Thermophilic and mesophilic methane production from anaerobic degradation of the cyanobacterium Spirulina maxima

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  • Resources, Conservation and Recycling, 1 (1988) 19-26 19 Elsevier Science Publishers B.V./Pergamon Press plc - - Printed in The Netherlands

    Thermophilic and Mesophilic Methane Production from Anaerobic Degradation of the Cyanobacterium Spirulina Maxima*

    V.H. VAREL, T.H CHEN and A.G. HASHIMOTO

    Roman L. Hruska U.S. Meat Animal Research Center, U.S. Department of Agriculture, Clay Center, NE 68933 (U.S.A.)

    (Received June 4, 1987; accepted in revised form September 9, 1987)

    ABSTRACT

    Methane production from fermentation of the cyanobacterium, Spirulina maxima, as the sole substrate was investigated in 200 mL working-volume anaerobic digesters maintained at 35 and 55C. Digesters were fed once-per-day with a feed concentration (Sto) of 22.5 g volatile solids (VS) per liter, at retention times (0) of 8, 12, and 16 days. Digester contents were mixed for one min before and after feeding. After 3 volume turnovers, effluent samples were obtained on four consecutive days. Methane production rate (L CH4/L-day ) and methane yield (B) ( L/g of COD fed) at 35C were 0.47 and 0.09, 0.41 and 0.15, and 0.31 and 0.15 at the respective 0 of 8, 12, and 16 days; at 55C they were 0.20 and 0.05, 0.31 and 0.11, and 0.19 and 0.09, respectively. The ultimate methane yield (Bo) after 105 days of batch fermentation was 0.22 L CH4 per g COD fed (0.33 L CH4/g VS fed). COD degradation at these retention times and temperatures was between 23 and 40%, ammonia nitrogen between 1.12 and 1.86 g/L, and alkalinity between 7.0 and 7.8 g CaCOJL. The concentration of total volatile acids at 35C was 4.07, 2.58 and 3.13 at 0 =8, 12, and 16 days, respectively; at 55 C they were 6.78, 4.18, and 4.07, respectively. These results indi- cate that the biomass of S. maxima can be used as a sole nutrient for methane production at mesophilic and thermophilic temperatures. However, the methane production rates at these re- tention times are higher at the mesophilic temperature. These rates are significantly lower than those obtainable with cattle or swine wastes because greater maximum loading rates can be ob- tained with these wastes before digester failure occurs.

    INTRODUCTION

    Spirulina maxima is a rapidly growing microscopic cyanobacterium (blue-green alga) that is easily separated from its growth medium by simple filtration, is highly digestible as a food source, and contains up to 70% protein of excellent quality [ 1,2 ]. It grows in a high alkaline medium, pH 9-10, which eliminates many potential contaminating microorganisms. Many studies sug-

    *Mention of commercial or proprietary products in this report does not constitute a recommen- dation or an endorsement of these products by the U.S. Department of Agriculture.

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    TABLE 1

    Composition of medium for growth of Spi ru l ina max ima a

    Component (g/L)

    NaHCO~ 16.8 K2HP04 0.5 NAN03 2.5 K~S04 1.0 NaC1 1.0 MgS04" 7H20 0.2 CaC12.2H20 0.04 FeSO4.7H20 0.01 EDTA 0.08 Solution A (mL/L) b 1.00 Solution B (mL/L) c 1.00

    aReferences [ 6 ] and [ 7 ] ; final pH was 9.5-10.0 bSolution A contained (g/L): H3B03, 2.85; MnC12-4H20, 1.81; ZnS04-7H2, 0.22; CuSO4-5H20, 0.08; Na2MoO4" 2H20, 0.015. cSolution B contained (g/L) : NH4V03, 0.023; KCr (S04) 2" 12H20, 0.096; NiS04- 7H20, 0.048; Na2WO4-2H20, 0.018; Ti2(SO4)a, 0.04; COC12"6H20, 0.044.

    gest that it is an excellent food source for man and animals because of its high protein content, low content of nucleic acids, high concentrations of vitamins and other growth factors, and the presence of a cell wall that is easily digestible when compared to that of yeast or algae. Because it has these qualities and the fact that it is easily harvested, due to its spiral shape and presence of gas vac- uoles, it is a potential feedstock or energy crop for the production of methane by anaerobic bacteria [ 3,4 ]. S. maxima is capable of using atmospheric carbon dioxide as a carbon source, has the potential to grow in sea water [ 5 ], or can use agricultural waste products as a growth substrate [ 6,7]. The objectives of these studies were to compare mesophilic (35 C) and thermophilic ( 55 C) fermentations at short retention times using biomass of the cyanobacterium, S. maxima as the sole substrate for methane production.

