Accepted Manuscript

Anaerobic co-digestion of sewage sludge and sugar beet pulp lixiviation in batch
reactors: Effect of temperature

Rocío Montañ és Alonso, Montserrat Pérez García, Rosario Solera del Río

PII: S0960-8524(14)01802-1
DOI: http://dx.doi.org/10.1016/j.biortech.2014.12.056
Reference: BITE 14378

To appear in: Bioresource Technology

Received Date: 31 August 2014

Revised Date: 11 December 2014
Accepted Date: 15 December 2014

Please cite this article as: Alonso, R.M., García, M.P., Río, R.S.d., Anaerobic co-digestion of sewage sludge and
sugar beet pulp lixiviation in batch reactors: Effect of temperature, Bioresource Technology (2014), doi: http://
dx.doi.org/10.1016/j.biortech.2014.12.056

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Anaerobic co-digestion of sewage sludge and sugar beet pulp lixiviation

in batch reactors: Effect of temperature

1* 1 1
Rocío Montañés Alonso ; Montserrat Pérez García ; Rosario Solera del Río

1
Department of Environmental Technologies. University of Cádiz.
*Corresponding author. Tel.: +34 660 92 12 71.
E-mail addresses: rocio.montanes@uca.es

Abstract

The feasibility of anaerobic co-digestion of sewage sludge (SS) and sugar beet pulp

lixiviation (SBPL) was assessed. Mesophilic and thermophilic batch assays of five

different SS/SBPL ratios were used to investigate the effect of temperature, providing

basic data on methane yield and reduction in total volatiles. Microbe concentrations

(Eubacteria and methanogenic Archaea) were linked to traditional parameters, namely

biogas production and removal of total volatile solids (TVS). The relationship between

Eubacteria and Archaea was analysed.

Given equal masses of organic matter, net methane generation was higher in the

mesophilic range on the Biochemical Methane Potential (BMP) test. Methane yield,

TVS removal data and high levels of volatile fatty acids provided further evidence of

the best bahaviour of the mesophilic range. At the end of testing the microbial

population under of the reactors consisted of Eubacteria and Archaea, with Eubacteria

predominant in all cases.

Keywords: Biochemical methane potential (BMP) test; mesophilic range; thermophilic

range; Anaerobic co-digestion; Sewage sludge; Sugar beet pulp lixiviation

Anaerobic co-digestion of sewage sludge and sugar beet pulp lixiviation

in batch reactors: Effect of temperature

Abstract

The feasibility of anaerobic co-digestion of sewage sludge (SS) and sugar beet pulp

lixiviation (SBPL) was assessed. Mesophilic and thermophilic batch assays of five

different SS/SBPL ratios were used to investigate the effect of temperature, providing

basic data on methane yield and reduction in total volatiles. Microbe concentrations

(Eubacteria and methanogenic Archaea) were linked to traditional parameters, namely

biogas production and removal of total volatile solids (TVS). The relationship between

Eubacteria and Archaea was analysed.

Given equal masses of organic matter, net methane generation was higher in the

mesophilic range on the Biochemical Methane Potential (BMP) test. Methane yield,

TVS removal data and high levels of volatile fatty acids provided further evidence of

the best bahaviour of the mesophilic range. At the end of testing the microbial

population under of the reactors consisted of Eubacteria and Archaea, with Eubacteria

predominant in all cases.

Keywords: Biochemical methane potential (BMP) test; mesophilic range; thermophilic

range; Anaerobic co-digestion; Sewage sludge; Sugar beet pulp lixiviation

1. Introduction

It is essential to develop sustainable energy supply systems to meet the demand for

energy from renewable sources. Reducing greenhouse gas emissions by increasing

production of renewable energy production is increasingly important. Biogas

production technology is critical to the sustainable use of biomass as a renewable

energy source. Biogas can be produced from a wide range of energy crops, animal

manures and organic wastes and therefore offers a flexible source of energy which can

be adapted to the specific needs of different locations and farming styles. The residues

of anaerobic digestion are a valuable fertiliser for agricultural crops.

Anaerobic digestion is a biological process in which a group of micro-organisms

biodegrade organic matter (substrate) in the absence of free molecular oxygen (O2).

This complex biological process converts organic matter into a mixture composed

mainly of methane (CH4), carbon dioxide (CO2) and new bacterial cells (Romano and

Zhang, 2008). Complete bioconversion of organic matter into stable end products is

accomplished by a series of interdependent metabolic reactions involving several

classes of micro-organisms.

