Chemical Engineering Journal 255 (2014) 492–499

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Chemical Engineering Journal

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Anaerobic mesophilic co-digestion of sewage sludge and sugar beet pulplixiviation in batch reactors: Effect of pH control

http://dx.doi.org/10.1016/j.cej.2014.06.0741385-8947/� 2014 Elsevier B.V. All rights reserved.

⇑ Corresponding author. Tel.: +34 956016158.E-mail address: rocio.montanes@uca.es (R. Montañés).

Rocío Montañés ⇑, Montserrat Pérez, Rosario SoleraDepartment of Environmental Technologies, Faculty of Sea and Environmental Sciences, University of Cádiz, 11510 Puerto Real, Cádiz, Spain

h i g h l i g h t s

� Accumulation of volatile fatty acids because of pH value.� Sugar beet pulp lixiviation (SBPL) improves cumulative net methane generation with initial pH control.� Different SS/SBPL ratios have been tested in biochemical methane potential tests.� Initial pH in batch test affects the biodegradability of anaerobic co-digestion.� A pH out of range for methanogenic microorganism, inhibits their growth.

a r t i c l e i n f o

Article history:Received 10 March 2014Received in revised form 10 June 2014Accepted 16 June 2014Available online 27 June 2014

Keywords:Biochemical methane potential (BMP) testpHAnaerobic co-digestionSewage sludgeSugar beet pulp lixiviation

a b s t r a c t

In this study, biochemical methane potential (BMP) tests were conducted to investigate the effect of pHcontrol on the co-digestion of sewage sludge (SS) and sugar beet pulp lixiviation (SBPL) at mesophilicrange (35 �C). Microbial concentrations (Eubacteria and methanogenic Archaea) are linked to traditionalparameters, biogas production and total volatile solids (TVS) removal. Also, the relationship betweenEubacteria and Archaea has been analysed. Organic matter being equal, higher net methane generationwas reported for the assay with pH adjustment at the beginning of the biochemical methane potential(BMP) test. This showed that there was inhibition of methane generation as measured by the BMP testin the absence of pH adjustment. Evidence of this inhibition was also supported by the methane yieldand TVS removal data. Microbial populations in the reactor at the end of both assays were composedof Eubacteria and Archaea, with a higher proportion of Eubacteria in all cases. It is noteworthy that in test1, when inhibition occurred due to a system pH that was not optimal for the activity of methanogenicArchaea, the analysis showed the largest number of Archaea present. In terms of productivity, it can besaid that methanogenic Archaea were inactive.

� 2014 Elsevier B.V. All rights reserved.

1. Introduction

Anaerobic digestion is a biological process in which a group ofmicroorganisms biodegrade organic matter (substrate) in theabsence of free molecular oxygen (O2). As a result of this complexbiological process, organic matter is mainly converted into a mix-ture of methane (CH4) and carbon dioxide (CO2), as well as newbacterial cells [1]. Throughout the process, complete bioconversionof organic matter into stable end products is accomplished by aseries of interdependent metabolic reactions in which differentclasses of microorganisms take part.

Efficiency of anaerobic digestion depends highly on wastecharacteristics, in addition to reactor configurations and other

operational parameters. Temperature, organic strength, bufferingcapacity, solids and nutrient content are considered crucialwaste characteristics affecting anaerobic biodegradation. If thecharacteristics of the waste are inappropriate for targeted treat-ment efficiency, some measurements can be taken to improve itsdigestibility.

The biochemical methane potential (BMP) test used to deter-mine methane potential is a batch procedure carried out over aperiod of time that is sufficient for the available carbon in the testsubstrate to be converted to biogas. This is normally considered tobe the point at which the biogas production from the test reactorsequals that from control reactors to which no substrate has beenadded. In practice, this can be in the range of 5–100 days, depend-ing on the degradability of the material. The approach has limita-tions and only provides an approximate basis for estimation ofthe biogas yield likely to be achieved in a continuous flow process.

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This will depend not only on the degradability of the substrate, butalso on factors such as process loading, retention time, tempera-ture and type of digester.

