Bioresource Technology 143 (2013) 221–230

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Bioresource Technology

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The steady state anaerobic digestion of Laminaria hyperborea – Effectof hydraulic residence on biogas production and bacterial communitycomposition

0960-8524/$ - see front matter � 2013 Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.biortech.2013.05.124

⇑ Corresponding author. Tel.: +65 65927902.E-mail address: jhinks@ntu.edu.sg (J. Hinks).

Jamie Hinks a,c,⇑, Stephen Edwards a, Paul J. Sallis a, Gary S. Caldwell b

a School of Civil Engineering and Geosciences, Cassie Building, Claremont Road, Newcastle University, Newcastle Upon Tyne NE1 7RU, UKb School of Marine Science and Technology, Ridley Building, Claremont Road, Newcastle University, Newcastle Upon Tyne NE1 7RU, UKc Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore

h i g h l i g h t s

� Batch digestion is an unreliable predictor of long term digester stability.� Anaerobic digestion was stable over extended operation periods.� Biogas and methane levels were comparable to terrestrial carbon sources.� Dilution rate (hydraulic residence) affected microbial community structure.

a r t i c l e i n f o

Article history:Received 18 March 2013Received in revised form 29 May 2013Accepted 30 May 2013Available online 5 June 2013

Keywords:Anaerobic digestionBiofuelBiogasBrown algaeSeaweed

a b s t r a c t

Methane production by anaerobic digestion (AD) of macroalgae (seaweed) is a promising algal bioenergyoption. Work presented here is primarily based on the AD of Laminaria hyperborea using batch and con-tinuously stirred tank reactors. Extrapolation of data from batch studies to long term continuous reactorswas unreliable. A conservative organic loading rate (OLR) of 1 g L�1 d�1 was used due to difficulties expe-rienced in achieving steady state performance at an OLR of 1.5 g L�1 d�1. Biogas composition and meth-ane yields (60–70%) were near to values expected from terrestrial feedstocks. Biomass washout, asimposed by the dilution rate (i.e., hydraulic residence), had considerable bearing on the biogas generationprofile, particularly at >3 hydraulic residences. Inhibition of methanogen growth was linked to nutrientdeficiency and potentially antimicrobial compounds associated with the feedstock. Anaerobic digestionof L. hyperborea proved feasible over extended operational periods.

� 2013 Published by Elsevier Ltd.

1. Introduction

The reliable supply of fossil fuels has become increasinglyconstrained resulting in volatility and uncertainty in global mar-kets. The combination of resource limitation, geopolitical instabil-ity and the threat posed by global climate change has greatlyenhanced investment in, and development of, scalable bioenergyresources. Of the feedstocks currently under evaluation algae areacknowledged as having the greatest potential for sustainableproduction and conversion to fuels (Singh et al., 2011) despite sig-nificant bottlenecks remaining in production and downstream pro-cessing. Unicellular microalgae are the focus of intensive researcheffort, primarily for conversion to biodiesel (Schenk et al., 2008);bioethanol (John et al., 2011) and production of hydrogen (Melisand Happe, 2001) and methane gas (Ras et al., 2011). In compari-

son, macroalgae (seaweed) has received little attention as aprospective feedstock.

Seaweeds have a number of properties that make them anattractive candidate feedstock, including: high carbohydrate con-tent; low lignocellulose content; high coastal biomass (Morandand Merceron, 2005); suitability for both subtidal and intertidalcollection (Dhargalkar and Verlecar, 2009); potential for large scalemariculture (Titlyanov and Titlyanova, 2010); the potential to mit-igate marine eutrophication both preventatively and responsively;potential availability of waste streams from established seaweedprocessing industries (Li and Zhang, 2010); as well as comparableor even higher productivity than terrestrial plants (Graham et al.,2007). Seaweeds have been investigated for bioenergy potentialvia a number of routes including; ethanol fermentation (Goh andLee, 2010; John et al., 2011); thermochemical processes (Zhouet al., 2010); and more commonly anaerobic digestion (AD)yielding biogas (Ghosh et al., 1981; Moen et al., 1997a,b; Verg-ara-Fernández et al., 2008).

222 J. Hinks et al. / Bioresource Technology 143 (2013) 221–230

The production of methane from AD of seaweed and seaweedby-products has been studied on a number of occasions (e.g.,Habig and Ryther, 1983; Habig et al., 1984; Carpentier et al.,1988; Kerner et al., 1991; Vergara-Fernández et al., 2008). Theprocess and application of AD is a well understood robust,low-tech and comparatively low cost technology for generatingbioenergy as biogas (a direct fossil fuel alternative). Biogas typi-cally contains a mixture of 40–60% methane with the balanceconsisting mainly of carbon dioxide and trace amounts of othergases such as sulphur dioxide. A number of studies have demon-strated that seaweeds can be degraded anaerobically to methanein batch tests (e.g., Ghosh et al., 1981; Moen et al., 1997a,b).Whereas batch test are useful to demonstrate biodegradabilityof seaweed biomass and describe their methane potential, theyreveal little about the long term feasibility of using seaweed incommercial scale anaerobic digesters. This is a highly pertinentpoint as it has been suggested that antimicrobial compoundse.g., polyphenols, produced by certain seaweeds may inhibitsustained anaerobic biodegradation (Moen et al., 1997b). Theinhibitory activity of such compounds would, in theory, limitthe scope for AD of a number of seaweed species particularlywithin the class Phaeophyceae (brown algae).

