ten evaluated microalgae biomass (Data shown below). As each of the biomass had varying percentages of ash as well as lipid, simple subtraction of the non-biodegradable ash allowed for this comparison between lipid content (% Ash Free Dry Weight) and SMP (L CH4/g VS Fed). While not a perfect relationship (SMP = 6.77  10 3LC + 0.296; R2 = 0.814), it is clear that one of the most important parameters dictating ultimate SMP was the whole cell or residual biomass lipid content. Presumably, as algal refineries come closer to reality, lipid-extraction processes will become more effective than done in this study, leading estimated SMP to be a bit lower within the developed range, but notably small in range for modeling purposes. More importantly, as BMP studies on microalgae biomass can be costly and time-consuming, such narrowing of the SMP range with a suitable linear equation for relationship to remaining lipid content can be important. Theoretical methane potentials (TMPT) were calculated from the elemental characterization of the microalgae biomass and compared to the generated SMP for determination of biodegradability factors. Biodegradability factors ranged from 52.36–75.09% with greater percentage biodegradability in general found within the lipid-extracted biomass, which is opposite to the findings related to VS% and SMP. While VS% and SMP reduced upon lipidextraction, presumably due to larger percentage of more recalcitrant organic material, the biodegradability ratio increased, presumably due to more efficient degradation of available type of organic material—perhaps a legacy of the aforementioned LCFA inhibition and its reduced intensity with lower lipid and LCFA content. 4. Conclusion The control of a viable I/S ratio (1.0 VS/VS) during BMP was instrumental in overcoming pH drop and LCFA inhibition, and providing for a more effective range of SMP (0.304–0.557 L CH4/g VS) and VS reduction (59.33–78.50%). The SMP was not so much related to microalgal species but more to lipid content. All biomass digested well with most achieving 95% methane accumulation prior to day 15. Methane content ranged from 60–75% while effluent TAN and VFA levels were quite low, indicating effective, complete digestion despite all samples having C/N ratios well below that ideally preferred. Acknowledgements This work (DOE Grant # 22902) was supported from the Office of Science of the U.S. Department of Energy to the National Renewable Energy Laboratory with sub-contract to Washington State University. Additional recognition is given to Ms. Cynthia Alwine for her assistance in analytical work. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014.03. 079.

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