Co-digestion is one of the options used for the enhancement ofanaerobic degradation of wastes with different characteristics.Anaerobic co-digestion is the simultaneous biodegradation of dif-ferent wastes in a reactor to establish positive synergism in thedigestion medium [2]. Merits of co-digestion include: creating asuitable ratio of required nutrients, diluting potential toxic com-pounds [3], supplying buffering capacity [4], sharing of equipment,establishing required moisture content and easing the handling ofwastes [2]. In addition, anaerobic co-digestion is advantageous ifthe amount of a single waste generated at a particular site is notsufficient to make anaerobic digestion cost effective [5]. Thereare numerous studies in the literature regarding the anaerobicco-digestion of various wastes including: food industry wastes[6,7], animal manure [8,9], municipal solid waste [10,11], waste-water sludge [1], fish wastes [4] and algal sludge [12]. In most ofthese studies, remarkable improvements were observed in bothtreatment efficiencies and biogas productions.

Besides the advantages listed above, the co-digestion method-ology of organic residues of different origin has proved successfulin both thermophilic and mesophilic regimes so that these vari-ants would be applicable to the co-digestion of said waste. Goodresults have been achieved from the co-digestion of sewagesludge (SS) with fruit and vegetable wastes [13]. Improvementshave also been achieved in the production of biogas slurry mix-ture or bovine manure and plant debris [14] with mixtures ofbovine and fruit and vegetable waste [15]. To date, no BMP testhas been performed with sewage sludge and sugar beet pulp lix-iviation (SBPL).

The pH of the system, as well as temperature, has a markedeffect on the rate of growth and in selecting the type of microor-ganisms predominant in the process. The growth of each type ofmicroorganism occurs only within a characteristic pH range. Themaximum growth rate occurs at an optimum pH value whichusually coincides with the average value of the interval (7-8)[16].

The system pH is one of the most important parametersinfluencing anaerobic digestion in reactors, as it affects both thechemical reactions and the activity of microbial consortia in thesludge [17,18]. The optimum pH for most anaerobic microorgan-isms is 7–7.5, except for hydrolysing/fermentative bacteria whoseoptimum pH is 5–7 [19,20]. Generally, a pH of 8.5 is consideredunfavourable for methanogenic microbes, and pH values lowerthan 5 are considered inhibitory.

The different microbial groups involved in the digestion processhave varying sensitivities to environmental changes. The acetogen-ic bacteria and methanogenic Archaea have more stringent require-ments (in the thermophilic pH range 7.5–8.5) and greater difficultyacclimating when a change in environmental conditions occursand, therefore, have lower growth rates. As a result of distortionsin the environmental conditions of the medium, acetogenic bacte-ria are inhibited temporarily. However, they continue to producevolatile organic acids, which are then converted to acetic acid,hydrogen (H2) and carbon dioxide. This causes a significantdecrease in pH of the medium and, therefore, inhibits the activityof the bacteria. This process is known as an acidification reaction[21,22]. In this regard, a neutral or basic feed, or even direct controlof medium pH should favour degradation activity and, therefore,the purification process.

There is no conclusive theory to explain the role of the systempH. Bacterial performance is associated with the concentration ofvolatile fatty acids (VFAs), redox potential, hydrogen partial pres-sure and alkalinity. When the pH of the residue fed to a reactordecreases, there is an associated decrease in alkalinity.

The organic fraction of the sewage sludge and beet pulp lixivi-ation is converted to methane and carbon dioxide by anaerobicco-digestion, which takes place by the coordinated action of differ-ent groups of microorganisms and passes through several interme-diate stages. The intermediates are the following volatile fattyacids: acetic acid, propionic acid and butyric acid. The conversionof acetate to methane by methanogenic bacteria becomes the lim-iting step in the production of biogas since microorganisms thatare known methanogens grow slowly, resulting in a relativelysmall population [23]. Methanogens are typically divided intotwo main groups based on their substrate conversion capabilities.Acetoclastic methanogens are capable of converting acetate intomethane and carbon dioxide. These microorganisms are consid-ered to play a dominant role in the production of methane sinceabout 70% of the methane produced in the digester comes fromacetate [23]. The H2-utilising methanogenic bacteria also play akey role in anaerobic digestion since they are responsible for main-taining the partial pressure of H2 at a very low level (<10 Pa). Thiscondition is needed for the operation of the intermediate group,which is responsible for the conversion of organic acids and alco-hols to methane [24].