The efficiency of anaerobic digestion is highly dependent on the characteristics of the

waste, the reactor configuration and other operational parameters. The temperature,

organic strength, buffering capacity and solid and nutrient content of the waste are

important influences on anaerobic biodegradation. Waste can be treated to improve

its digestibility.

Assay of biochemical methane potential (BMP) is a procedure developed to determine

how much methane is produced by anaerobic decomposition of a given organic

substrate. BMP assay has proved to be a relatively simple, reliable method for

determining the extent and rate at which organic matter is converted to methane

(Chynoweth et al., 1993).

Co-digestion can be used to enhance anaerobic degradation of wastes with certain

characteristics. Anaerobic co-digestion is the synergistic simultaneous biodegradation

of different wastes (Mata-Alvarez et al., 2008). The merits of co-digestion include

creation of a suitable nutrient ratio, dilution of potentially toxic compounds (Sosnowski

et al., 2003), provision of buffering capacity (Mshandale et al., 2004), equipment

sharing, establishment of the required moisture content and easier waste-handling

(Mata-Alvarez et al., 2008). Anaerobic co-digestion is also advantageous if the amount

of a given waste generated at a particular site is not sufficient to make anaerobic

digestion cost effective (Parawira et al., 2004). There have been numerous studies of

anaerobic co-digestion of various wastes including food industry wastes (Garucci et al.,

2005, Murto et al., 2004), animal manure (Gungo-Demirci and Demirel, 2004),

municipal solid waste (Zuparicic et al., 2008), waste water sludge (Romano and Zhang,

2008), fish wastes (Mshandale et al., 2004) and algal sludge (Yen and Brune, 2007);

most showed a remarkable improvement in both treatment efficiency and biogas

production in comparison with single-waste anaerobic digestion.

Both thermophilic and mesophilic co-digestion regimes have been used successfully,

making the technique more flexible than conventional anaerobic digestion.

Methane is formed over a wide range of temperatures; however anaerobic digestion

processes are highly temperature dependent. Most BMP assays have been performed

at mesophilic temperatures. Both pH and temperature have a marked effect on the

rate of growth and the composition of the micro-organism population during the

digestion process (Callaghan et al., 1999).

The majority of methanogens (micro-organisms that produce methane from organic

matter) are mesophiles, growing quickly and converting a higher proportion of organic
matter in the mesophilic temperature range. Mesophilic systems are more stable than

thermophilic systems, which has implications for the design of biogas plants. The

stability and growing conditions in a mesophilic digester make the process more

balanced, more resistant to chemicals that inhibit digestion (e.g. ammonia) and also

more capable of treating a variety of types of biomass and waste.

Only a minority of methanogens are thermophilic, preferring higher temperatures.

Reaction rates are higher in a thermophilic system and the microbial population grows

faster. This means that thermophilic digesters can be smaller (which means lower

manufacturing costs) whilst maintaining very high biogas yields. Thermophilic

anaerobic digestion also destroys a higher proportion of the pathogenic bacteria

present in organic wastes.

Despite the advantages of the thermophilic process most biogas plants continue to use

mesophilic anaerobic digestion systems. This choice can be justified on the grounds

that it is more difficult to control and optimise the thermophilic process. Thermophilic

methanogens are extremely sensitive to changes in the environment for anaerobic

digestion; even a small change in operating parameters can have a negative impact, for

example changes of more than 1-2°C in temperature greater significantly reduce

biogas yield. Anaerobic thermophilic conditions are suitable for a smaller range of

waste materials than mesophilic conditions, mainly because of the chemical

composition of wastes and the greater impact of certain digestion inhibitors on the

digestion process.

Anaerobic co-digestion converts the organic fraction of sewage sludge (SS) and sugar

beet pulp lixiviation (SBPL) to methane and carbon dioxide. It involves coordinated

action of several groups of micro-organisms and is a multi-stage process. The outputs

 of the intermediate stages are volatile fatty acids (VFAs): acetic acid, propionic acid

and butyric acid. The conversion of acetate to methane by methanogenic bacteria is

the limiting step in the production of biogas as known methanogens grow slowly

meaning that populations remain relatively small (Zinder 1993). Methanogens are

typically divided into two main groups based on their substrate conversion capabilities.