Using seaweed as sole feedstock has previously not been suffi-ciently optimised or convincingly for anaerobic digestion demon-strated to perform stably and reliably over extended periods. Thisstudy demonstrated indefinite stability for the AD of a single spe-cies feedstock of Laminaria hyperborea. Indefinite stability wasdetermined by operating several reactors beyond steady state – de-fined here as three hydraulic residences i.e., the number of cyclesof hydraulic residence completed. How the different treatments af-fected prokaryotic community composition was also explored. Thisis the first study to report steady state anaerobic digestion using L.hyperborea as the sole feedstock.

2. Methods

2.1. Seaweed collection processing and storage

L. hyperborea and Laminaria digitata used in the initial batchtests were collected from shallow water during low tide at Culler-coats Bay, Tyne and Wear, UK (NZ3572) on 16 July 2009. Additionalsamples of L. hyperborea used in the continuous reactor tests werecollected as beach-cast specimens at low tide from Boulmer Beach,Northumberland UK (NZ268148) on 3 February, 5 March and 7April 2010.

Only frond material was used in the preparation of reactor feed-stocks; the stipe and holdfast were discarded. The rationale for thiswas in the event of scalable mariculture, harvesting only the frondwould allow for further biomass production through regrowthwithout having to reseed entire new plants. To prepare feedstockslurry suitable for direct addition to the reactors, the fronds wereroughly chopped by hand to about 10 mm particle size. Approxi-mately 300 g was placed in a domestic food blender and liquidisedwith 300 ml distilled water, until a thick, consistent slurry wasproduced (particles generally <2 mm diameter). The entire batchof freshly collected seaweed (2–10 kg) was processed in this man-ner and the pooled slurry samples were then mixed thoroughlyand aliquots placed into 500 ml plastic food containers and frozenat �20 �C until required.

2.2. Experimental design

The investigation was carried out in three phases: (1) a batchreactor study; (2) a trial study; (3) a long-term continuous reactorstudy. The batch study consisted of four batch reactors (BR) to

determine the methane potential of both kelp species in duplicate.The trial reactor study (TR) consisted of a Continuous Stirred TankReactor (CSTR) operated under continuous daily feeding with noexternal nutrient supplementation, to assess the requirement fornutrient amendments during subsequent continuous reactor stud-ies. The continuous reactor study (CR) comprised a series of threeidentical CSTRs operating under different continuous daily feedingregimes over 179 d. Only data obtained from the third phase arepresented here in detail, although some information from thebatch study and the trial study has been included to complementthe main results.

2.3. Reactor systems

Batch tests (phase 1) were carried out in unstirred 0.5 L Duran�

bottles fitted with tubing connectors in the caps allowing the reac-tor to be connected by PVC tubing to a bubble counter system forbiogas flow measurement (Chelliapan et al., 2006). The batch reac-tors were fed by unscrewing the cap, sliding it momentarily side-ways to expose part of the bottle neck, and immediatelywithdrawing 20 ml of the contents with a 20 ml plastic samplingladle, then, in a single operation, adding 20 ml of prepared seaweedfeedstock directly into the flask from a modified plastic syringe.The reactors were operated with a working volume of 400 mland maintained at 35 (±1) �C in a water bath, with daily agitationby hand for 30 s before and after feeding in order improve homo-geneity of removed samples and to assist in mixing.

The trial study (phase 2) and the continuous study (phase 3)were performed in 6 L Quickfit� reactor vessels (5 L workingvolume) with wide ground-glass necks. A Quickfit� flanged, mul-ti-port headplate was fitted to the reactor with a spring-clamp.Five, 19/26 ground sockets on the headplate allowed gas linesto be fitted, and the impeller drive shaft to pass into the reactor,the latter through a Quickfit� glass stirrer gland with water-sealto ensure the reactor remained gas tight. Unused sockets weresealed with ground glass 19/26 stoppers. Mixing was achievedwith a 40 � 80 mm rectangular impeller rotating at 200 rpm.Vacuum grease (Dow Corning, USA) was used to maintain theintegrity of all ground glass seals. The trial study ran for 81 d,with daily feeding of seaweed feedstock at a hydraulic residenceof 20 d. The continuous study ran for 179 d of continuous opera-tion with intensive monitoring over the first 108 d, followed by afurther 71 d of continual feeding, but with only periodic monitor-ing. The continuous reactor study employed three parallelreactors operating with hydraulic residences of 20, 25 and 30 dfor CR1, CR2 and CR3, respectively. Reactor conditions are shownin Table 1.

2.4. Inoculation and operation

The reactors were inoculated with a mixed methanogenicsludge pooled from three full-scale anaerobic digesters operatingon different waste input materials: paper sludge: sugar processingsludge: sewage sludge (2:2:1 by volume). The solids content ofthese seed sludge samples was not characterised in detail, but eachcontained 5–20% dry solids content. The reactors were inoculatedto exactly 28% of their working volume with a mixed anaerobicsludge (11.2% from the sugar processing digester, 11.2% from thepaper mill wastewater anaerobic digester, 5.6% from the sewagesludge anaerobic digester). The total suspended solids concentra-tion in each reactor was determined to be between 32,500 and35,000 mgTSS L�1 at the start of the experiment after the reactorshad been made up to working volume with distilled water andthe first feed of seaweed.

