Anaerobic co-digestion of the marine microalga Nannochloropsis salina with energy crops

Bioresource Technology 148 (2013) 428–435

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Anaerobic co-digestion of the marine microalga Nannochloropsis salina with energy crops Sebastian Schwede ⇑, Alexandra Kowalczyk, Mandy Gerber, Roland Span Ruhr-University Bochum, Institute of Thermo- and Fluid Dynamics, Universitaetsstr. 150, D-44801 Bochum, Germany

h i g h l i g h t s  Anaerobic co-digestion of corn and Nannochloropsis salina biomass was investigated.  At higher organic loading rates process failure occurred in corn mono-digestion.  Nannochloropsis salina biomass enhanced process stability in co-digestion with corn.  Algal biomass induced enhanced alkalinity and balanced nutrient composition.  Results suggest Nannochloropsis salina as convenient feedstock for co-digestion.

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Article history: Received 19 June 2013 Received in revised form 21 August 2013 Accepted 23 August 2013 Available online 6 September 2013 Keywords: Microalgae Anaerobic digestion Biogas Nannochloropsis salina Corn silage

a b s t r a c t Anaerobic co-digestion of corn silage with the marine microalga Nannochloropsis salina was investigated under batch and semi-continuous conditions. Under batch conditions process stability and biogas yields significantly increased by microalgae addition. During semi-continuous long-term experiments anaerobic digestion was stable in corn silage mono- and co-digestion with the algal biomass for more than 200 days. At higher organic loading rates (4.7 kg volatile solids m3 d1) inhibition and finally process failure occurred in corn silage mono-digestion, whereas acid and methane formation remained balanced in co-digestion. The positive influences in co-digestion can be attributed to an adjusted carbon to nitrogen ratio, enhanced alkalinity, essential trace elements and a balanced nutrient composition. The results suggest that N. salina biomass is a suitable feedstock for anaerobic co-digestion of energy crops, especially for regions with manure scarcity. Enhanced process stability may result in higher organic loading rates or lower digester volumes. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Biomass from conversion of solar into chemical energy via photosynthesis is a favourable alternative for fossil fuels (Schenk et al., 2008). On average, 10–15% of global overall energy demand is covered by biomass resources (Braun et al., 2008). Biomasses, e.g. industrial, agricultural and municipal solid wastes, wastewaters and energy crops, are especially promising as feedstocks for anaerobic digestion to produce biogas (Ward et al., 2008). All organic biomass components (soluble carbohydrates, proteins, lipids, cellulose, hemicelluloses) except strong lignified organic substances are suitable for anaerobic degradation (Weiland, 2010). Energy crops, with corn as the dominating plant, are cultivated exclusively for energy production via anaerobic digestion due to high methane yields and storage possibilities through ensiling of the biomass (Comino et al., 2012; Ward et al., 2008). Nevertheless, ⇑ Corresponding author. Tel.: +49 234 32 26390; fax: +49 234 32 14163. E-mail address: [email protected] (S. Schwede). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.08.157

nutritional deficiencies and insufficient alkalinity in energy crops may lead to process imbalance (Demirel and Scherer, 2009). The availability of the trace elements cobalt, nickel, iron, zinc and molybdenum is especially limited in mono-digestion of energy crops (Hinken et al., 2008). Stable anaerobic digestion can be realised by co-digestion of suitable feedstocks (Nges et al., 2012). Co-digestion of energy crops with manure was reported to considerably improve process stability and methane yield (Comino et al., 2010). Furthermore, livestock effluents, in contrast to energy crops, are characterised by low carbon to nitrogen ratios (C/N) and methane yields resulting in mutual benefits during co-digestion of both feedstocks (Giuliano et al., 2013). However, for economically viable anaerobic digestion continuous feedstock supply and consistent quality is required (Nges et al., 2012). Both may be problematic in regions with scarcity or periodical lack of biomass waste (Lebuhn et al., 2008). Moreover, the increasing energy crop production on arable land leads to a competition with global food or feed supply (Schenk et al., 2008).