Acetoclastic methanogens convert acetate into methane and carbon dioxide; they are

the primary methane producers: about 70% of the methane produced in digesters

comes from acetate (Zinder 1993). Methanogenic bacteria which use H2 also play a

critical role in anaerobic digestion since they are responsible for maintaining the partial

pressure of H2 at the very low level (< 10 Pa) required by the intermediate group,

which is responsible for the conversion of organic acids and alcohols to methane

(Pauss et al., 1990)

In this study anaerobic batch reactors were used to determine the anaerobic

biodegradation and biogas generation potential (Owen et al., 1979) of SS and SBPL.

Both substrates were subjected to anaerobic biodegradation in batch reactors. Sugar

beet pulp is a waste product of sugar beet processing plants and is known to be

suitable for biological degradation so this study investigated the potential benefits of

co-digesting SS and SBPL as well as separate digestion of these wastes. This study was

also the first systematic investigation of the effects of variations in temperature on

anaerobic digestion of SS and SBPL.

Abbreviations

1 BMP biochemical methane potential 4 CODt total chemical oxygen demand

2 OLR organic loading rate 5 H-Ac acetic acid

3 COD soluble chemical oxygen demand 6 H-Bu butyric acid

 7 H-Pr propionic acid 19 1-2 75% SBPL-25% SS series 1

8 TS total solids 20 1-3 50% SBPL- 50% SS series 1

9 TVS total volatile solids 21 1-4 25% SBPL- 75% SS series 1

10 VFA volatile fatty acid 22 1-5 100% SS series 1

11 SS sewage sludge 23 2-i Inoculum series 2

12 SBPL sugar beet pulp lixiviation 24 2-1 100% SBPL series 2

13 Series 1 Assays under thermophilic 25 2-2 75% SBPL-25% SS series 2

14 conditions 26 2-3 50% SBPL- 50% SS series2

15 Series 2 Assays under mesophilic 27 2-4 25% SBPL- 75% SS series 2

16 conditions 28 2-5 100% SS series 2

17 1-i Inoculum series 1 29 Subscripts

18 1-1 100% SBPL series 1 30 t: total; s: soluble

2. Materials and Methods

2.1. Feedstock

The substrates used in the tests were sugar beet pulp, from Azucarera Ebro company

in Jerez de la Frontera (Cádiz), and SS from the municipal waste water treatment plant

of San Fernando-Cádiz (Spain). Sugar beet pellets were subjected to physical pre-

treatment before the co-digestion process to promote hydrolysis and solubilisation of

the organic matter and thus improve anaerobic digestion, biogas yield and possibly

also the agronomic value of the final residue (Montañés et al., 2013).

2.2. Inoculum

In both series of tests primary sludge from the San Fernando-Cádiz waste water

treatment plant was used as the inoculum.

Both final methane yield and methane production rate are dependent on the

substrates and inoculum. Large inoculation volumes ensure high rates of microbial

activity and reduce the risks of overloading and inhibition (Angelidaki and Sanders,

2004). The seed culture was the effluent of a completely mixed anaerobic digester

having an Hydraulic Retention Time (HRT) of 20 days and was capable of operating at

mesophilic and thermophilic ranges.

Mesophilic and thermophilic inocula with 1.45% or 2.70% Total Solids (TS) were added

to the assays, until the desired conditions were achieved. Table 1 shows the pH, TS,

Total Volatile Solids (TVS), Total and soluble Carbon Oxygen Demand (CODt, COD),

alkalinity, VFA content and microbial characterisation of inocula used.

2.3. Experimental set-up and procedures

Separate and co-digestion of SS and SBPL were studied in 250ml serum bottles with an

effective volume of 130ml.

The digesters were loaded with a mixture of inoculum and substrate, resulting in a

final inoculum concentration of 40% w/w, which is considered optimal for biogas

production and substrate acclimatisation, leading to establishment of a TVS

concentration of 8.58 or 19.8 kg/m3 in the mesophilic and thermophilic test systems

respectively. Then different amounts of the wastes were added to the reactors to give

SS/SBPL ratios of approximately 0.25, 0.5 or 0.75 (Table 2). Control reactors containing

only anaerobic inocula were also incubated to determine background gas production.

All reactors were run in duplicate and the data presented here are average values.

NaOH was added to reactors prior to incubation, to adjust the pH at the beginning of

the BMP test and then all 24 reactors were purged with 100% N2 for 3-4 min to
maintain anaerobic conditions at the appropriate pH. Subsequently the reactors were

sealed with natural rubber stoppers and plastic screw caps. Prepared reactors were

incubated in a temperature-controlled bath at 35°C or 55°C in mesophilic and

thermophilic systems respectively. Reactor contents were manually mixed three times

a day during testing.