Process imbalances explain the formation and accumulation ofvolatile fatty acids (acetic, propionic and butyric acid), hydrogenand carbon dioxide in gaseous and liquid effluents when theirproduction by acidogenic bacteria are not balanced with theirdegradation.

In this study, anaerobic batch reactors were used to determinethe anaerobic biodegradation and biogas generation potential [25]of sewage sludge and sugar beet pulp lixiviation. For this purpose,both substrates were subjected to anaerobic biodegradation inbatch reactors. Since sugar beet pulp is a waste product of sugarbeet processing plants and is known to be suitable for biologicaldegradation, this study aimed to investigate the potential benefitsof co-digestion with sewage sludge as well as separate digestion ofthese wastes. The effect of pH on sewage sludge and sugar beetpulp lixiviation using the BMP test was examined in this studyfor the first time, as it has not previously been reported in theliterature.

Notations

BMP, biochemical methane potential

1-1, 100% SBPL test 1 OLR, organic loading rate 1-2, 75% SBPL–25% SS test 1 COD, chemical oxygen demand 1-3, 50% SBPL–50% SS test 1 H-Ac, acetic acid 1-4, 25% SBPL–75% SS test 1 H-Bu, butyric acid 1-5, 100% SS test 1 H-Pr, propionic acid 2-i, inoculum test 2 TS, total solids 2-1, 100% SBPL test 2 TVS, total volatile solids 2-2, 75% SBPL–25% SS test 2 VFA, volatile fatty acids 2-3, 50% SBPL–50% SS test 2 SS, sewage sludge 2-4, 25% SBPL–75% SS test 2 SBPL, sugar beet pulp lixiviation 2-5, 100% SS test 2 Test 1, assay without pH control Subscripts Test 2, assay with pH control t, s, total, soluble 1-i, inoculum test 1

2. Materials and methods

2.1. Feedstock

The substrates that were used in batch tests are sugar beet pulp,from Azucarera Ebro company in Jerez de la Frontera (Cádiz) andsewage sludge from the municipal wastewater treatment plant ofSan Fernando-Cádiz (Spain). Pellets were subjected to biologicalpretreatment before the co-digestion process in order to promotehydrolysis and solubilisation of the organic matter and therefore,improve anaerobic digestion in the generation of biogas andpossible final residue agronomic valorisation [26].

Table 1Inocula characteristics.

Mesophilic inocula

pH 7.4CODt (kg/m3) 21.3CODs (kg/m3) 1.2ST (kg/m3) 14.50SV (kg/m3) 8.58ST (%) 1.45SV (%) 0.86Alkalinity (mg CaCO3/l) 2480VFA t (mg H-Ac/l) 45.5H-Ac (mg/l) 45.5H-Pr (mg/l) 0.0H-Bu (mg/l) 0.0Total microorganism (cell/ml) 6.5�108

% Eubacteria 59.4% Archaea 40.6

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2.2. Inoculum

Primary Sludge from the WWTP of San Fernando-Cádiz wasused as inoculum in both tests.

The ultimate methane yields as well as the methane productionrates are dependent on the specific substrates and inoculum. Largeinoculations volumes ensure high microbial activity, low risk foroverloading and low risk of inhibition [27]. In this study, the anaer-obic seed was obtained from the municipal wastewater treatmentplant of San Fernando-Cádiz (Spain). The seed culture was theeffluent of a completely mixed anaerobic sludge digester havingan SRT of 30 days and operating at mesophilic range.