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Microalgae can be produced continuously with consistent nutritional quality independent of farm land. The biomass productivity is estimated to be 50 times higher than the fastest growing terrestrial plants (Li et al., 2008). Lipids and carbohydrates for example derived from algal biomass can be converted to biodiesel or ethanol (Ghasemi et al., 2012). The residual or total biomass can be alternatively used as feedstock for biogas production. For anaerobic digestion of microalgae three main bottlenecks are identified: the low biodegradability depending on the biochemical composition and the nature of the cell wall, the presence of sodium in marine algae and unbalanced nutrient composition due to high protein contents (Sialve et al., 2009). The low C/N ratio of 6/1 in algal biomass especially excludes efficient degradation and can result in ammonia inhibition (Yen and Brune, 2007). On the other hand the released ammonia is capable of increasing the alkalinity and stabilizing the pH in the system (Gerardi, 2003). Yen and Brune (2007) reported a doubled methane yield during co-digestion of algal sludge and high carbon paper waste by increasing the C/N ratio. Additionally, most of the trace elements with positive effects on the anaerobic biodegradability (Fe, Co, Zn) occur in microalgae (Grobbelaar, 2004). The mentioned characteristics suggest microalgal biomass as suitable feedstock for co-digestion with energy crops resulting in mutual benefits. The aim of the present study was to investigate the potential of the marine microalga Nannochloropsis salina as feedstock for the co-digestion with corn in the context of batch and semi-continuous experiments. 2. Methods 2.1. Feedstocks and inocula N. salina as algal biomass for the batch assay was obtained from Phytolutions Ltd. (Bremen, Germany). Algal sludge was harvested by centrifugation to a total solids (TS) content of 32% (w/w) and volatile solids (VS) content of 92% of TS (w/w). For the semi-continuous study N. salina biomass was obtained from BlueBioTech Ltd. (Büsum, Germany). TS content was between 15% and 30% (w/w) and VS content between 78% and 90% of TS (w/w). Corn silage (CS) is the main feedstock for biogas production in Germany and is made of the whole corn plant. TS content was 35% (w/w) and VS content 96% of TS (w/w) in the batch assay. Corn-cob-mix (CCM) is a concentrate in pig fattening consisting of the corn cob. TS content was 62% (w/w) and VS content 98% of TS (w/w). For the semi-continuous study TS and VS content of corn silage were 27–33% (w/w) and 96% of TS (w/w), respectively. Digestate as inoculum for the batch assay was taken from an agricultural biogas plant (Neurath, Germany, operated by RWE Power (Essen, Germany)) fed with corn silages and cattle dung. Digestate as inoculum for the semi-continuous study was obtained from an agricultural biogas plant operated with corn silage and cattle dung as feedstock (Bottrop, Germany). The inocula were not adapted for algal biomass prior to the experiments. 2.2. Batch assays Anaerobic digestion was carried out as batch assays according to the standard VDI 4630. Inoculum and algal biomass were mixed to attain an inoculum to substrate ratio of 2, with 7 g VS from the digestate and 3.5 g VS from the algal biomass in 0.5 L glass bottles. The difference to 0.4 L was adjusted with tap water. The algal biomass (A) was mixed with CS in the ratios 1/2 (1A/2CS), 1/6 (1A/ 6CS) and 2/1 (2A/1CS) and with CCM in the ratio 1/3 (1A/3CCM) referred to the fresh mass (FM). In addition all feedstocks and the inoculum without feedstock were mono-digested as control.

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Anaerobic conditions were obtained by flushing the headspaces with helium for 1 min. All approaches were investigated in triplicate at 40 °C for a period of 36 days. The produced biogas volume was recorded by measuring the displacement of a confining liquid (55.2 g/l sulphuric acid; 200 g/l sodium sulphate decahydrate) in 400 mL eudiometers. Total biogas was collected in gas sampling bags (Ritter, Bochum, Germany) and analysed by gas chromatography (see Section 2.4). Dry gas volume was corrected to standard conditions (0 °C, 101.325 kPa). An equation (Eq. (1)) was fitted to every sample to illustrate the daily gas production over retention time.