Biogas production and biogas composition were determined daily during the digestion

period. At the end of the digestion period data on pH, TS, TVS, VFA, alkalinity and CODt

and COD were collected for all reactors to enable calculation of treatment efficiency

and microbiological analyses.

The parameters of the digestion process were: the degradative capacity of the system

(measured as percentage TVS removal), carbon oxygen demand (COD) and biogas

productivity (in terms of cumulative net generation of methane). The mesophilic and

thermophilic inocula were characterised in terms of microbial activity at the start and

end of the BMP tests.

2.4. Analytical methods

Two types of analysis were performed: analysis of the physical and chemical

parameters of the degradation process and quantification of the microbial population

of the reactors.

Total and volatile solids, total and soluble chemical oxygen demand, pH and alkalinity

were assessed using the standard method (APHA, 1995). VFA content and biogas

composition were determined by gas chromatography. The gases analysed were H2,

CH4, CO2, O2 and N2 (GC-2010 Shimadzu). Those first five components were analysed
using a thermal conductivity detector (TCD) using a Supelco Carboxen 1010 Plot

column. Samples were taken using a 1ml Dynatech Gastight gas syringe.

The efficiency of organic matter removal was calculated as the percentage difference

between the TVS content of the initial and final substrates in the assays. Total acidity

was calculated by summing the values for individual fatty acids.

Ideal gas balance was calculated daily for each reactor to determine the amount of

methane generated from the stabilisation of the waste, net methane generation for

experimental reactors was calculated as the difference between the amount of

methane generated in the experimental reactor and the control reactor (Alkaya and

Demirel, 2011).

Biogas production was determined indirectly, by measuring the cumulative pressure

inside the bottles via pressure transducers. Pressure data were used to infer the

volume of biogas at standard temperature and pressure, according to the ideal law of

gases:

P•V = n•R•T

where P is absolute pressure (kPa), V is volume (m3), n is amount of substance (moles),

T is temperature (K) and R is the universal gas constant (8.3145 L kPa/Kmol).

2.5. Microbial analyses

The main steps in fluorescence in situ hybridisation (FISH) of whole cells using 16S

rRNA-targeted oligonucleotide probes are cell fixation, consequent permeabilisation

and hybridisation with the desired probe(s).

Samples from batch reactors were collected into sterile universal bottles at the end of

BMP tests. Absolute ethanol was added to the bottles in a 1:1 v/v ratio. The samples
were stored at -20°C until they were fixed. Further details of this procedure are given

in Montero et al. (2009).

The technique used for fixing and permeabilising cells was based on the one described

by Amann et al. (1990). The following 16S rRNA-targeted oligonucleotide probes were

used in this study: bacteria-universal probe EUB338 (Amann, 1990a, Amann et al.,

1990b), Archaea-universal probe ARC915 (Speece, 1996), H2-utilising methanogens

probe MB1174 (specific to Methanobacteriaceae; Stahl and Amann, 1991).

The cellular concentration and percentages of Eubacteria, Archaea and H2-utilising

methanogens were obtained using FISH. Total microbial population was estimated as

the sum of the populations of Eubacteria and Archaea, because most anaerobic micro-

organisms in anaerobic reactors belong to these two groups (Stahl and Amann,

1991).The population of acetate-utilising methanogens was calculated as the

difference between the populations of Archaea and a H2-utilising methanogens.

Samples were examined visually and the cells were counted using an Axio Imager

Upright epifluorescence microscope (Zeiss) with a 100W mercury lamp and a 100x oil

objective lens. The filter used depended on the identity of the labelled probe, a B-2A

filter (DM 510, Excitation 450–490 and Barrer 520) was used for 6-FAM; a G-2A (DM

580, Excitation 510–560 and Barrer 590) filter was used for Cy3.

3. Results and Discussion

3.1. Evolution of biogas generation

Reactors were operated until significant biogas production was no longer taking place.

The methane production curves for all assays following removal of the inoculum are

presented in Figure 1. Cumulative methane production followed similar time courses in

mesophilic and thermophilic reactors, but the thermophilic curves are shorter and

asymptote was reached before the lag phase.

In anaerobic treatment systems waste stabilisation is achieved by methane production

(Speece, 1996) and so the rate of methane production is directly related to the stabilisation

rate, which is a crucial factor in the design and operation of anaerobic treatment systems.

