Importance of the methanogenic archaea populations in anaerobic wastewater treatments

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Process Biochemistry 45 (2010) 1214–1225

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Review

Importance of the methanogenic archaea populations in anaerobic wastewatertreatments

Meisam Tabatabaeia,b,∗, Raha Abdul Rahimc, Norhani Abdullahd, André-Denis G. Wrighte,Yoshihito Shirai f, Kenji Sakaig, Alawi Sulaimanh, Mohd Ali Hassanb,h

a Microbial Biotechnology and Biosafety Department, Agricultural Biotechnology Research Institute of Iran (ABRII), Seed and Plant Improvement Institute’s Campus,31535-1897, Mahdasht Road, Karaj, Iranb Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysiac Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysiad Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysiae Department of Animal Science, University of Vermont, Burlington, VT, USAf Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu 808-0196, Japang Laboratory of Soil Microorganisms, Department of Plant Resources, Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University, 6-10-10 Hakozaki,Higashi-ku, Fukuoka 812-8581, Japanh Department of Food and Process Engineering, Faculty of Engineering, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

a r t i c l e i n f o

Article history:Received 18 January 2010Received in revised form 1 May 2010Accepted 17 May 2010

Keywords:BiomethaneBiomassMethanogensAnaerobic treatmentWastewater

a b s t r a c t

Methane derived from anaerobic treatment of organic wastes has a great potential to be an alternativefuel. Abundant biomass from various industries could be a source for biomethane production wherecombination of waste treatment and energy production would be an advantage. This article summarizesthe importance of the microbial population, with a focus on the methanogenic archaea, on the anaerobicfermentative biomethane production from biomass. Types of major wastewaters that could be the sourcefor biomethane generation such as brewery wastewater, palm oil mill effluent, dairy wastes, cheese wheyand dairy wastewater, pulp and paper wastewaters and olive oil mill wastewaters in relevance to theirdominant methanogenic population are fully discussed in this article.

© 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12152. Types of waste materials and their dominant methanogenic population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1215

2.1. Brewery wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12152.2. Palm oil mill effluent (POME) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12162.3. Dairy waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12172.4. Cheese whey and dairy wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12182.5. Pulp and paper wastewater . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12182.6. Olive oil mill wastewater. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1219

3. Anaerobic reactors: designs and operation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12204. Molecular methods for microbial ecosystem studies during anaerobic digestion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12215. A look to the future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1221

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223

∗ Corresponding author at: Microbial Biotechnology and Biosafety Department, Agricultural Biotechnology Research Institute of Iran (ABRII),Seed and Plant Improvement Institute’s Campus, 31535-1897, Mahdasht Road, Karaj, Iran. Tel.: +98261 2703536; fax: +98261 2704539.

E-mail address: meisam [email protected] (M. Tabatabaei).

1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.procbio.2010.05.017


M. Tabatabaei et al. / Process Biochemistry 45 (2010) 1214–1225 1215

1. Introduction

To date, global energy requirements are heavily dependent onfossil fuels such as oil, coal and natural gas. As the exhaustion of lim-ited fossil fuels is to be anticipated, there is a necessity to searchfor replacement source of energy [1]. On the other hand, thereis a growing amount of organic waste and wastewater producedannually. Anaerobic digestion technology is an ideal cost-effectivebiological means for the removal of organic pollutants in waste andwastewater which simultaneously produces gaseous methane asan energy resource [2,3]. The many applications of this digestiontechnology are the high-rate treatment of high-strength industrialorganic wastewater [1,4], low-strength organic wastewater [5],complex wastewater containing persistent chemical compounds[4], sulfate-rich wastewaters [6], wastewater discharged at tem-peratures ranging from psychrophilic to thermophilic [2,7] as wellas offering potentials for the removal of metals [8], nitrates [9], andtoxic substances [10].

The biomethane produced by anaerobic digestion is an odorless,colorless and non-poisonous gas [11]. The process by which anaer-obic bacteria decompose organic matter into biomethane, carbondioxide, and a nutrient-rich sludge involves a step-wise series ofreactions requiring the cooperative action of several organisms. Itoccurs in three basic stages as the result of the activity of a varietyof microorganisms. Initially, a group of microorganisms convertsorganic material to a form that a second group of organisms uti-lizes to form organic acids. Methane-generating (methanogenic)anaerobic archaea utilize these acids and complete the decompo-sition process. Table 1 presents the classification of methanogenicarchaea as outlined by Demirel and Scherer [12]. In the first stage,a variety of primary producers (acidogens) break down the rawwastes into simpler fatty acids. In the second stage, a different groupof organisms (methanogens) consumes the organic acids producedby the acidogens, generating biogas as a metabolic byproduct. Onaverage, acidogens grow much more quickly than methanogens.Finally, the organic acids are converted to biogas [13]. Moreover,compared with ethanol or other liquid biofuels, biomethane iseasily separated from liquid phase, which can contribute to thereduction of the process costs [14].

Renewable biomass is the most versatile non-petroleum basedresource that is generated from various industries as waste mate-

rials. Animal manure, agricultural waste, municipal solid waste,sewerage, food industry waste, and forest industry residues—allof these sources can be used for production of biogas especiallybiomethane [15] and it would be estimated that at least 25% ofall bioenergy can in the future originate from biogas producedfrom waste [16]. The conversion of the waste to biomethane isnot only an alternative cost-effective way of energy production,but it also contributes to very large overall reductions of green-house gas emissions as leakages of methane into the atmosphereare avoided. Although biomass energy is more costly than fossil-fuel-derived energy, trends to minimize carbon dioxide and otheremissions through emission regulations, carbon taxes, and subsi-dies of biomass energy would make it cost competitive [17].