Vbiogas ðtÞ ¼ a þ b=2 ðtanhðcðt  dÞÞ þ 1Þ þ e=2 ðtanhðf ðt  gÞÞ þ 1Þ; ð1Þ where Vbiogas is the dry biogas volume under standard conditions, t is the time and a–g are empirically fitted parameters used to describe the progression. Eq. (1) was used to combine the triplicate approaches and to deduct the gas production of the inoculum. 2.3. Semi-continuous digestion Double glass shell digesters with a working volume of 22 L were used for anaerobic digestion under mesophilic conditions (38 °C) with continuous mixing. The temperature was maintained by circulating water through a water jacket. Feeding was semi-continuously once a day by mixing the feedstock with effluent from the digester. The first digester was fed with corn silage (CS), the second with N. salina biomass and corn silage in the ratio 1/6 (1A/6CS) on VS basis. Before the first feeding both digesters were filled with 20 L of the same inoculum. After 124 days thermally pretreated algal biomass was used as feedstock in the co-digestion. For thermal pretreatment 0.20 L of concentrated microalgae suspension was filled in 0.25 L pressure resistant sealed glass bottles. The heating and pretreatment time in a drying cabinet was 2.25 and 2 h at 120 °C, respectively. pH value (Orion 3-Star, Thermo Scientific, Germany), electrical conductivity (EC Testr.11, Eutech Instruments, Holland), total/volatile solids, ammonium, total volatile acids and the total alkalinic carbonate were measured periodically. The biogas volume was measured continuously using a drum type gasmeter (TG 05, Ritter Gas, Germany) and was corrected to dry gas under standard conditions (0 °C and 101.325 kPa). Gas composition was analysed on a daily basis before feeding. The initial organic loading rate (OLR) was 2.0 kg VS m3 d1. After 188 and 218 days OLR was gradually increased to 3.7 kg VS m3 d1 and 4.7 kg VS m3 d1, respectively. 2.4. Analytical methods TS and VS content in feedstocks and digesters were measured according to DIN EN 12879 and DIN EN 12880. The elementary composition required to calculate the C/N ratio of the feedstock was determined using quantitative elementary analysis (Vario el, Elementar Analysensysteme Ltd., Germany). The biogas composition was determined by gas chromatography (Focus GC, Axel Semrau Ltd., Germany) equipped with a micropacked column (ShinCarbon ST 100/120, Restek Ltd., Germany) and a thermal conductivity detector with helium as carrier gas. External calibration allowed the detection of O2, N2, CH4 and CO2. Total volatile acids (TVA) and total alkalinic carbonate (TAC) were estimated by titration (Nordmann, 1977). The diluted samples were titrated with 0.1 M HCl till pH 5.0 for the TAC determination and till pH 4.4 for TVA determination (TitroLine 6000, SI Analytics, Germany). The organic composition of the feedstock (crude ash, crude protein, crude fiber, crude fat; standard analysing procedures) and the

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concentration of ammonium (photometrical according to DIN 38406 E5-1), volatile fatty acids (acetic acid, propionic acid, butyric acid, iso-butyric acid, valeric acid, iso-valeric acid, hexanoic acid; in-house method with flame ionisation detector equipped gas chromatograph) and trace elements (aqua regia extraction, detection via inductively coupled plasma optical emission) in the digester were externally examined (Wessling Ltd., Germany).

3. Results and discussion 3.1. Chemical composition of N. salina biomass and corn silages Table 1 shows the chemical composition and the C/N ratio of the feedstocks used in this study. For N. salina biomass (A) and corn silage (CS) two different samples are presented. The suppliers of the biomasses were changed between the different studies due to operational reasons (larger amount of biogas required). Sample 1 shows the composition of the biomasses used for the batch experiments, whereas sample 2 represents the composition from the semi-continuous study. The chemical composition of both biomass resources depends on environmental conditions and for energy plants on harvesting time and conservation technology (Amon et al., 2007; Brown et al., 1997). Consequently, certain variations can be observed between different batches. Between the two samples of A differences were determined in the lipid (crude fat; difference 6%), soluble carbohydrate (Nitrogen-free extractives, Nfe; difference 4%) and inorganic fractions (crude ash; difference 4%). The C/N ratio is approximately two times higher in sample 2 (12.2). For the two CS samples differences were determined in the protein (2%), lipid (6%) and Nfe (9%) fractions. Accordingly, the C/N ratio in sample 1 is lower (32.6) than in sample 2 (44.5). The C/N ratio for corn-cob-mix (CCM) was 26.2. The average C/N ratio for marine microalgae is approximately 7 (Geider and La Roche, 2002), whereas the ratio for terrestrial plants is 36 (Elser et al., 2000). In general, the organic composition of CS and CCM significantly differed from the composition of A. 29–33% of the TS in N. salina belonged to the soluble carbohydrates compared to 57–66% in CS and 81% in CCM. Furthermore, A contained higher amounts of proteins (21% of TS) and lipids (36–42% of TS) compared to CS (7–9% proteins and 2–7% lipids) and CCM (10% proteins and 4% lipids). The rate limiting step in anaerobic digestion depends on the composition of the feedstock (Weiland, 2010). Soluble carbohydrates are degraded within few hours. Increasing organic acid concentrations may inhibit methanogenic bacteria. However, hydrolysis of undissolved components like cellulose, proteins or lipids takes several days. In this case methane production is limited by the hydrolytic stage. Accordingly, based on the chemical composition hydrolysis is probably the rate-limiting stage in anaerobic

Table 1 Chemical composition of N. salina, CS and CCM.