As is commonly observed in biogas production, cumulative net methane generation in

the BMP test was higher at mesophilic temperature (series 2). This explained the

relatively poor co-digestion of SS and SBPL at 55°C. The high VFA levels (Table 3) were

consistent with this interpretation.

The treatment efficiencies of reactors with similar substrate composition (SS/SBPL

ratio) operating at mesophilic and thermophilic temperatures can be used to compare

the biodegradability of various SS/SBPL ratios. The highest methane yield (544.4 ml/g

TVS added) and greatest reduction in TVS (63.5%) were observed in reactor 2-1, which

was fed with SBPL only. It is widely accepted that SBPL is highly biodegradable because

it is made up of soluble carbohydrates, mainly sucrose (Iza et al, 1990). More

interestingly, cumulative methane production was greatest in reactor 2-4.

Methane yield, CODt removal and TVS reduction were lower in series 1. These results

reflect the amount of VFAs present at the end of the test. The high values of VFA could

be due to the amount of VFA in the thermophilic inocula (Table 1). This implies a pH

reduction in all reactors tested in series 1.

Figures 1a and 1b show that methane production was lower in series 1 than in series 2

at all SS/SBPL ratios tested. Biodegradation was inhibited in series 1 reactors; there

was some methane production, but it appears that the high VFA content of the
thermophilic inoculum limits the biodegradability of different mixtures of SS and SBPL.

Table 3 gives physical and chemical parameters for the substrate at the end of the

biodegradability test; methane production was lower in series 1 than series 2 for all

reactors and all SS/SBPL ratios investigated reflecting lower biodegradability caused by

the high concentration of VFAs in the digesters during the degradation test and the

resultant decrease in pH. A higher pH is needed for development of methanogen

populations in anaerobic digestion systems.

The following figures compare cumulative net methane generation for all substrates

tested in both assays and show how temperature affected methane generation and

the productivity of anaerobic co-digestion of SS and SBPL.

It is remarkable that although methane production was significantly greater in series 2,

carried out in mesophilic conditions, the pattern of results for different SS/SBPL ratios

was the same under thermophilic conditions. In both regimes the adding SBPL to the

system as a co-substrate increased methane production relative to separate digestion

of the two types of waste, except in reactor 1-4.

Figure 2 shows clearly that the choice of inoculum had a marked effect on the

cumulative production of methane; this effect was stronger when SS and SBPL were

digested separately (reactors 1-1 and 1-5 respectively). These results indicate that

even when conditions are not optimal for the inoculum co-digestion of SS and SBPL

improved biodegradation of the wastes due the synergy between them except in

reactor 1-4.

3.2. Alkalinity and VFAs

VFAs, also known as short chain fatty acids, are widely used as an indicator of stress in

anaerobic digestion processes. Accumulation of VFAs results in a drop in pH and may

even lead to reactor failure or severely compromised digestion (Ahring et al., 1995,

McCarty, 1964).

The reduction in pH produced by VFAs is normally offset by the activity of

methanogens, which have an alkalising effect as they produce carbon dioxide,

ammonia and bicarbonate. The pH of a digestion system is determined by CO2

concentration in the gas phase and by HCO3- concentration in the liquid phase. Given a

constant CO2 concentration in the gas phase the addition of HCO3- will increase the pH

in a digester (Turovski and Mathai, 2006). Figure 3 shows the relationship between

acidifying and alkalising substances in the digesters; it is clear that the digesters in

series 2 were operating with good buffering capacity as the quantity of VFAs in the

digester remained low or negligible. The reduction in VFA levels and alkalinity over

time in series 2 did not affect methanogenic activity as the methane concentration in

the digesters did not fall.

The ratio VFA/Alk demonstrated that the acetogens and methanogens were able to

cope with the fluctuations in VFAs and alkalinity in the digesters, indicating that

conditions were stable and the risk of methanogen inhibition was low. The data on the

composition and cumulative production of biogas in series 1 reactors showed that

VFAs nevertheless affected the anaerobic digestion process.

In series 1 total acidity values were very high at the beginning of the tests owing to the

characteristics of the inoculum used. The concentration of VFAs in the effluent from

the anaerobic digesters was often high, owing to overloading of the digester, entrance

of toxic compounds or changes in the temperature or pH. Low pH stimulates

acidogenic activity (VFA production) and inhibits methanogenic activity (VFA

consumption) which may explain the high VFA levels at the end of the tests (Figure 4).