Mesophilic inocula with 1.45% of TS were added to the assayswithout and with pH control, until the desired conditions wereachieved. Table 1 shows the pH, TS, TVS, CODt, CODs, Alkalinity,VFA and microbial characterisation of inocula used.

2.3. Experimental set-up and procedures

Separate and co-digestion of sewage sludge and sugar beet pulplixiviation were studied in 250 ml serum bottles with effective vol-ume of 130 ml.

The digesters were initially loaded with a mixture of inoculumand substrate, resulting in a final concentration of 40% w/w of inoc-ulum, which is considered optimum for biogas production and sub-strates acclimatize, establishing a TVS concentration of 8.58 kg/m3

for test without and with pH control, respectively. Then the wasteswere added to the reactors at different amounts to give SS/SBPLratios in the range of 0.25, 0.5 and 0.75 (Table 2). Control reactors,containing only anaerobic inoculums, were also incubated todetermine the background gas production. All reactors were runin duplicates and presented data composed of the averaged values.

Table 2Initial characteristics from substrates in bottle serum.

1-1 1-2 1-3 1-4

pH 4.8 5 5.1 5.5CODt (kg/m3) 31.6 32.1 31.3 35.3CODs (kg/m3) 13.4 12.2 9.9 8.3ST (kg/m3) 14.9 17.5 18.4 19.1SV (kg/m3) 12 12.8 13.5 13.6ST (%) 1.6 1.7 1.8 1.9SV (%) 1.2 1.2 1.3 1.3Alkalinity (kg CaCO3/m3) 1.4 1.5 1.5 1.7VFA t (mg H-Ac/l) 5441 3921 3435 3521H-Ac (mg/l) 1734 936 1104 1153H-Pr (mg/l) 727 758 780 957H-Bu (mg/l) 1671 1155 693 531

Prior to incubation, all of the 24 reactors were purged with100% N2 for 3–4 min in order to maintain anaerobic conditionswith proper pH. Then the reactors were sealed with natural rubberstoppers and plastic screw-caps. Prepared reactors were incubatedin a temperature controlled bath at 35 �C. Manual mixing wasapplied three times a day during the test.

During digestion period, biogas productions and biogas compo-sitions were determined daily. After the digestion period wasended, all reactors were subjected to pH, TS, TVS, VFA, alkalinityand COD total and soluble determinations, in order to analysethe treatment efficiencies, as well as the microbiology analyses.

The process control parameters were: the degradative capacityof the system, measured as a percentage TVS removal and carbonoxygen demand (COD) and as well as the productivity of biogas,especially quantifying the cumulative net methane generation.Initially, mesophilic inoculas were characterised regarding theactivity of the microorganisms presented. At the end of the tests,the same parameters were analysed. Volume and composition ofbiogas were measured daily.

2.4. Analytical methods

Methods of analysis performed in this study can be groupedinto two categories: physical and chemical parameters of theprocess control of degradability and methods for quantificationof the microbial population in the reactors.

The determination of pH, total and volatile solids, total and sol-uble chemical oxygen demand and alkalinity will be establishedaccording to the standard method [28]. Volatile fatty acids andthe composition of the biogas are determined by gas chromatogra-phy. Gas chromatography was used to analyse the different com-ponents of the biogas. The gases analysed were: H2, CH4, CO2, O2

and N2 (GC-2010 Shimadzu). The first five components were ana-lysed by means of a thermal conductivity detector (TCD) using aSupelco Carboxen 1010 Plot column. Samples were taken using a1 ml Dynatech Gastight gas syringe.

Organic matter removal was calculated as the percentage differ-ence between the TVS of the initial and final substrates in theassays. Total acidity was calculated by addition of the individualfatty acids.

In order to compute methane generations, ideal gas balanceevaluations were carried out for each reactor, calculations wereperformed daily. During calculations, methane, generated in con-trol reactor was subtracted from that of reactor of interest, to beable to determine the net methane generation, resulted from thestabilisation of the waste in the corresponding reactor [29].