The CSTRs were operated in semi-continuous batch mode, eachdaily feeding event being initiated by the removal of an appropriate

Table 1Summary of operating and monitoring conditions for Batch Reactors (BR), Trial Reactor (TR) and Continuous Reactors (CR) in phases 1, 2 and 3 using either Laminaria digitata(digitata) or L. hyperborea (hyper).

Parameter Reactor conditions

Phase 1 Phase 2 Phase 3

Reactor name BR1b BR2b TR1 CR1 CR2 CR3Reactor type Batch Batch CSTR CSTR CSTR CSTRVolume (L) bed/working 0.5/0.4 0.5/0.4 6/5 6/5 6/5 6/5Inoculuma (source) 28 (mixed) 28 (mixed) 28 (mixed) 28 (mixed) 28 (mixed) 28 (mixed)Make-up water Fresh water Fresh water Fresh water Fresh water Fresh water Fresh waterWorking temp �C 35 (±1) 35 (±1) 35 (±1) 35 (±1) 35 (±1) 35 (±1)Feedstock Hyper Digitata Hyper Hyper Hyper HyperHydraulic residence 1 1 20 20 25 30OLR (gTSS L�1 d�1) 1–2 1–2 0.5–2.0 1 1 1Feeding Batch Batch Daily Daily Daily DailyNutrient amendment No No No Yes N/P & trace Yes N/P & trace Yes N/P & traceDuration (d�1) n/a n/a 81 179d 179d 179d

Reactor analysis

pH n/a n/a Daily Daily Daily DailyBiogas volume Batch Batch Continuous Continuous Continuous ContinuousBiogas composition Batch Batch Weekly Weekly Weekly WeeklyVFA n/a n/a Weeklyc Weeklyc Weeklyc Weeklyc

COD n/a n/a Weekly Weekly Weekly WeeklySolids n/a n/a Weekly Weekly Weekly Weekly

a Indicates % of reactor working volume.b Reactors operated in duplicate.c Monitoring undertaken more regularly than weekly when biogas data showed a reduction in reactor efficiency.d Final 71 days of operation with only intermittent monitoring.

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volume (Reactor Volume/hydraulic residence) of mixed liquorsfrom the reactor using a 12 mm diameter siphon tube connectedto a 60 ml plastic syringe (stirrer mixing continued during samplingto prevent settling and fractionation of the reactor solids). The sea-weed feedstock was defrosted as required. An experimentallydetermined quantity (expressed as dry weight (g L�1)) was madeup to a specified volume with water (water volume dependent onhydraulic residence, Table 1), to replace exactly the sample volumethat had been removed from the reactor, and added manuallythrough a headplate port. During phase 3 (hydraulic residencestudy), the feedstock was amended with a commercial trace ele-ments supplement, Nutromex TEA 310 (Omex, UK), at 20 ll pergram of carbon in the feed, and nitrogen (urea) and phosphorus(potassium phosphate) were added to maintain a C:N:P ratio of200:5:1. For this purpose, 1 g of carbon was assumed to be equiva-lent to 1 g seaweed dry weight; however, error in that assumptionmeant that the C:N:P ratio would have contained relatively more Nand P than the ideal ratio. For the phase 3 study, reactors receiveddaily feeds, excluding weekends – this feeding regime accountsfor the stepped curve in Fig. 3.

2.5. Chemical and molecular analysis

The pH was taken from the removed liquors at each feedingevent using a pHep� handheld pH probe (Hanna instruments, UK).

Chemical oxygen demand (COD) analysis was carried out usingcommercially available COD kits (Merck, UK). Liquors were centri-fuged at 3600g for five minutes, and the supernatant liquid passedthrough a 0.12 lm syringe filter (VWR, UK). This sample was addedto COD tubes, and digested at 150 �C for 2 h. If necessary, thesupernatant was diluted to ensure it was within the range coveredby the COD kit. COD values were determined by spectroscopicabsorbance using a Spectroquant Nova 60 (VWR, UK) colorimeter.

Total suspended solids content of the reactor liquors was deter-mined by passing a 3 ml aliquot of the stirred liquor through adried and pre-weighed 70 mm diameter glass-fibre filter paper,having a nominal pore size of 8 lm (Whatman GFC, UK). The filterpaper plus retentate were then dried at 105 �C until constantweight, and the TSS expressed in g L�1.

Analysis of volatile fatty acids (VFA) was performed on a DionexICS 1000 Ion Chromatograph system with Chromeleon� acquisitionsoftware, and an AS40 auto sampler (Dionex, USA). Separation wasperformed on an ionpac ICE-AS1 4 � 250 mm analytical columnunder the following conditions: flow rate, 16 ml min�1; eluent,1.0 mM heptafluorobutyric acid; suppressant regenerant, 5 mMtetrabutylammonium hydroxide; 10 ll injection loop. Sampleswere prepared by centrifuging liquors at 3600g for five minutes,and passing the supernatant liquor through a 0.12 lm syringe filter(VWR, UK). Samples were then diluted 1:1 with octanesulfonicacid, and sonicated (FS200B Sonic Bath, Decon Laboratories, Sus-sex, UK) for 45 min to remove carbonate, which caused interfer-ence. The prepared samples were then transferred to 0.5 mltubes with filter caps (Dionex, USA) before analysis.