    MATERIALS AND METHODS

    S. maxima was cultured at room temperature (25C) in 38-L aquariums using the medium listed in Table 1 [6,7]. The organism was harvested and concentrated to a slurry containing 12% total solids with an Amicon DC2 hol- low-fiber ultra-filtration system using HIP-100-20 filter cartridges (Amicon Corporation, Danvers, MA ), placed on a 38 pm( 400 mesh) screen, rinsed with distilled water, bottled at a specific VS concentration, and stored at -20C until used.

    Daily fed digesters consisted of 500 mL aspirator bottles operated with a 200

  • 21

    mL working volume. They were placed in chambers maintained at 35 and 55 C. Inocula for the digesters came from cattle waste digesters maintained at the same temperatures. Digesters were fed once-per-day with S. maxima biomass at 2.25% VS and at retention times (0) of 8, 12, and 16 days. Digester contents were mixed with a magnetic stir bar for one min before and after feeding.

    Slurries fed and withdrawn from the digesters were analyzed for total solids, VS, alkalinity (to pH 3.7), pH, and total volatile acids (TVA or actic acid, silicic acid method) using standard methods for waste water analyses [8]. Organic nitrogen (Kjeldahl) was determined as described by Wael and Gehrke [9]. Individual volatile fatty acids were measured using a Hewlett Packard Model 5840A gas chromatograph with dual flame ionization detectors. Coiled glass columns {0.32 cm ID by 183 cm) packed with 15% SP-1220 and 1% H~PO4 on 100/200 mesh Chromosorb WAW (Supelco, Inc., Bellefonte, PA) were used for the separation of acids. Nitrogen carrier-gas flow was 40 mL/min and injector, oven and detector temperatures were 200, 125 and 250 C, respec- tively. Ammonia was determined colorimetrically using phenol hypochlorite [ 10 ]. Chemical oxygen demand (COD) was determined using the Standard Ampule Method (O.I. Corporation, Ampule C.O.D. Operating Procedure Manual, Dallas, TX).

    Biogas from digesters was collected in gas-impermeable bags. Gas volume was measured by solution displacement (water saturated with NaC1) and cor- rected for temperature and pressure to standard conditions. Gas composition was analyzed by gas chromatography as previously described [ 10 ].

    Digesters were assumed to be in steady-state conditions after three volume turnovers. Samples were analyzed on four consecutive days once steady state was achieved. Retention time was then reduced to the next lower level. Each temperature (35 and 55 C ) and 0 (8, 12, 16 days ) were replicated.

    Batch digesters maintained at 35 C were used to determine ultimate meth- ane yield (Bo). These digesters, which were run in duplicate, consisted of 120 mL serum bottles with a 50 mL working volume. They were started with in- oculum from the daily fed digesters. A mineral-salts solution [ 11 ] was added to control digesters in place of the cyanobacterial substrate. The 35 C diges- ters were incubated for 105 days, during which time gas volume was periodi- cally determined as described by Owen et al. [ 12 ], and composition as described above. The ultimate methane yield (Bo) was calculated by subtracting the average accumulated methane volume of the inoculum from the average ac- cumulated methane volume of the cyanobacterium plus inoculum, and the dif- ference was divided by the mass of cyanobacteria fermented.