2.5. Microbial analyses

The main steps of fluorescence in situ hybridisation (FISH) ofwhole cells using 16S rRNA-targeted oligonucleotide probes are

1-5 2-1 2-2 2-3 2-4 2-5

6 7.3 7.4 7.3 7.5 7.833.9 29.1 40.3 47.2 63.2 72.16.5 12 10.3 7.8 5.9 1.520.5 17.3 22.6 25.6 31.2 34.514 10.7 15.3 18.5 23.4 26.32 1.7 2.3 2.6 3.1 3.41.5 1.1 1.5 1.8 2.3 2.62 2.2 3.9 3.4 3 2.53743 0 20.2 8.5 0 171154 0 0 0 0 01246 0 0 0 0 0347 0 0 0 0 0

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cell fixation, consequent permeabilisation and hybridisation withthe desired probe(s).

The samples were collected from batch reactor at the end of theassays into sterile universal bottles. Absolute ethanol was added tothe bottles in a volume ratio of 1 sample: 1 ethanol. The sampleswere stored at �20 �C until they were fixed. The experimental pro-cedure was conducted according Montero et al. [30].

The technique used for fixation and permeabilisation of cellswas based on the one used by Amann et al. [31]. The following16S rRNA-targeted oligonucleotide probes were used in this study:Bacteria-universal probe EUB338 [31,32], Archaea-universal probeARC915 [34], H2-utilising methanogens probe MB1174 (specifi-cally Methanobacteriaceae) [33].

The cellular concentration and percentages of Eubacteria,Archaea, H2-utilising methanogens were obtained by FISH. Thetotal population was calculated as the sum of the relative amountsof Eubacteria and Archaea, because the main anaerobic groups inthe anaerobic reactors are contained within these two domains[33]. Acetate utilising methanogens were calculated as thedifference in the relative amounts of Archaea and a H2-utilisingmethanogens. The main steps of FISH of whole cells using 16SrRNA-targeted oligonucleotide probes are cell fixation, consequentpermeabilisation, and hybridisation with the desired probe(s).

The samples were examined visually and cells counted using anAxio Imager Upright epifluorescence microscope (Zeiss) with a100 W mercury lamp and an 100� oil objective. According oflabelled probe, if the fluorochrome was 6-FAM, the filter was usedB-2A (DM 510, Excitation 450–490 and Barrer 520) and Cy3, thefilter was G-2A (DM 580, Excitation 510–560 and Barrer 590).

3. Results and discussion

3.1. Evolution of biogas generation

Reactors were operated until no significant biogas productionwas detected. Fig. 1 illustrates the cumulative net methane

Fig. 1. Cumulative net methane gen

Table 3Final characteristics from substrates in bottle serum.

1-i/2-i 1-1 1-2 1-3

pH final 7.5 4.7 5.4 7.4CODs (kg/m3) 3.8 15.5 14.3 12.4% CODt removal 8.3 0 0 0TVS (kg/m3) 6.7 11.9 10.2 10.8% TVS removal 22.1 3.1 20.6 20Final VFA (mg Ac/l) 21.6 5417 6103 5117Alkalinity (mg CaCO3/l) 4737 2997 1727 2682ml CH4/g VS added – 0 0 0% CH4 62.9 10.2 9 42.2% of biogas produced in first 10 days 61.2 – – 100

generation in all reactors. Cumulative biogas production isdepicted for 20 and 35 days of operation in test 1 and 2,respectively.

In anaerobic treatment systems, waste stabilisation is achievedby methane production [34]. Therefore, the rate of methane produc-tion directly reflects the rate of process stabilisation, which is crucialinformation for the design and operation of anaerobic treatmentsystems. Thus, determining the rate-limiting step, as well as analys-ing the overall biodegradation rate, is of fundamental importance.

As observed in biogas production, higher net methane genera-tion was reported for assay 2 when NaOH was added to adjustthe pH at the beginning of the BMP test. This explained the inhibi-tion in the first test compared to the second test. This effect wasalso supported by methane yield records and TVS removal data(Table 3).