TS VS Crude fat Crude protein Crude fiber Crude ash Nfe C/N ratio

% % % % % % % –

of of of of of of of

FM TS TS TS TS TS TS

N. salina 1

N. salina 2

CS 1

CS 2

CCM

31.8 92.0 42.2 21.0 2.0 6.0 28.8 6.5

26.5 90.2 36.1 20.8 0.0 9.8 33.3 12.2

34.5 95.7 1.7 6.7 21.2 4.4 66.0 32.6

30.7 95.8 7.4 8.8 22.4 4.2 57.2 44.5

64.2 98.3 4.1 10.0 3.6 1.7 80.6 26.2

FM = fresh mass, samples 1 were used for batch and samples 2 for semi-continuous experiments.

digestion of A, whereas methanogenesis becomes rate-limiting for corn silages. 3.2. Batch co-digestion of N. salina biomass and corn silages Fig. 1 shows the kinetics of biogas produced during anaerobic batch digestion of A, CS, CCM and mixtures of A with both corn feedstocks. The biogas volume produced by the inoculum was deducted via regression according to Eq. (1). Mono-digestion of CS and CCM exhibited significant differences to the mono-digestion of A and all mixtures. For CS and CCM slight inhibition occurred after 3 and 2 days, respectively. Inhibition was time-limited and resulted in decelerated degradation. After 10 days biogas volume increased linearly in both samples. Anaerobic digestion of A and the mixtures was characterised by a steep increase in accumulated biogas volume without inhibition or retarded degradation. In Fig. 2 the biogas and methane volume produced is referred to the applied amount of VS to evaluate biogas and methane yields of the examined feedstocks. Despite a low biogas yield in mono-digestion of A (0.28 m3 kg VS1), biogas yields in codigestion of A with CCM (ratio 1/3:0.61 m3 kg VS1) and CS (ratio 1/6:0.66 m3 kg VS1) increased about 7% and 9% compared to the mono-digested feedstocks (CS: 0.60 m3 kg VS1; CCM: 0.56 m3 kg VS1), respectively. Additionally, biogas yields of the mixtures were calculated from the values measured in mono-digestion by a linear mixing approach on VS basis (see black bars in Fig. 2). The actual biogas yield of all mixtures was substantially underestimated by this calculation. For 2A/1CS the difference between calculation and measurement was about 9%, for 1A/2CS 11%, for 1A/3CCM 14% and for 1A/6CS 15%. In other words, the addition of A is beneficial for process stability, degradability and biogas yield of rapidly hydrolysable feedstocks. The methane yield obtained in the CS mono-digestion (0.38 m3 kg VS1) corresponds to observations that were reported in literature under similar conditions. Amon et al. (2007) found an average methane yield of 0.40 m3 kg VS1 for various corn varieties. Zhong et al. (2012) obtained a methane yield of 0.20 m3 kg VS1 in the mono-digestion of cyanobacterial biomass. The degradability was limited by the resistance of the microalgal cell wall to hydrolysis. The methane yield was significantly increased in the co-digestion with corn straw (0.33 m3 kg VS1) due to an adjusted C/N ratio and increased alkalinity. The optimal C/N ratio in the mixture was 20.0 compared to 6.0 in the algal biomass and 71.0 in the corn straw. The C/N ratios in the mixtures in this study ranged from 9.1 in 2A/1CS to 21.2 in 1A/6CS (14.4 in 1A/2CS and 17.6 in 1A/3CCM). Regarding the biogas yields of all samples the optimum C/N ratio is 21.2. Higher C/N ratios in the mono-digestion of CS (32.6) and CCM (26.2) led to lower biogas yields. These results correspond to investigations by Yen and Brune (2007), who suggested an optimal C/N ratio for the co-digestion of algal sludge and paper waste in the range of 20–25. Despite a low C/N ratio of A inhibition of anaerobic digestion due to released ammonia was not observed in this study. Hence, anaerobic degradation is limited in hydrolysis stage due to robust cell wall structures. Schwede et al. (2013) showed that biodegradability and biogas yield from N. salina biomass was significantly increased by physical pretreatment. The digestate that was taken as inoculum in this batch assay showed a deficit in essential trace elements, resulting in increasing volatile fatty acids (VFA) in the further operation of the biogas plant. Certain trace elements like Co or Ni function as co-factors of enzymes in methanogenic pathways. Deficiencies of such co-factors cause imbalances in anaerobic degradation with decreasing methanogenic activities and accumulation of VFAs (Lebuhn et al., 2008). Addition of a trace element cocktail recovered process

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3,000 1A/6CS CS

2,500

2A/1CS 1A/3CCM CCM 1A/2CS

Biogas volume (mL)

2,000

1,500

1,000

A

500

0 0

10

20 Digestion time (days)

30

Fig. 1. Kinetics of biogas production during anaerobic batch digestion. Comparison of mono-digestion and mixtures of corn silages and algal biomass. CCM = corn-cob-mix, CS = corn silage, A = Nannochloropsis salina biomass.