    RESULTS

    Preliminary studies indicated that the cyanobacterial biomass as harvested from the growth medium (Table 1 ) generated a pH greater than 8.0 in the 35

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    TABLE 2

    Data from fermentation of Spirulina maxima at 35 C a

    Parameter Retention time (days)

    8 12 16

    Methane production L CH4/L-day 0.47 + 0.01 0.41 _+ 0.008 0.31 _+ 0.003 L CH4/g COD fed 0.09 _+ 0.002 0.15 _+ 0.005 0.15 +__ 0.001

    CH4 (%) 61.2 ___0.2 65.6 _+1.4 61.7 _+0.3 COD reduction (%) 27.8 _+1.3 39.8 _+2.0 37.4 _+ 1.9 Total VFA's (g/L) 4.07 _+ 0.06 2.58_+ 0.08 3.13 _+ 0.03

    Acetate 2.87 _+ 0.10 2.16_+ 0.07 2.67 _+ 0.03 Propionate 0.40 _+ 0.03 0.11 _+ 0.008 0.16 _+ 0.006 Butyrate 0.09 _+ 0.003 0.01 _+ 0.002 0.15 _+ 0.001 Isobutyrate 0.21 _ 0.001 0.04 _+ 0.003 0.58 _+ 0.001 Valerate 0.01 -+ 0.001 0 0 Isovalerate 0.47 _+ 0.01 0.26 -+ 0.005 0.22 _+ 0.001

    Organic nitrogen (g/L) 2.65 _+ 0.08 2.52 _+ 0.10 Ammonia nitrogen (g/L) 1.55 _+ 0.17 1.12 _+ 0.10 Alkalinity (g CaCOJL) 7.00 _ 0.07 7.03 _+ 0.05 7.03 _+ 0.06 pH 7.05 _+ 0.01 7.27 _+ 0.02 7.20 _+ 0.01 /(0 1.6 1.5 2.1

    aMean _+ S.E. from four analyses on four consecutive days. Feed concentration (Sto) was 22.5 g VS/L. bKinetic parameter [ 14].

    and 55 C digesters, which eventually caused the fermentations to fall. Thus, it was necessary to rinse the biomass with water after it had been harvested to dilute the mineral salts which created the high alkaline conditions. Once this was done no further problems were experienced with high pH.

    Results from Table 2 indicate that the maximum amount of methane pro- duced from fermentation of S. maxima at 35 C was 0.47 L CH4/L-day at 0 -- 8 days. This compares to 0.31 L CHt/L-day at 0-12 days for the 55C digesters (Table 3). The percentage of CHt in the biogas was slightly higher for the 35C digesters (maximum 65.6% at 0=12 days) than the 55C digesters (maximum 63.2% at 0=12 days). The biogas in the 55C digesters at 0=8 days contained 48.7% CHt, which suggeted that some form of inhibition was occurring at this 0. COD reduction ofbiomass was greater in the 35 C digesters with a maximum reduction ( 39.8% ) occurring at 0 = 12 days.

    The total VFA's in both the 35 and 55 C digester contents were much higher than what might be expected from digesters containing an equivalent amount of organic matter in the form of animal waste [ 11,13 ]. The contents from the 55 C digester, 0 = 8 days, contained the highest level of acids, 678 g/L, which again suggested that some factor was inhibiting the fermentation. The concen-

  • TABLE 3

    Data from fermentation of Spirulina maxima at 55 C a

    23

    Parameter Retention time (days)

    8 12 16

    Methane production L CHJL -day 0.20 0.005 0.31 0.10 0.19 4- 0.004 L CHJg COD fed 0.05 0.001 0.11 _ 0.003 0.09 0.001

    CH4 (%) 48.7 _+0.2 63.2 _+0.9 57.2 0.8 COD reduction (%) 22.6 1.2 31.8 1.9 30.9 1.6 Total VFA's (g/L) 6.78 ___ 0.11 4.18 0.11 4.07 0.09

    Acetate 4.17 _ 0.08 1.81 0.08 1.83 0.07 Propionate 1.17 ___ 0.01 1.30 0.01 1.15 0.03 Butyrate 0.26 0.006 0.08 0.007 0.06 0.004 Isobutyrate 0.36 0.006 0.31 0.003 0.20 0.005 Valerate 0.01 ___ 0.001 0.02 0.002 0.03 0.001 Isovalerate 0.78 0.01 0.59 0.008 0.82 0.01

    Organic nitrogen (g/L) 2.66 0.08 2.56 0.10 Ammonia nitrogen (g/L) 1.86 0.17 1.20 0.06 Alkalinity (g CaCOJL) 7.80 0.07 7.85 0.03 7.83 0.05 pH 6.97 0.04 7.47 0.02 7.45 0.03 /~ 13.7 6.4 11.4

    aMean + S.E. from four analyses on four consecutive days. Feed concentration (Sto) was 22.5 g VS/L. bKinetic parameter [ 14].