Organic matter being equal, the reactors with the same sub-strate composition (SS/SBPL ratio) in both assays can be evaluatedin terms of treatment efficiencies in order to compare relative bio-degradability of SS/SBPL ratios with and without pH adjustment.The highest values of methane yield (544.4 ml/g TVS added[TVSadd]) and TVS reduction (63.5%) were observed in reactor 2-1, which was fed only with sugar beet pulp lixiviation. In fact, itis widely accepted that sugar beet pulp lixiviation is highly biode-gradable because it is made up of soluble carbohydrates, mainlysucrose [35,36]. Lower methane yield, chemical oxygen demand(COD) removal and TVS reduction were observed in test 1. Theseobservations are a direct result of inhibition in the assay due to asystem pH that is out of the methanogenic range and the high val-ues of VFAs in the initial substrates.

Thus to SS/SBPL ratio, initial pH was influential on thetreatment performance of the reactors (Table 4). Still, in allSS/SBPL ratios tested with pH adjustment, treatment efficiencies(48–61.5% total COD removal and 47–63.5% TVS reduction) areindications of high biodegradability for both substrates.

Fig. 1a and b shows that, in test 1, only reactors with a high per-centage (75% and 100%) of sewage sludge in the feed generated

erations: (a) test 1; (b) test 2.

1-4 1-5 2-1 2-2 2-3 2-4 2-5

7.1 7.1 7.7 7.7 7.6 7.6 7.69.7 8 13.3 12.3 10.7 8.4 6.90 0 47.8 49.9 56.1 59 61.510.5 10.9 3.9 6.4 9.6 12.3 11.665.3 27 63.5 57.8 48 47.3 562975 2459 0 20.2 8.5 0 172915 2895 1185 2322 2987 2880 29175.6 34.8 544.4 520.8 403.4 358.8 25560.9 56.2 67 63 61.6 63.3 57.571.3 3 43.2 47.2 48.4 47 47.2

Table 4Concentrations and percentages of Eubacteria, Archaea, H2-utilising methanogens and acetate-utilising methanogens in test 1 and in test 2.

Test 1

1-i 1-1 1-2 1-3 1-4 1-5

Total microorganism (cell/ml) 1.1�109 7.3�108 6.3�108 9.5�108 6.9�108 5.1�108

% Eubacteria 52.4 68.1 74.8 82.4 74.5 61.5% Archaea 47.6 31.9 25.2 17.6 25.5 38.5% H2-utilising methanogens 71.2 100 95.2 72.1 62.7 86.8% Acetate-utilising methanogens1 28.8 0 4.8 27.9 37.3 13.2

Test 2

2-i 2-1 2-2 2-3 2-4 2-5

Total microorganism (cell/ml) 1.2�108 7.3�107 6.5 107 1.3 108 1.1 108 1.1 108

% Eubacteria 52.6 76.1 61.5 64.3 60.3 53% Archaea 47.4 23.9 38.5 35.7 39.7 47% H2-utilising methanogens 100 100 100 100 100 100% Acetate-utilising methanogensa 0.0 0.0 0.0 0.0 0..0 0..0

a Acetate-utilising methanogens has been calculated in relation with Archaea.

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biogas. The rest of the reactors had no biogas production orremoval of organic matter due to low pH values and highquantities of VFAs.

Table 4 shows the parameters measured at the end of thebiodegradability test, in which very low methane productions wereobserved in all reactors assayed in test 1 for the different SS/SBPL

Fig. 2. Comparative cumulative net methane generatio

ratios tested. The cause of these results in test 1 was inhibitionof the process as a result of inappropriately low pH in the digesterduring the degradation test. The development of methanogens inanaerobic digestion requires higher pH values. In addition, thesubstrates analysed in test 1 had a high quantity of VFAs, whichinhibits methane generation.

ns in test 1 and test 2 at different SS/SBPL ratios.

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The following figures compare cumulative net methanegeneration for all substrates tested in both assays and show howpH adjustment affects methane generation and prevents systeminhibition.