The accumulation of VFAs has long been associated with disturbances in the conditions

in anaerobic digestion systems (Harper and Pohland, 1986); accumulation of VFAs was

reported to be more pronounced at thermophilic temperatures (Gray et al., 2006),

probably because aceticlastic methanogens are temperature-sensitive (Ahring et al.,

2001; Leven et al., 2007)

Levels of VFAs were low at the end of testing in series 2 reactors reflecting the

complete biodegradation of wastes that occurred in all series 2 reactors.

The ratios of total acidity/ total alkalinity (VFA/Alk) were very low in series 2 reactors

and high in series 1, with the exception of reactor 1-i (reactor with inoculum only).

Maintenance of an appropriate pH is essential for proper operation of the digester and

optimal VFA degradation; in a strong system the VFA/Alk ratio will be between 0.0 and

0.1, values between 0.1 and 0.4 indicate favourable operating conditions and low or no

risk of acidification (Sanchez et al., 2005).

3.3. Microbial population dynamics

Data were collected on micro-organism concentrations in the reactors at the end of

the BMP tests. Absolute quantities and proportions (%) of the main microbial groups

are shown in Table 4.

At the end of testing the microbial populations in both reactor series consisted of

Eubacteria and Archaea, with Eubacteria in the majority in all cases. Stable anaerobic

reactors have a much larger population of Eubacteria than Archaea (Zahedi et al,
2013). It is noteworthy that in the reactors there were more H2-utilising methanogens

than acetate-utilising methanogens.

Biodegradation was limited in all series 1 reactors as pH values were not optimum for

the methanogenic Archaea as a consequence of the quantity of VFAs produced.

Analysis revealed that although the Archaea populations were higher in series 1

reactors they were less active than in series 2 reactors.

The pattern of activity and composition of the microbial population were similar in all

series 2 reactors indicating that all substrates tested had biodegraded.

The relationship between organic loading rates and microbial activity which was

calculated as the ratio of the volume of CH4 generated to the number of Archaea

inside the reactor (assessed using FISH staining) (Montero et al., 2009). Table 5 shows

microbial activity in both series of reactors; microbial activity was greater in series 2

reactors, although the pattern of activity for different SS/SBPL ratios was similar under

thermophilic and mesophilic conditions, just as the pattern of change in

physicochemical parameters was similar in both series. The best results were obtained

when SBPL was added to SS as a co-substrate.

To evaluate the biochemical activity on initial OLR (in terms of g TVSadd), it has been

considered the parameter to measure methanogenic activity. These data are

presented in Figure 5.

Figures 5a and 5b show the relationship between Archaea population density and the

productivity of the reactor for all SS/SBPL ratios under both thermophilic (series 1) and

mesophilic (series 2) conditions respectively.

Productivity in terms of ml CH4/g TVSadd was lower under thermophilic conditions

although Archaea population densities were of the same order of magnitude.

Figure 5b shows that the size of the Archaea population is indirectly related to

productivity measured in terms of ml CH4/g TVSadd; this relationship is mediated by the

proportion of total volatile solids in the initial substrate, which is higher. Productivity

was not proportional to the size of the Archaea population.

Previous studies have demonstrated links between digester operating conditions,

physical and chemical performance parameters and microbial population dynamics

(Montero et al, 2009). These results suggest that the composition and density of

microbial population may be more closely related to initial organic load than to the

activity of anaerobic micro-organisms during digestion.

4. Conclusions

Biodegradation was limited under thermophilic conditions, because the VFA

concentration increased, but under mesophilic conditions complete biodegradation of

the test substrates occurred.

More methane was produced in reactors containing a substrate made up of a mixture

of SS and SPBL (i.e. reactors 1-2, 1-3, 1-4, 2-2, 2-3 and 2-4), indicating that combining

these wastes had a synergistic effect on anaerobic digestion.

Anaerobic co-digestion of SS and SBPL is a promising waste-processing procedure;

addition of SBPL to SS significantly increases the rate of biomethanation of SS under

suitable pH conditions.

Acknowledgments
The authors wish to express their gratitude to Junta de Andalucía, specifically to

Proyecto de Excelencia financed through FEDER funds, with reference P09-TEP-5275,

called ‘‘Codigestión anaerobia de lodos de depuradora y residuos de cultivos vegetales

energéticos. Estrategias para mejorar la producción de biogás y la valorización

agronómica del residuo final’’.