Ammoniacal nitrogen (NH3-N) was determined using a Vapo-dest 30S steam distillation apparatus (C Gerhardt Lab Supplies,UK). Fifty millilitres of sample was placed in a Kjehdahl tube alongwith a few drops of phenolphthalein indicator and adjusted toabove pH 8.3 using NaOH. Borate buffer (3 ml) was added andthe mixture was distilled into 50 ml of boric acid indicator. The dis-tillate was titrated to a pale lavender end point with 0.02 N H2SO4.A reagent blank was distilled and titrated in the same way and sub-tracted from the sample titer to calculate the NH3–N of the sample.All reagents were prepared to the manufacturer’s specification.

Total Kjeldahl Nitrogen (TKN) was determined using Turbo-therm acid digestion and Vapodest 30S steam distillation appara-tus (C Gerhardt Lab Supplies, UK). A 10 ml sample of reactoreffluent was digested by the Turbotherm in Kjehldahl tubes withH2SO4 and a K2SO4/CuSO4 Kjeltab catalyst tablet (C Gerhardt LabSupplies, UK). The digestate was then neutralised and steam dis-tilled as described above for ammoniacal nitrogen analysis. All re-agents were prepared to the manufacturer’s specification.

Biogas production rate was measured using commercial bubblecounters, (AER-208 Respirometer, Challenge Technology, ArkansasUSA) with real-time data recording by desktop PC. Flow rate deter-minations were made on wet biogas taken directly from the reac-tor headspace, biogas cooling to room temperature (22 �C ± 1 �C)occurring before the point of flow measurement. Unless otherwisestated, biogas production rate is reported as the volume produced

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in the first 12 h immediately after each feeding event. This waschosen as it was found in the trial study that the majority of biogaswas produced in the first few hours after feeding, but mainly be-cause it provided directly comparable data when the interval be-tween feeding events was not exactly 24 h.

Biogas was collected for compositional analysis directly fromthe reactor biogas line either in evacuated 3.7 ml glass vials (Labco,UK) for the phase 1 study, or Tedlar� gas bags (VWR, UK), to collectbulk gas samples, for phase 2 and 3 studies. Biogas methane con-tent was determined using a HRGC 51600 gas chromatograph (Car-lo Erba Instruments, Italy) fitted with a flame ionisation detector.To achieve separation, duplicate 100 ll samples were injected ontoa 30 m � 0.32 mm i.d HP-PLOT-Q capillary column with a station-ary Q-phase film thickness of 20 lm (Agilent Technologies Ltd.,USA) using a 100 ll Sample Lock syringe (Hamilton, USA). The oventemperature was 35 �C isothermal and the injection port tempera-ture was 300 �C. Hydrogen was used as the carrier gas at a split ra-tio of 1000:1. Atlas data acquisition software was used for datacollection. Methane standards were prepared prior to each analysisfrom 100% analytical grade CH4 (BOC Gases, UK), and injected induplicate to make a four-point standard curve in the range 20–80% CH4.

Direct microbial cell counts of autofluorescent methanogenicarchea were performed using a Neubauer chamber and epifluores-cence microscope according to Uyanik et al. (2002). DNA was ex-tracted from frozen liquor samples suspended in ethanol (50/50v/v) using a FastDNA� Spin Kit for soil. The DNA extract was ampli-fied by PCR using primers 1 and 3 targeting the V3 region of the16S RNA gene (Muyzer et al., 1993). The extraction of DNA of thecorrect length (233 bp) was confirmed by agarose gel electrophore-sis. The amplified DNA samples were loaded directly onto a 10%polyacrylamide gel with a 30–60% denaturing gradient and runfor 900 volt hours at 60 �C in TAE buffer. DGGE gels were stainedusing SYBR gold, developed for 30 min and visualized using aFluor-S Multimager using Quantity One software. DGGE fingerprintanalysis was carried out using Bionumerics software package(version 3.5, Applied Maths, USA) and the band data from thiswas analysed and ordinated statistically using Primer 6 for win-dows (Version 6.15, Primer-E Ltd., UK).

3. Results and discussion

3.1. Batch trial studies

The phase 1 batch study indicated that the biogas potential of L.hyperborea, 0.08 (±0.009) L gTSS

�1, was higher than L. digitata, 0.041(±0.0022) L gTSS

�1. The biogas also had higher methane content(82% ± 10.53 versus 53% ± 2.24). Consequently, L. hyperborea wasselected as the feedstock material for all subsequent experiments.

Reactors in the trial experiments (phase 2) which were notsupplemented with macronutrients or trace minerals showed signsof instability within less than 3 hydraulic residences. Fig. 1 showsresults for one trial reactor (TR1), which was operated in a similarway to the continuous reactor CR1 (Table 1), except withoutnutrient amendment. At day 65 (equivalent to 1.3 hydraulicresidences), VFAs started to accumulate rapidly from a baselineof <1 mg L�1 to around 1250 mg L�1 (Fig. 1a). This transition inperformance corresponded to the step-increase in OLR from 1250to 2000 mgTSS L�1 d�1, and was preceded by a decline in biogasvolume and quality (Fig. 1b). The increase in OLR was intendedto achieve an OLR similar to commercial anaerobic digestersoperating on food or agricultural wastes (about 1–5 g L�1 d�1)(Nagao et al., 2012). However, the reactor became unstable beforethe target OLR could be reached. The pH reduction observed duringthe same period (Fig. 1a) was neutralised by amendments of