0.80 biogas

methane

calculation (biogas)

Biogas/methane yield (m3 kg VS-1)

0.70

0.60

0.50

0.40

0.30

0.20

0.10

0.00 A

CS

CCM

2A/1CS

1A/2CS

1A/3CCM

1A/6CS

Fig. 2. Biogas and methane yields after 36 days anaerobic batch digestion of corn silages and algal biomass. Black bars represent the biogas yield of mixtures calculated by a linear mixing approach of yields obtained in the mono-digestions. CCM = corn-cob-mix, CS = corn silage, A = Nannochloropsis salina biomass.

stability. Therefore, positive effects of A addition during the batch assay can be attributed to the essential trace elements cobalt and nickel, which were detected in A, or to the contribution of protein degradation on alkalinity. The reproduction of the anaerobic co-digestion of A and CS with inoculum that showed high buffer capacity and no trace element deficiency revealed only a minor impact on the biogas yield (6% improvement of the biogas yield calculated from the mono-digestion of A and MS in 1A/6CS, data not shown). The biogas production

of CS mono-digestion exhibited unlike in the batch series mentioned above no inhibition. On the other hand, total inhibition without biogas production occurred in anaerobic digestion of CS and in co-digestion with A using inoculum with high amounts of VFA (>10,000 mg L1 acetic acid equivalents, data not shown). However, under these conditions the mono-digestion of A and a protein standard (bovine serum albumin, BSA) exhibited no inhibition. These results suggest that the positive effect of protein degradation on alkalinity is the major factor for process stability in

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anaerobic digestion of protein rich microalgae. During protein degradation ammonia is released from the amino group of the amino acids. Under mesophilic conditions and neutral pH 99% of the ammonia dissociates to ammonium. The formation of ammonium bicarbonate enhances the solubility of carbon dioxide and thereby the alkalinity of the digester due to the availability to react with VFA (Gerardi, 2003). Nevertheless, the explanation for the positive effects on the process stability in the co-digestion of A and CS could not be clarified explicitly in the context of batch assays. The investigation of the effects during semi-continuous long-term studies is more convenient due to frequent analysis possibilities. Accordingly, co-digestion of the most beneficial mixture of A and CS (1A/6CS) obtained from the batch experiments were investigated in semicontinuous digestion in comparison to the mono-digestion of CS. The anaerobic digestion of CCM was not further investigated. The utilisation of the whole corn plant appears to be more sustainable than using only the corn cob. 3.3. Semi-continuous co-digestion of N. salina and corn silage Semi-continuous digestion of corn silage (CS) and co-digestion of corn silage with N. salina biomass (1A/6CS) in the ratio 6/1 on VS basis was carried out for 245 days. The initial organic loading rate (OLR) was 2 kg VS m3 d1. Fig. 3 shows the development of OLR during the experimental period. In both digesters the OLR was in the same order of magnitude and slightly increased during the first 180 days due to decreasing volumes in the digesters caused by high degradability of CS under the low OLR. After 180 days the OLR was raised to 3.7 kg VS m3 d1 and after 220 days to 4.7 kg VS m3 d1 to investigate OLR limits. 3.3.1. Biogas productivity and methane content The biogas productivity (Fig. 3) is indicated as produced biogas in relation to the effective digester volume per day in m3 m3 d1 due to different volumes in both digesters. Biogas productivity proceeded in both digesters in the same order of magnitude. In the first 124 days biogas productivity in the CS digester was slightly higher than in the co-digester. After 124 days thermally pretreated algal biomass (120 °C for 2 h) was used in the co-digestion. The hydrolysis of N. salina biomass in anaerobic digestion is limited by the robust cell wall structure (Schwede et al., 2013). Consequently, the biogas productivity after application of pretreated