    "~ 0 ,25 . 2 c l 0 o 0.20,

    I 0.15. r,D

    .-J

    d O.lO. ..J w >-

    w z 0.05-

    I

    0.00 0 2'o 4'o do go 15o

    Doy Fig. 1. Ultimate methane yield (Bo) from batch fermentation of S. maxima at 35 C.

    trations of branched-chain acids, isobutyrate and isovalerate, were proportion- aly higher than the concentration of these acids found in typical animal waste fermentations, and higher than the respective straight-chain acids, presum-

  • 24

    ably because of the high protein content of the algal biomass. Organic and ammonia nitrogen varied little between the contents of the 35 and 55 C diges- ters. The ammonia nitrogen was approximately 3 to 5 times higher in these digesters than in digesters fed swine manure at a similar substrate concentra- tion [14]. The pH of the digesters remained in a range (6.97 to 7.47) satisfac- tory for methanogenic fermentation after washing the biomass. The washing reduced the pH of the substrate from 9.5 to 8.0.

    Figure 1 shows the change in ultimate methane yield with time during the batch fermentation of S. maxima at 35 C. Approximately 90% of the methane is produced in the first 20 days of the fermentation. After 105 days, the meth- ane yield was 0.22 L CH4/g COD fed or 0.33 L CH4/g VS fed.

    DISCUSSION

    The data in Tables 2 and 3 indicate that biomass from S. maxima can serve as the sole substrate for methane production at 35 or 55 C. No advantages were found with the thermophilic fermentation. The methane yield from S. maxima at 0 = 12 and 16 days is approximately 80 to 90% that of beef cattle manure at 35C, but only 50 to 60% at 55C [15]. This suggests that some nutrients may be lacking, a poor C/N ratio exists, or toxic compounds are present or are generated in the fermentation at 55 C. Less stability at 55 C was observed as indicated by the higher concentration of volatile fatty acids and pH below 7.0 at 0 -- 8 days. The kinetic parameter (K) was calculated for each set of conditions by the procedures described elsewhere [ 14 ] and shown in Tables 2 and 3. The K values ranged from 1.5 to 2.1 for the 35C fermen- tations and from 6.4 to 13.7 for the 55 C fermentations. The higher K values for the thermophilic fermentations indicate that they were inhibited much more than the mesophilic fermentations. Thus, a mesophilic fermentation may be the preferred temperture for methane production from S. maxima. This agrees with the study of Samson and LeDuy [ 4 ] in which they found 35 C to be more efficient than 52 C in degradation of this alga to methane.

    Although the ultimate methane yield at infinite hydraulic retention time from the cyanobacterial biomass at 35 C may be comparable to that obtained from cattle waste [ 15 ], the maximum methane production rate is significantly lower than that obtainable with cattle or swine waste because loading rates can be greater with these wastes before digester failure occurs. It was not possible to successfully operate the S. maxima digesters at 0 = 6 days, whereas cattle waste digesters are routinely operated at this 0 and lower [ 10,16,17]. Methane production rates of 4.5 [15] and 6.1 L CHt/L-day [14] have been obtained with cattle waste and 3.2 L CHt/L-day with swine waste [13]. The highest methane production rate we obtained in this study and that obtained by Sam- son and LeDuy [ 4 ] with S. maxima biomass was 0.47 and 0.40 L CH4/L-day, respectively. Thus, from a practical viewpoint, a much greater volume of meth-

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    ane can be produced from cattle or swine waste in a shorter period of time if the digester size is constant. Therefore, with the high protein content of S. maxima, future studies may be warranted to look further into feeding the bio- mass to livestock. Some studies have already shown promising potential in this area [6,7].

    Comparing results from this study and that of Samson and LeDuy [ 4 ], sug- gest that loading rate appears to be a sensitive parameter governing perform-...

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