bicarbonate (on days 51 and 76). Feeding was interrupted at day81, and the VFA concentration declined to its former baseline level(Fig. 1a). However, upon resumption of feeding (1 gTSS L�1 d�1),VFA accumulated rapidly, and the experiment was terminated(Fig. 1a). Another reactor (TR2, data not shown) operated withmarine makeup water (seawater) became unstable much sooner(0.6 hydraulic residences), and despite repeated attempts at recov-ery by partial dilution of up to 50% of the reactor volume with freshseawater, recovery could not be achieved (data not shown). Salin-ity is a known inhibitor of AD, and sodium concentrations of<0.5 g L�1 have been shown to inhibit the AD of microalgae (Sialveet al., 2009; Hierholtzer and Akunna, 2012).

Ammonia and total Kjehldahl nitrogen (TKN) analysis of dige-state samples from TR1 showed no detectable free ammonia, withonly low levels of organic nitrogen present (74 mg L�1). Given theabsence of free ammonia in the reactor liquor, it is possible thatnitrogen may have been growth limiting for the microbial consor-tia, and probably contributed to the instability of these reactors, assimilar effects have been observed in other nutrient-limited reac-tors (Chen et al., 2008). However, it is not know to what extentinhibitory compounds such as phlorotannins, which are well de-scribed components of phaeophytes (Connan et al., 2006), or sul-phide may have contributed to the instability of TR1.

The mean specific methane yield of L. hyperborea in TR1 wasapproximately 0.25 L gTSS

�1, considerably greater than the yieldof 0.080 (±0.009) L gTSS

�1 found in the batch experiments. The dif-ference observed between biogas production in batch reactors andCSTRs, coupled with the observed failure of TR1 well before reach-ing steady state, indicates that any extrapolation of data fromshort-term batch studies to long-term continuous reactors shouldbe made with caution. Based on the results from TR1, a second ser-ies of continuous reactors was operated to steady state with morefavourable nutrient conditions and a more conservative OLR(1 g L�1 d�1).

3.2. Continuous reactor studies

Fig. 2a shows the 12 h biogas production data for CR1 (20 dhydraulic residence), which remained almost constant for the first40 days, but then declined increasingly after day 80 when the reac-tor had reached steady state (3 hydraulic residences). The biogasquality, as proportion of methane (%) in the total biogas, increasedsteadily up to a maximum of about 80% at day 50. The two minimain biogas quality at day 8 and day 92 (Fig. 2a) were due to leaks inthe gas collection bag, and do not reflect the biogas quality onthose days. Fig. 2b shows biogas production and reactor pH, andindicates the reactor was stable until about day 50 when pHstarted to increase steadily; at about the same time a downwardtrend was observed in biogas production. The increase in alkalinityis likely due to bicarbonate additions or ammonia, the latter re-leased from the urea added as a nutrient amendment. Althoughreduction in biogas volume would have arisen as a result of greaterCO2 solubility at the higher pH (Liu et al., 1995), the 12 h methanevolume also declined, suggesting CR1 biogas production had de-clined substantially in real terms during the 108 d of monitoring.The soluble COD profile (Fig. 2c) is difficult to interpret as substan-tial fluctuations are apparent over the observation period, probablydue to experimental errors, as a daily sequence from day 92 to 95,all analysed on the same day, clusters around 700 mg L�1. Thesoluble COD does appear to show an increasing trend from approx-imately 100 mg L�1 at day 0, to approximately 700 mg L�1 at day95.

The total solids in CR1 declined steadily from 32.6 gTSS L�1 (theconcentration achieved by the seed inoculum and the initial feed),past the feed concentration of 20 g L�1 (equivalent to the 2% solidsof the seaweed feedstock), to a stable concentration of 8–9 gTSS L�1

Fig. 1. Performance of Reactor TR1: (a) volatile fatty acid (VFA), organic loading rate (OLR); (b) 12 h biogas production and methane volume, and (c) COD and VFAconcentration and mean biogas production.

J. Hinks et al. / Bioresource Technology 143 (2013) 221–230 225

at steady state (>3 hydraulic residences) on day 86 (Fig. 2c). By tak-ing the average of the last four data points for both TSS and solubleCOD, the proportion of the feedstock solids that were available formethanogenesis can be estimated in order to calculate a specificmethane yield. The mean steady state total suspended solids con-centration for CR1 was 8240 (±90) mg L�1, or approximately 41% ofthe applied seaweed mass of 20,000 mg L�1. Given that approxi-mately 700 mg L�1 of the soluble fraction was retained as COD, itcan be estimated that approximately 55% of the feedstock solidshad been converted to biogas (assuming that 1 g COD equates

approximately to 1 g solids). The conversion rate of 55% in CR1compares well with conventional AD, and also conversion rates re-ported in the literature for seaweed of about 40–60% (Briand andMorand, 1997; Hanssen et al., 1987; Chynoweth et al., 2001).