material was enhanced in the co-digestion. The increase of the OLR initially resulted in increasing biogas productivity in both digesters. The further enhancement of the OLR to 4.7 kg VS m3 d1 led to a more distinctive increase in the biogas productivity of the codigestion and eventually a significant decrease in the CS digestion. Similarly, the methane content (Fig. 3) decreased substantially in the CS digester, indicating that the anaerobic degradation process was inhibited. On an average the methane content was approximately 1 vol.% higher in the co-digestion. After the application of pretreated algal biomass the methane content increased in the co-digestion due to higher amounts of degradable protein and lipid compounds from the algal biomass. Hence, the average methane content (54.45 vol.%) was approximately 3 vol.% higher than in the CS mono-digestion. 3.3.2. Total volatile acids and total alkalinic carbonate The ratio of total volatile acids (TVA) to total alkalinic carbonate (TAC) is an indicator for process stability in anaerobic digestion. TVA/TAC ratios below 0.4 are typical for systems with balanced acid and methane formation (Gomez et al., 2011), whereas increasing ratios occur in systems where acid formation exceeds methane formation resulting in inhibition of methanogenesis. Fig. 4 shows the development of pH, TVA, TAC and TVA/TAC ratio during the experimental period. TVA/TAC ratio was for both digesters constant at 0.23 for 200 days indicating that volatile fatty acid production and methane formation were in equilibrium. The increase of OLR led to decreasing alkalinity and volatile fatty acid accumulation in CS mono-digestion. As a consequence, pH and TAC decreased and TVA increased significantly to values (>10,000 mg L1), which are critical for process stability, resulting in decreased methane content and biogas productivity (see Fig. 3). Methanogens are especially inhibited at pH below 7.0 (Weiland, 2010). On the contrary, process stability regarding pH, TVA and TAC was not affected by the increasing OLR in the co-digestion. 3.3.3. Total and volatile solids Besides methane content and biogas productivity the decreasing process stability in the CS mono-digestion influenced the overall degradability in the digester. The development of TS and VS content in the digesters are shown in Fig. 5. TS and VS content increased distinctly from 8.2% to 9.5% (TS) and from 77.5% to 84.5% of TS (VS) under higher OLR. In the co-digester TS content was on

5.0

50.0 3.0 40.0

2.0

30.0

1.0

0.0

Methane content (Vol.%)

Biogas productivity (m3 m-3 d-1); OLR (kg VS m-3 d-1)

60.0 4.0

20.0 0

25

50

75

100

125

150

175

200

225

Digestion time (days) Methane content (1A/6CS) Methane content (CS)

Biogas production (1A/6CS) Biogas production (CS)

OLR (CS) OLR (1A/6CS)

Fig. 3. Biogas productivity, organic loading rates (OLR) and methane content during semi-continuous anaerobic corn silage mono- (CS) and co-digestion with Nannochloropsis salina biomass (1A/6CS).

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20,000

15,000 6.0 10,000 4.0

5,000

2.0

0

TVA/TAC (-); pH (-)

TVA, TAC (mg L-1)

8.0

0.0 0

25

50

75

100

125

150

175

200

225

Digestion time (days) pH (CS) pH (1A/6MS)

TAC (CS) TAC (1A/6CS)

TVA (CS) TVA (1A/6CS)

TVA/TAC (CS) TVA/TAC (1A/6CS)

Fig. 4. Total volatile acids (TVA), total alkalinic carbonate (TAC), TVA/TAC ratio and pH during semi-continuous anaerobic corn silage mono- (CS) and co-digestion with Nannochloropsis salina biomass (1A/6CS).

85.0

9.0 8.0 7.0

80.0

6.0 5.0 4.0 75.0

3.0

Volatile solids (% TS)

Total solids (% FM); NH4+(g kg FM-1)

10.0

2.0 1.0 0.0

70.0 0

25

50

75

100

125

150

175

200

225

Digestion time (days) Total solids (CS)

Volatile solids (CS)

Ammonium (CS)

Total solids (1A/6CS)

Volatile solids (1A/6CS)

Ammonium (1A/6CS)

Fig. 5. Total and volatile solids and ammonium contents during semi-continuous anaerobic corn silage mono- (CS) and co-digestion with Nannochloropsis salina biomass (1A/ 6CS).

average (8.7%) higher than in mono-digestion, whereas VS content was in the same order of magnitude (77.6% of TS). With application of algal biomass higher amounts of inorganic components, mostly sodium from the nutrient broth (see Fig. 6), accumulated in the digester without influencing the degradability, but increasing the TS content. Under higher OLR TS (9.5%) and VS content (80% of TS) increased slightly due to increasing available inorganic and organic compounds that are mostly degraded.

3.3.4. Influence of co-digestion on organic composition The difference during CS mono- and co-digestion with algal biomass was on one side the result of the different organic composition of the alga and on the other hand based on the variation of inorganic compounds. The higher amounts of soluble carbohydrates in CS resulted in fast hydrolysis and fermentation to volatile fatty acids. The accumulation of fatty acids indicates that the methane production became the rate limiting step of anaerobic digestion under high OLR. The alkalinity initially prevented the system from inhibition by stabilizing the pH for a concise period of time.