From the graphs above it is noteworthy that the inocula usedwas able to generate the biggest quantity of cumulative biogascompared with the rest of the reactors in test 1, and the lowestcompared with reactors in test 2. In the rest of the reactors tested,those in assay 2 have higher biogas production than reactors inassay 1, as shown in Fig. 2.

3.2. Alkalinity and volatile fatty acids

The monitoring of volatile fatty acids, also known as shortchain fatty acids, is widely applied as a stress indicator in anaer-obic digestion processes. Allowing their accumulation leads todrops in pH and even subsequent reactor failures [37–39] asshows Fig. 3.

This pH reduction is normally counteracted by the activity ofthe methanogens, which produce alkalinity in the form of carbondioxide, ammonia and bicarbonate. The system pH is controlledby the CO2 concentration in the gas phase and the HCO3� alkalinityof the liquid phase. If the CO2 concentration in the gas phaseremains constant, the addition of HCO3� alkalinity will increasethe digester pH [40]. Fig. 4 shows the relationship between totalacidity and alkalinity. It is clear that the digesters in test 2 wereoperating with good buffering capacity, indicated by the low ornull amounts of VFAs. In test 2 the reduction in VFA levels andalkalinity over time did not affect methanogenic activity as themethane concentration in the gas did not drop. This demonstratedthat the acetogens and methanogens were able to cope with the

Fig. 3. (a) Individual volatile acid (as mg/l) levels in test 1

Fig. 4. (a) Ratio total acidity/alkalinity in test 1

fluctuations in the VFAs and alkalinity in the digester, indicatingthat the conditions were stable and the possibility of methanogeninhibition was low. Nevertheless, in test 1 reactors the productionand composition of biogas shows that VFAs affected the anaerobicdigestion process.

Total acidity values in test 1 were very high at the beginningand end of the process due to methanogen inhibition in the assay.High concentrations of volatile fatty acids were observed fre-quently in the effluent from anaerobic digesters as a result oforganic causes, such as overloading of the digester, entrance oftoxic compounds and changes in the temperature or pH. Low pHstimulates acidogenic activity (VFA production) and inhibits meth-anogenic activity (VFA consumption). This could explain the obser-vation of VFAs at the end of the assays.

Acetate has been described as the least toxic fatty acid [41],while an increase in propionate concentration has been shown tobe associated with system failure [42]. Propionate is even moreinhibitory than butyrate.

At the end of the test 2 assay, the level of VFAs decreased con-siderably. This was a consequence of the total biodegradability thatoccurred in all test 2 reactors.

During the entire operating time of the digester, alkalinityremained constant at around 3000 mg CaCO3/l, except for reactorswhere inhibition occurred because of low pH values. As shown inFig. 4, the total acidity/alkalinity ratio was very low in test 2 reac-tors and high in test 1 reactors, except for reactor 1-i (reactor withinocula). The pH balance is essential for proper operation and opti-mal degradation of VFAs. A ratio of total acidity/alkalinity between0.0 and 0.1 is desirable and results in a strong system. Valuesbetween 0.1 and 0.4 indicate favourable operating conditionswithout the risk of acidification [43,44].

; (b) individual volatile acid (as mg/l) levels in test 2.

; (b) ratio total acidity/alkalinity in test 2.

Fig. 5. (a, b) Analysis of productivity and microbial activity respectively in allreactors tested. (c) Relation between total Archaea population and initial organicloading rate in terms of g TVSadd in test 2.

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3.3. Microbial population dynamics

The microorganism concentrations in the effluent at the end ofthe BMP tests were studied. The amounts and relative percentagesof the main microbial groups are shown in Table 4.

Microbial populations in the reactor at the end of both assaysconsist of Eubacteria and Archaea, with higher proportions ofEubacteria in all cases. Stable anaerobic reactors contain aEubacteria population much larger than that of Archaea [45]. It isnoteworthy that in most cases studied, the H2-utilising methano-genic subpopulations are dominant over the acetate-utilisingmethanogens.