For reactors CR2 and CR3 (Fig. 2d–i), the trends for biogas pro-duction, biogas quality and pH were broadly similar to CR1, espe-cially for pH and the 12 h biogas production rates (Fig. 2d, e, g andh). However, whilst CR1 and CR2 showed a substantial decline in12 h biogas to around 0.06 (±22%) and 0.07 (±17%) L L�1 12 h�1

(expressed as the mean 12 h production on days 104–108), the

Fig. 2. Performance of reactors CR1 (a–c), CR2 (d–f) and CR3 (g–I). Charts a, d, g: 12 h biogas production (broken black line and diamonds), methane content (grey line andtriangles) and methane fraction (%) (black line and squares) the flat, broken line indicates the 12 biogas production as an average of day 156 and 158 which is 0.076, 0.076 and0.031 for CR1–CR3 respectively. Charts b, e, h: 12 h biogas production (broken line and diamonds) and pH (black line and squares). Charts c, f, i: total suspended solids (brokenline and diamonds) and soluble COD (black line and squares) the flat grey line indicates reactor solids equilibrium concentration in g.L�1.

226 J. Hinks et al. / Bioresource Technology 143 (2013) 221–230

equivalent 12 h biogas production for CR3 was higher at around0.08 (±12%) L L�1 12 h�1. No data are shown for VFA, as VFAconcentrations were below the detection limit of 5 ppmthroughout the study, except during the final analysis on day 108when the concentrations were 28.45, 48.54 and 20.27 mg L�1 inCR1–CR3, respectively. These results confirm that VFA weremaintained at very low levels in all three reactors by balanced pop-ulations of the anaerobic guilds responsible for acidogenesis andmethanogenesis.

The 12 h gas data has been used to present the biogas resultsbecause, initially, the majority of biogas production occurred inthe first few hours after feeding, and as the reactors were not fedat weekends, or at the same time of day, this parameter gave a bet-ter indication of gas production from each individual batch of feed.Although this simplification of daily gas production was of littleconsequence during the early part of the experiment, towardsthe latter stages it was evident that biogas production pattern dur-ing a 24 h period had changed, with greater proportions beingevolved over the second part of the day (12–24 h). This observationraises questions about the characteristics of microbial consortiapresent within each reactor, and their dependence on the hydraulicresidence.

Fig. 3. Cumulative biogas production for Reactors CR1 (dashed grey line), CR2(dotted grey line) and CR3 (solid black line).

3.3. Biogas production profiles

The total cumulative biogas production for all reactors over108 days was similar at 102, 106 and 108 L for CR1–CR3 respec-tively (Fig. 3). The gradients of the curves in Fig. 3 declined steadilythroughout the experiment showing the biogas production rate re-duced steadily with time. However, this does not follow the samesubstantial reduction in 12 h biogas production rates shown inFig. 2, with 24 h production rates (slope of the line) being about

0.1, 0.12 and 0.13 L L�1 d�1 containing 57%, 54% and 72% CH4 forCR1, CR2 and CR3 respectively. This suggests that the 12 h biogasmetric was not always a good surrogate for total biogas production,particularly towards the end of the experiment when 35–42% ofthe biogas production appeared in the final 12 h of the day. Fig. 4shows examples of real-time flow-rate profiles for each reactorover different 24 h periods. When the reactors had been operatingfor a relatively short period of time (<1 hydraulic residence), thepeak biogas production rate was reached very quickly after feed-ing, and the majority of the daily biogas was produced in the first6 h after feeding. However, the biogas production-rate profilechanged considerably as the reactors reached steady state condi-tions (operation for 3 hydraulic residences), and the initial earlypeak of intense biogas production changed to broader peaks with

J. Hinks et al. / Bioresource Technology 143 (2013) 221–230 227

lower maximum rates, and more sustained production occurringtowards the end of the 24 h feeding cycle. The biogas profile ofCR1 shows the most pronounced differences as the reactor pro-ceeded towards steady state, characterised by a decrease in peakbiogas production immediately after feeding, and a lower but moresustained biogas flow over a 24 h period (Fig. 4). This trend appearsless pronounced in CR2 and CR3; these reactors retaining more ofthe rapid biogas production immediately after feeding, even duringthe latter stages of the experiment (Fig. 4). Despite the dramaticchange in the daily cycle of biogas flow of in CR1, it produced morebiogas than CR2 and CR3: 0.26, 0.17 and 0.1 L L�1 d�1 for CR1–CR3respectively at 3 hydraulic residences. These biogas productiondata suggest that the breakdown of some readily biodegradablecomponents of the seaweed took longer as the reactors progressedtowards steady state. This is likely to reflect temporal changes inthe community composition that occurred as the reactor sludgeacclimatised to the substrate composition. Shortly after start-upin all three reactors, almost all the biogas was produced soon afterfeeding when the microbial consortium would have been com-posed almost entirely of populations from the seed inoculum. Incontrast, at 3 hydraulic residences, when the biomass would havebeen almost totally replaced with new biomass (by growth/wash-out), the biogas production became more evenly distributed overthe 24 h cycle.