Complete inhibition was observed after alkalinity was consumed and pH significantly decreased. In the co-digestion lower amounts of soluble carbohydrates and higher amounts of proteins and lipids were introduced into the process with the algal biomass. Under these conditions acid formation and methane production were balanced and hydrolysis maintained the rate-limiting step. Therefore, the variation of the organic composition equally influenced the digestibility of both feedstocks in the co-digestion. Consequently, biogas productivity in the co-digestion remained in the same order of magnitude compared to the CS mono-digestion, despite low biogas productivity of A mono-digestion. Similarly, the methane content in the co-digestion was enhanced due to the degradation of proteins and lipids from the algal biomass.

3.3.5. Influence of co-digestion on inorganic composition In addition to the organic composition, the inorganic fraction was influenced by addition of A in co-digestion in two different ways. Firstly, the degradation of proteins released ammonia that contributed to the alkalinity of the system (see Section 3.1). The

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1,500

0.8 1,000 0.6 0.4

500

Na (mg L-1)

Co, Mo, Ni (mg L-1)

1.0

0.2 0.0 0

50

100

150

200

0 250

Digestion time (days) Co (CS)

Mo (CS)

Ni (CS)

Na (CS)

Co (1A/6CS)

Mo (1A/6CS)

Ni (1A/6CS)

Na (1A/6CS)

Fig. 6. Selected trace element concentrations during semi-continuous anaerobic corn silage mono- (CS) and co-digestion with Nannochloropsis salina biomass (1A/6CS).

ammonium concentration during the study is shown in Fig. 5. With application of thermally pretreated algal biomass ammonium levels increased on average by 16% in the co-digestion, inducing enhanced alkalinity. Secondly, selected trace elements (Co, Mo, Ni, Na) exhibited a distinctive shift during the experimental period in both digesters (see Fig. 6). The trace element concentrations detected in the codigestion are assumed as optimal due to high process stability of anaerobic digestion. Trace element deficiencies can occur in biogas plants operated with energy crops and manure as feedstocks (Schattauer et al., 2011). Cobalt, molybdenum, nickel and selenium are especially considered as essential cofactors of various enzymes in methanogenesis. A large variation of minimum or optimal requirements for trace elements in anaerobic digestion is described in the recent literature (Schattauer et al., 2011). However, the measured trace element concentrations do not indicate whether the metals are available as free metal ions for uptake by microorganisms. Metal precipitation, basically by sulphide, carbonate or phosphate, and metal chelation or complexing with inorganic or organic ligands occur in anaerobic digestion and substantially affect the availability of metals as nutrients (Callander and Barford, 1983). Additionally, trace element requirements depend on the particular methanogenic species and reaction pathways in the actual anaerobic system (Takashima and Speece, 1990). Consequently, only variances during the experimental period in the particular digester were evaluated and compared to the development in both systems. This consideration excluded zinc, copper, manganese and selenium. Zinc and copper slightly decreased in both digesters in the same order of magnitude. However, inhibitory effects only occur in CS mono-digestion. Manganese admittedly decreased in mono-digestion and remains constant in co-digestion, but has not been reported as a relevant limiting-factor in anaerobic digestion. Furthermore, the concentration was distinctly higher (7.71 mg L1) than the minimum requirement (0.005 mg L1) described in literature (Schattauer et al., 2011). Selenium was below the detection limit in both digesters for the whole experimental period. Fig. 6 displays the total metal concentration of the most relevant trace elements in both digesters. Nickel and molybdenum increased in both digesters due to the occurrence in CS, whereas the amount of cobalt and sodium significantly decreased in monodigestion. Cobalt was identified by Lebuhn et al. (2008) as the most limiting element in CS mono-digestion causing acidification even under a low OLR. Sodium concentrations of 350 mg L1 are benefi-

cial for the growth of methanogens (Vrieze et al., 2012). Methanogenesis is the least exergonic process in anaerobic environments with only 15% of energy release compared to aerobic degradation (Schink, 1997). Sodium ions play a vital role in the bioenergetics of various microorganisms as an important substitute for protons in osmotic, chemical and mechanical membrane-linked reactions (Ferry, 1993). In CS mono-digestion sodium concentration decreased below 60 mg L1 suggesting that vital energy recovery reactions became rate-limiting. Conversely, the addition of algal biomass in the co-digestion caused increasing sodium concentrations (ca. 1250 mg L1) that are still far below inhibition levels beginning at 3500 mg L1 (McCarty, 1964).