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 700 700

600 600

500 500
1-i 2-i
400 400
ml CH4 1.1

ml CH4
2.1

300 1.2 2.2

300
1.3 2.3
200 1.4 200 2.4

100 1.5 2.5

100

0 0
1 2 3 4 5 6 7 8 9 10 111213 14 15 16 17181920 21 22 23242526 27 28 29 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
a days b days

Figure 1: Cumulative net methane production a) thermophilic series; b) mesophilic

series
 70 600

60 500
50
400
40
ml CH4

ml CH4
300
30 2-i 2.3
1-i 200 1.3
20

10 100

0 0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
days days

600 700

500 600

500
400
400

ml CH4
ml CH4

300
2.1 300 2.4
200 1.1 1.4
200
100 100

0 0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
days days

700 600

600
500
500
400
400
ml CH4

ml CH4

300
300 2.2 2.5
1.2 200 1.5
200

100 100

0 0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35
days days

Figure 2: Cumulative net methane production at various SS/SBPL ratios in thermophilic

conditions (series 1) and mesophilic conditions (series 2)

 3 0,01
0,009
2,5 0,008

mg Ac-H/mg CaCO3
mg Ac-H/mg CaCO3
0,007
2
0,006
1,5 0,005
0,004
1 0,003
0,002
0,5
0,001
0 0
1-i 1-1 1-2 1-3 1-4 1-5 2-i 2-1 2-2 2-3 2-4 2-5

a VFA/Alkf b VFA/Alkf

Figure 3: Ratio of total acidity to total alkalinity (VFA/Alk) in a) thermophilic conditions

(series 1); b) mesophilic conditions (series 2)

 10000 2500
9000
8000 2000
7000
mg/L 6000 1500

mg/L
5000
4000 1000
3000
2000 500
1000
0 0
1-i 1-1 1-2 1-3 1-4 1-5 2-i 2-1 2-2 2-3 2-4 2-5

a H-Ac H-Pr H-Bu b H-Ac H-Pr H-Bu

Figure 4: Levels of individual volatile fatty acids (mg/l) in a) thermophilic conditions; b)

mesophilic conditions.
 180 1,00E+08 700 6,00E+07
160 9,00E+07
600 5,00E+07
140 8,00E+07
7,00E+07 500
120 4,00E+07
6,00E+07 400
100
5,00E+07 3,00E+07
80 300
4,00E+07
60 2,00E+07
3,00E+07 200
40 2,00E+07
100 1,00E+07
20 1,00E+07
0 0,00E+00 0 0,00E+00
1-i 1-1 1-2 1-3 1-4 1-5 2-i 2-1 2-2 2-3 2-4 2-5
a ml CH4/g TVSadd Archaea (cell/ml)
b ml CH4/g TVSadd Archaea (cell/ml)

Figure 5: Relationship between physicochemical parameters and microbial

concentrations. Archaea population density (cells/ ml) is shown alongside methane

yield (ml CH4/g TVSadd) in a) thermophilic conditions and b) mesophilic conditions.

Table 1: Inocula characteristics

Mesophilic inocula Thermophilic inocula

pH 7.4 7.49
CODt (kg/m3) 21.3 45.9
COD (kg/m3) 1.2 7.8
TS (kg/m3) 14.50 27
TVS (kg/m3) 8.58 19.8
TS (%) 1.45 2.70
TVS (%) 0.86 1.96
Alkalinity (kg CaCO3/m3) 2.5 6.6
VFA t (mg H -Ac/l) 45.5 7477
H-Ac (mg/l) 45.5 649
H-Pr (mg/l) 0.0 2909
H-Bu (mg/l) 0.0 839
Total micro-organism 1.6·108
6.5·108
content (cell/ml)
% Eubacteria 59.4 57.6
% Archaea 40.6 42.4
Table 2: Initial characteristics from substrates in bottle serum