If the cumulative biogas production is expressed as a function ofhydraulic residence, then the cumulative biogas volumes appearquite different between reactors. For example, after 2 hydraulicresidences, the cumulative biogas volume was 61.3, 75.4 and91.4 L reactors CR1–CR3 respectively. The reason for the large

Fig. 4. Variation in biogas production rate during a single 24 h period after reactors had bhydraulic residences had elapsed (left to right); and for reactors CR1, CR2 and CR3 (top

difference in these values simply reflects the number of feedsrequired to bring each reactor to steady state. The total mass ofseaweed which had been fed to each reactor for a given hydraulicresidence value followed the order CR3 > CR2 > CR1 (61, 50 and40 gTSS

�1 respectively after completing 2 hydraulic residences).Expressed as biogas yield, the values at 2 hydraulic residencesare all similar, at approximately 0.3 L gTSS

�1. If the methane pro-duction volume is expressed in terms of volatile solids loading(i.e., the total dry solids minus the ash content), then the methaneyield for CR1 and CR2 was 0.23, and 0.14 L gVS

�1 (Table 2). Themethane yield, expressed in terms of substrate degradation (Biode-gradable Total Solids) in each reactor, gives specific methane yieldsof 0.41 and 0.25 L gBTS for CR1 and CR2, respectively, meaning thatthe specific methane yield is greater when a reactor is operated atshorter hydraulic residences. A value has not been calculated forCR3 as the reactor did not reach steady state before the end ofthe monitored experiment. The methane yield of 0.14 and0.23 L gVS

�1 (equivalent to 0.25 and 0.41 L gBTS) for CR2 and CR1,respectively, is comparable to the data reported in the literaturefor Laminaria spp. (Moen et al., 1997a,b; Adams et al., 2011; Hans-son, 1983).

Although the main experiment terminated on day 108, the reac-tors were run for a further 71 days with the same feeding patternbut less frequent data monitoring. Results from this extended per-iod of operation confirmed that the reactors all had a stable pH andcontinued to produce biogas until day 179 (representing hydraulicresidences of 6.45, 5.1 and 3.8 for CR1, CR2 and CR3 respectively).Furthermore, average gas analysis data at day 155 and 157 showeddaily biogas production rates of 0.143, 0.155 and 0.06 L L�1 d�1

een in in operation for a different number of hydraulic residences: after 0, 1, 2 and 3to bottom).

Fig. 5. NMDS plot of the similarity between microbial communities recovered fromthe liquors of seaweed anaerobic digesters operated at different hydraulicresidences at different points proceeding towards steady state and showing theeffect of hydraulic residence on eubacterial community structure. Calculated usingthe Bray–Curtis similarity index from band intensity data of 16S rRNA DGGEprofiles using the Bionumerics software package and ordinated in Primer 6 forwindows.

228 J. Hinks et al. / Bioresource Technology 143 (2013) 221–230

with a methane content of 64.7 (±2.98%), 44.9 (±3.24%) and 62.6%(±3.68%) for CR1, CR2 and CR3 respectively, equating to a methaneyields of 0.12, 0.09, and 0.05 L gVS. The equivalent 12 h biogasproduction rates are shown by the broken line in Fig. 2A, D andG demonstrate that 12 h biogas yields for CR1 and CR2 were sim-ilar to the biogas yield on day 108 but for CR3 the biogas yieldwas lower during the final days of the experiment (Fig. 2A, D andG). This suggests that a steady state performance was maintainedwell beyond 3 hydraulic residences, up to 6.45 and 5.1 hydraulicresidences for the 20 (CR1) and 25 (CR2) day hydraulic residencereactors, respectively, but not in CR3 operating with a 30 dayhydraulic residence.

The cumulative biogas production in this experiment was notinfluenced by operating hydraulic residence, but the 24 h biogasproduction and the specific methane yield was higher for CR1 thanCR2 and CR3 after 3 hydraulic residences. It can therefore be con-cluded that biogas yield was related to the reactors’ exposure tothe seaweed material, or seaweed breakdown products. This isnot unexpected, as the solids residence time (SRT) is equal tohydraulic residence in this study, which means the exposure time

Table 2Summary of continuous reactor performance and operation.

Total biogas (L)Total methane (L)Total feed (gTS)Total feed (gVS)Mean specific methane yield (L gvs

�1)Specific methane yield (L gvs

�1) at reactor start upSpecific methane yield (L gvs

�1) after 1 hydraulic residenceSpecific methane yield (L gvs

�1) after 2 hydraulic residencesSpecific methane yield (L gvs

�1) after 3 hydraulic residencesFinal specific methane yield (L gBTS

�1)

Total feed (gvs) is expressed as the difference between the total solids of the feedstock ansteady state under the continuous monitoring regime). Units (L gBTS

�1) is defined as thea R3 shown at 2.5 hydraulic residences.

of the active biomass to seaweed components increased withhydraulic residence. In addition, as discussed above, standardisingthe OLR in each reactor meant that the feed solids concentrationfollowed the order CR3 > CR2 > CR1. The biogas production rateprofile of CR1 after 3 hydraulic residences (Fig. 4), is very differentfrom that of CR2 and CR3, pointing to differences in the microbialconsortia in each reactor at a given hydraulic residence. Conse-quently, the performance of the reactors appears to be governedby a dynamic relationship between washout (brought about bythe dilution rates imposed by hydraulic residence), as well as otherfactors related to the length of time the microbial consortia hadbeen exposed to the feedstock. Longer exposure might producegreater negative effects on the microbial community from poten-tially toxic compounds such as polyphenols (Adams et al., 2011;Moen et al., 1997a,b). In order to investigate whether the differingexposure times had caused differing effects on the biomass, preli-minary characterisation of the microbial communities of eachreactor was undertaken.