In test 1, inhibition occurred in reactors 1, 2, 3, 4 and 5 due tosystem pH values that were not optimum for the activity of meth-anogenic Archaea. The analysis showed the largest number ofArchaea present, however, in terms of productivity it can be saidthat methanogenic Archaea were inactive.

All the reactors in test 2 showed the same behaviour, and therelationships between all microbial populations were similar. Forthis reason, all substrates tested showed biodegradability.

To evaluate the biochemical activity on initial OLR (in terms of gTVSadd), it has been considered the parameter to measure metha-nogenic activity. The relation between those parameters was cal-culated as the ratio of CH4 volume generated to the number ofArchaea inside the reactor by FISH staining [30]. Fig. 5a and b com-pares productivity and microbial activity, respectively, in tests 1and 2. The productivity values of reactors in test 1 showed nullto low productivity in terms of ml CH4/g TVSadd, as well as null tolow microbial activity, due to the inhibition in the system. InFig. 5b inhibition is observed in test 1 as seen by the resultsobtained in terms of ml CH4/cell. This is because the Archaeapopulation present in the reactor did not generate methane dueto bacterial inactivity. However, Fig. 5b shows good results for test2 regarding microbial activity. A more detailed analysis is reflectedin Fig. 5d, which shows a linear relationship between the initialorganic loading, in terms of g TVSadd, and the total Archaea popula-tion at the end of the assay in batch reactors, except for inoculumreactor 2-i.

According to Fig. 5d, a high amount of microorganisms in theeffluent is directly related to higher levels of organic matter inthe initial substrates as defined by TVS.

Fig. 6a and b shows the relationship between the amount ofArchaea in the effluent and productivity obtained in all SS/SBPLratios studied in test 1 and test 2, respectively.

Compared to test 2 (Fig. 6b), test 1 (Fig. 6a) showed no or lowvalues of productivity in terms of ml CH4/g TVSadd in reactorstested, except for inoculum reactor 1-i, although the Archaeapopulation is higher in this test.

Fig. 6b shows that the size of the Archaea population has anindirect relationship with productivity in terms of ml CH4/g TVSadd,

Fig. 6. Relations between physicochemical parameters and microbial concentrations. (a,test 1 and 2 respectively.

due to the increased amount of volatile solids in the initial sub-strates. Comparing Fig. 5d with Fig. 6b shows that the results ofproductivity in terms of ml CH4/g TVSadd are not proportional tothe initial organic loading for the reason mentioned above.

Previous studies demonstrated links between digester operat-ing conditions, physical and chemical performance parametersand microbial population dynamics [30]. As discussed, the results

b) Compare Archaea population with methane yield in terms of ml CH4/g TVSadd in

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obtained in this study show that the initial organic loading in thereactors could be more related to microbial concentrations thanto the activity of anaerobic microorganisms.

4. Conclusions

Recent studies have indicated that in addition to sewage sludge,sugar beet pulp lixiviation has huge potential to be used as arenewable energy source by using anaerobic co-digestion. Basedon the experimental results, the following conclusions were made:

In tests without pH adjustment, inhibition occurred in anaero-bic digestion, except in reactor 1 with inocula, because the opti-mum pH of the methanogens was not reached and there was anaccumulation of volatile fatty acids. In the second test, after an ini-tial pH adjustment total biodegradability of the substrates testedoccurred. In reactors 2-2, 2-3 and 2-4, where the substrates weresewage sludge and sugar beet pulp lixiviation in different concen-trations, more methane was produced, which was an indication ofhigh biodegradability for both wastes.

Owing to the above mentioned points, the anaerobic co-digestion of SS and SBPL is a promising application, since additionof SBPL significantly increases the rate of biomethanation of SSwhen an optimum pH is reached.

Acknowledgments

The authors wish to express their gratitude to Junta de And-alucía, especifically to Proyecto de Excelencia with reference P09-TEP-5275, financed by FEDER funds, called ‘‘Codigestión anaerobiade lodos de depuradora y residuos de cultivos vegetales energéti-cos. Estrategias para mejorar producción de biogas y la valorizaciónagronómica del residuo final’’.

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