1-1 1-2 1-3 1-4 1-5 2-1 2-2 2-3 2-4 2-5
pH 8.1 8.1 7.4 7.6 7.3 7.3 7.4 7.3 7.5 7.8
CODt (kg/m3) 41.4 47.6 48.1 50.7 58.2 29.1 40.3 47.2 63.2 72.1
COD (kg/m3) 11.1 10.8 9.1 6.8 3.9 12 10.3 7.8 5.9 1.5
TS (kg/m3) 33.1 36.6 36.4 35.7 36.9 17.3 22.6 25.6 31.2 34.5
TVS (kg/m3) 24.0 25.0 27.6 27.3 29.9 10.7 15.3 18.5 23.4 26.3
TS (%) 3.31 3.66 3.64 3.57 3.69 1.7 2.3 2.6 3.1 3.4
TVS (%) 2.40 2.5 2.76 2.73 2.99 1.1 1.5 1.8 2.3 2.6
Alkalinity
(kg 4.9 6.8 5.4 4.2 4.1 2.2 3.9 3.4 3 2.5
CaCO3/m3)
VFA t
4248 5431 3582 2824 1705 1503 1271 830 346 152
(mg H-Ac/l)
H-Ac (mg/l) 1536 1518 792 564 163 371 313.6 209.7 34.5 380
H-Pr (mg/l) 1180 1353 948 837 498 75.6 62.7 34.5 0 0
H-Bu (mg/l) 640 664 439 357 266 696 585 380 175 91
Table 3: Characteristics of substrates in bottle serum at the end of the biogas methane

production test

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

pH final 7.6 7.7 6.6 7.7 6.8 6.8 7.5 7.7 7.7 7.6 7.6 7.6

COD
3 16.8 22.2 13.3 13.4 21.0 21.0 3.8 13.3 12.3 10.7 8.4 6.9
(kg/m )

% CODt
11.7 1.5 34.4 35.3 16.9 21.6 8.3 47.8 49.9 56.1 59 61.5
removal

TVS
3 7.8 15.3 12.4 10.5 19.4 22.5 6.7 3.9 6.4 9.6 12.3 11.6
(kg/m )

% TVS
60.6 36.4 50.6 61.9 28.9 24.8 22.1 63.5 57.8 48 47.3 56
removal

Final VFA 1509 1543 1190

6552 7840 8120 21.6 0 20.2 8.5 0 17
(mg Ac/l) 1 8 2

Alkalinity
(kgCaCO3/ 7.1 5.8 7.6 7.8 6.5 6.6 4.7 1.1 2.3 3.0 2.9 2.9
m3)

ml CH4/g
- 52.8 173.1 149 50.7 28.2 - 544.4 520.8 403.4 358.8 255
VS added

%CH4 59.0 47.6 51.4 59.8 57.4 42.1 62.9 67 63 61.6 63.3 57.5

% of
biogas
produced 45.4 77.5 44.1 43.0 92.5 95.5 61.2 43.2 47.2 48.4 47 47.2
in first 10
days
Table 4: Concentrations and percentages of Eubacteria, Archaea, H2-utilising

methanogens and acetate-utilising methanogens in series 1 and series 2

Series 1
1-i 1-1 1-2 1-3 1-4 1-5
Total micro-
8 8 8 8 8 8
organism 1.6 10 3.1 10 2.7 10 2 10 2 10 1.3 10
(cell/ml)
% Eubacteria 57.7 69.5 74.5 71.5 68.8 77.6
%Archaea 42.3 30.5 25.5 28.5 31.2 22.4
% H2-utilising
100 100 100 100 100 100
methanogens
% acetate-
utilising 0.0 0.0 0.0 0.0 0.0 0.0
methanogens1
Series 2
2-i 2-1 2-2 2-3 2-4 2-5
Total micro-
organism 1.2 x 108 7.3 x 107 6.5 x 107 1.3 x 108 1.1 x 108 1.1 x 108
(cell/ml)
% 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
100 100 100 100 100 100
methanogens
% acetate-
utilising 0.0 0,0 0,0 0,0 0,0 0,0
methanogensa
a
Figures for acetate-utilising methanogens have been calculated relative to Archaea
 Table 5: Microbial activity.

Series 1
1-i 1-1 1-2 1-3 1-4 1-5
Microbial
Activity 1.1 10-11 1. 10-11 5.1 10-11 4.7 10-11 1.5 10-11 1.9 10-11
(L CH4/cell)
Series 2
2-i 2-1 2-2 2-3 2-4 2-5
Microbial
-11 -10 -10 -10 -10 -11
1.1 10 2.1 10 2 10 1 10 1.2 10 7.9 10
Activity
(L CH4/cell)
Highlights:

Methane productivity is higher under a mesophilic than thermophilic regimen.

Sugar beet pulp lixiviation (SBPL) improves cumulative net methane

generation.

Several sludge/SBPL ratios were tested in biochemical methane potential assays.

Initial volatile fatty acid (VFA) content of inocula affects BMP test results.

High VFA content reduces microbial activity