3.4. Microbial community analysis

From observed changes in biogas production profiles (Fig. 4),both dilution rate (1/hydraulic residence) and time of exposureto suspected inhibitory compounds in the feedstock were sus-pected to have influenced the reactor microbial community com-position over the course of the experiment. Direct cell countsshowed that the number of metabolically active methanogensdropped by about half in reactor CR1, from 7.58 � 106 to3.54 � 106 cells ml�1, between the completion of 1 hydraulic resi-dence and end of the experiment. No real change was detected inthe number of metabolically active methanogens for the sameinterval in CR3, with counts of 4.53 � 106 and 4.93 � 106 cell ml�1

after 1 hydraulic residence, and at end of the experiment, respec-tively. The relative losses of active methanogens, and biomass ingeneral, in CR1 and CR3 can be explained by the greater washoutof the slow growing methanogens at the shorter hydraulic resi-dence. However, while washout of methanogens would explainabsolute changes in methane production, it does not explain thechange in biogas production profiles. A possible explanation forthe change in biogas production profiles (Fig. 4) is that the eubac-terial components of the microbial population underwent changesthat limited hydrolysis and acidogenesis, and this ultimately af-fected the conversion of complex substrates to the hydrogen andacetate used by methanogens.

Comparison of the bacterial community structure using non-metric multidimensional scaling (NMDS) plots (Clarke, 1993)(Fig. 5) shows that the microbial composition in each reactor overseveral hydraulic residences is characterised by four main ordina-tions, or clusters with 40% similarity to one another. The NMDSplot consists of the following clusters: the inoculum (at the bottom

CR1 CR2 CR3

102 106 10866 64 69385 385 385288 288 2880.23 0.22 0.260.19 0.10 0.150.22 0.21 0.280.27 0.17 0.280.23 0.14 0.10a

0.41 0.25 –

d the ash. Final specific methane yield calculated at steady state (R3 did not achieveCH4 produced compared to solids degraded in the reactor.

J. Hinks et al. / Bioresource Technology 143 (2013) 221–230 229

of the diagram sharing 80% similarity); CR3 at different hydraulicresidence (to the left side of the diagram which share 60% similar-ity); and two clusters which appear to show a shift in the microbialcommunity composition of reactors CR1 and CR2 related to theirhydraulic residence (at the top and right of the diagram). In termsof operating hydraulic residence (20 d, 25 d and 30 d), shorterhydraulic residence subjects the microbial population to the mostwashout, therefore the most dynamic community compositionwould be expected in reactors subjected to the shortest hydraulicresidence, i.e., CR1 and CR2, as shown in Fig. 5. Conversely, R3,which has the longest hydraulic residence, remains distinct fromthe other reactors, and the community composition appears rela-tively conserved over three hydraulic residences. Consequently,the influence of the 20 and 25 day hydraulic residence on microbialcomposition appears pronounced, but the 30 day residence time isnot sufficient to elicit overall obvious changes in microbial compo-sition, even though the biomass would have undergone three cellgenerations (on average) to have remained in the system. TheSimpson’s reciprocal index (1/d) was 8.54, 9.27 and 8.69 at thestart of the experiment and 8.28, 7.77 and 6.74 at the end of theexperiment for CR1, CR2 & CR3, respectively, showing that the pro-karyotic diversity was lower at the end of the experiment in allcases, and follows the order of diversity CR1 > CR2 > CR3. Themicrobial community in CR3 remained relatively conservedthroughout the course of the experiment even though its methano-genic activity declined. This evidence suggests that possible inhib-itory factors contained in the original inoculum, or added throughcontinuous feeding with seaweed, rather than loss of competentbiomass through washout, had limited the methanogenic capabil-ity of the reactors by the end of the experiment. Additionally, themethanogens appear to remain relatively intact in CR3 but are sub-ject to washout in CR1. The loss in performance, therefore, wouldappear to be due in part to an inhibition of the hydrolytic and fer-mentative guilds. The combination of high washout and lowerexposure to the seaweed feedstock in CR1, resulted in a transfor-mation of the CR1 microbial community to a population whichcould be sustained indefinitely in this reactor, and one which isable to confer higher specific methane yields than the populationsin reactor CR2 and CR3. The microbial data presented here are notexhaustive, and further work is required to extend the chemicaldata, and obtain DNA sequence data of both Archeal and Eubacte-rial populations in the reactor samples. This will allow any shifts incommunity structure to be linked to changes in specific chemicalfactors over time, and the consequential impact on reactor perfor-mance can be described and understood.

4. Conclusion

Batch tests proved unreliable in predicting the performance ofcontinuous anaerobic reactors during long-term steady state stud-ies. Stable anaerobic digestion of L. hyperborea as sole feedstockover extended operation periods (beyond steady state), is shownunequivocally for the first time by running reactors in excess of6.5 hydraulic residences for three different operational hydraulicresidences. Microbial community structure altered substantiallyat shorter hydraulic residences and was conserved at longerhydraulic residences. Future research should focus on achievinghigher OLR, higher methane yields, and on quantifying the inhibi-tory factors in seaweed so that strategies can be identified to elim-inate or manage them.

Acknowledgements

This work was funded as part of the ITI Energy/ScottishEnterprise Seaweed Anaerobic Digestion Research Programme.

Additional work was carried out by Pranali Sherkar and the authorsgratefully acknowledge her kind contributions. The microbiologicalstudy was funded by Newcastle University. The authors thank Dr.Angie Sherry for her assistance with NMDS plots.

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