3.3.6. Benefits from co-digestion Co-digestion of at least two suitable feedstocks enables a balanced nutrient composition, an appropriate C/N ratio and stable pH adjustment (Mata-Alvarez, 2003). The co-digestion of algal biomass and CS accomplished the requirements for mutual positive effects. Organic and inorganic nutrient composition was balanced due to high amounts of fast degradable carbohydrates in CS and addition of slower degradable proteins and lipids in the algal biomass as well as essential trace elements and alkalinity for process stability. Equivalent amounts of carbohydrates, fats and proteins improve the process performance in anaerobic digestion (Nges et al., 2012). High C/N ratios cause nitrogen deficiency, whereas low ratios induce ammonia accumulation and inhibition. C/N ratios in the feedstock between 15 and 30 are considered to be optimal for anaerobic digestion (Weiland, 2010). Both feedstocks used in this study were not optimal regarding the C/N ratio in the biomass. The C/N ratio in the algal biomass was 12.2 due to high protein contents, whereas CS exhibited a ratio of 44.9. Yen and Brune (2007) showed that biogas production of carbon-rich paper waste and algal biomass was enhanced in co-digestion significantly by adjusting the unbalanced nutrient composition. The mixture of A and CS used in the semi-continuous study led to a C/N ratio of 30.6 unlike the optimal value obtained during the batch assays (21.2). The suppliers of the biomasses were changed between the different studies due to operational reasons (larger amount of biogas required). Nevertheless, the beneficial effects observed in the batch studies were reproduced during the semi-continuous longterm experiments. These findings suggest that the proportion of A in the co-digestion can be increased according to the optimal C/N ratio.

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Similar to algal biomass livestock effluents contain low C/N ratios due to high ammonia concentrations (Giuliano et al., 2013). Despite the low methane yield, nutrients and buffer capacity positively influence the digester performance, whereas the biogas yield obtained in co-digestion of livestock effluents and CS (1.3 m3 m3 d1) under comparable conditions by Giuliano et al. (2013) was considerably lower than in this paper (2.2 m3 m3 d1). Nevertheless, the requirements for a balanced nutrient composition are fulfilled by co-digestion of energy plants with algal biomass or livestock effluents. The higher methane yield from algal feedstock results in improved utilisation of the digester volume. Therefore, by co-digestion with algal biomass it is possible to decrease the digester volume in new biogas plants or to exploit the digester volume with increasing OLR in existing plants. The results suggest that N. salina biomass is a suitable feedstock for the co-digestion of energy plants, especially in regions with manure scarcity (Lebuhn et al., 2008). Algal biomass can be provided continuously with the same nutritional quality due to consistent and controlled conditions in photobioreactors independent from agricultural resources. This avoids low biogas productivity and inhibitory heavy metals using livestock wastes as feedstock for anaerobic digestion (Sakar et al., 2009). The controlled addition of macro- and micronutrients in algal biomass production maintains a controlled addition of these nutrients into the digester and via the digestate into the environment as well. Currently, biomethane production from microalgae is not competitive with corn due to high production costs and low production capacity for algal biomass (Schenk et al., 2008). The production costs for microalgae are assumed to range between 210 and 810 € t TS1 (Ghasemi et al., 2012), whereas the production costs for corn are 114 € t TS1 (Comino et al., 2012). Consequently, codigestion of microalgae with energy crops can be a first step to enhance the feasibility of biogas production from microalgae during operation by designing advanced photobioreactors and cost-effective technologies for biomass harvesting. 4. Conclusions In this work algal biomass from the marine microalga N. salina was co-digested with corn-cob-mix and corn silage in batch experiments and with corn silage under semi-continuous long-term conditions. The co-digestion led to a balanced nutrient composition with mostly soluble carbohydrates from the silages and more complex proteins and lipids from the algal biomass. Ammonium release during protein degradation and higher trace element concentrations resulted in enhanced process stability under higher OLR in co-digestion. The results suggest N. salina biomass as a suitable feedstock for co-digestion with energy crops. Acknowledgement This work was partly supported with a Grant by the Graduate School of Energy Efficient Production and Logistics (Bochum and Dortmund, Germany). References Amon, T., Amon, B., Kryvoruchko, V., Machmüller, A., Hopfner-Sixt, K., Bodiroza, V., Hrbek, R., Friedel, J., Pötsch, E., Wagentristl, H., Schreiner, M., Zollitsch, W., 2007. Methane production through anaerobic digestion of various energy crops grown in sustainable crop rotations. Bioresour. Technol. 98, 1–9. Braun, R., Weiland, P., Wellinger, A., 2008. Biogas from energy crop digestion. IEA Bioenergy Task 37 (Energy from Biogas and Landfill Gas), 1–20, . Brown, M.R., Jeffrey, S.W., Volkman, J.K., Dunstan, G.A., 1997. Nutritional properties of microalgae for mariculture. Aquaculture 151, 315–331.

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