In order to take full advantage of renewable biomass throughanaerobic digestion technology, one the most advanced fields asso-ciated with the technology which is the microbiology of anaerobicdigestion processes should be fully understood. The knowledgeof the ecology and function of the microbial community in theseprocesses is required to better control the biological processesas the process is ultimately dependent on an active biomassfor operational efficiency. Therefore considerable attempts havebeen made to understand the microbial community structureby using culture-dependent and culture-independent molecularapproaches [2,18,19]. Through these analyses, particularly thosetargeting the 16S rRNA gene, comprehensive pictures of the com-munity compositions have been documented.

In this review, we focus on microbiological aspects of anaero-bic digestion of various renewable biomasses with a focus on theirdominant methanogenic population, the leading factor of their suc-cessful anaerobic treatment, and update the recent findings in thisfield. In addition, we highlight the importance of molecular tech-niques in moving from the conventional monitoring systems ofanaerobic digesters to biomonitoring procedures.

2. Types of waste materials and their dominantmethanogenic population

2.1. Brewery wastewater

The brewing process generates a unique, high-strength wastew-ater as a byproduct. Even though substantial technological

Table 1Classification of methanogenic archaea as outlined by Demirel and Scherer [12].

Class I. Methanobacteria Order I. MethanobacterialesFamily II. Methanothermaceae

Genus I. MethanobacteriumGenus II. MethanobrevibacterGenus III. MethanosphaeraGenus IV. Methanothermobacter

Family II. Methanothermaceae Genus I. Methanothermus

Class II. Methanococci Order I. MethanococcalesFamily I. Methanococcaceae

Genus I. MethanococcusGenus II. Methanothermococcus

Family II. MethanocaldococcaceaeGenus I. MethanocaldococcusGenus II. Methanotorris

Class III. Methanomicrobia

Order I. MethanomicrobialesFamily I. Methanomicrobiaceae

Genus I. MethanomicrobiumGenus II. MethanoculleusGenus III. MethanofollisGenus IV. MethanogeniumGenus V. MethanolaciniaGenus VI. Methanoplanus

Family II. Methanocorpusculaceae Genus I. MethanocorpusculumFamily III. Methanospirillaceae Genus I. Methanospirillum

Order II. Methanosarcinales Family I. Methansarcinaceae

Genus I. MethanosarcinaGenus II. MethanococcoidesGenus III. MethanohalobiumGenus IV. MethanohalophilusGenus V. MethanolobusGenus VI. MethanomethylovoransGenus VII. MethanimicrococcusGenus VIII. Methanosalsum

Family II. Methanosaetaceae Genus I. Methanosaeta


1216 M. Tabatabaei et al. / Process Biochemistry 45 (2010) 1214–1225

Table 2List of the abbreviations and definitions.

Abbreviation Definition Abbreviation Definition

ABR Anaerobic baffled reactor LH-PCR Length heterogeneity PCRAFB Anaerobic fluidized bed M. concilii Methanosaeta conciliiBOD Biological oxygen demand MAS Membrane anaerobic systemCDT Closed digester tank MOSTI Ministry of Science, Technology and Innovation, MalaysiaCOD Chemical oxygen demandCSTR Completely stirred tank reactor OMW Olive mill wastewaterDGGE Denaturing gradient gel electrophoresis POME Palm oil mill effluentEGSB Expanded granular sludge blanket RISA Ribosomal intergenic spacer analysisFBL Fixed-bed loop SEC Sulfite evaporator condensateFELDA Federal Land Development Authority SRT Sludge retention timeFISH Fluorescent in situ hybridization SS Suspended solidsGAC Granular activated carbon SSCP Single-strand conformation polymorphismGRABBR Granular bed baffled reactor T-RFLP Terminal restriction fragment length polymorphismHHW Household waste UASB Upflow anaerobic blanket reactorHRT Hydraulic retention time UASFF Upflow anaerobic sludge fixed film reactorJSPS Japan Society for the Promotion of Science VFA Volatile fatty acidsLCFA Long chain fatty acids VOL Volumetric organic loading

improvements have been made in the past, it has been estimatedthat for each liter of beer produced in breweries approximately3–10 l of waste effluent is generated [20]. The high level of sol-uble biological oxygen demand (BOD) (Table 2) and the warmtemperature (>37 ◦C) make brewery wastewater an ideal substratefor anaerobic treatment. Brewery wastewater is characterized byhigh-strength soluble organic pollutants and suspended solids (SS)[21]. Therefore, aerobic treatment due to the need for an inten-sive amount of energy for aeration and a large amount of wastedsludge produced is not a favorable choice [22]. Hence, anaero-bic digestion using high-rate anaerobic reactors such as upflowanaerobic blanket reactor (UASB) [23], anaerobic granular bed baf-fled reactor (GRABBR) [24] and anaerobic fluidized bed (AFB)[25]have been reported to treat brewery wastewater with a satisfac-tory chemical oxygen demand (COD) reduction. Díaz et al. foundMethanosaeta the dominant genus (between 75 and 95% of totalarchaeal cells) in a UASB reactor treating brewery wastewater, withMethanosaeta concilii accounting for 70% of the archaeal clones[26] (Fig. 1A and B). Methanosarcina mazei and Methanospiril-lum hungatei were also present. This could be explained by thefavorable concentration of acetate in brewery wastewater [27], asMethanosarcina has a higher maximum growth rate, but a loweraffinity for acetate (�max, 0.21 day−1; Ks, 4 mM) than Methanosaeta(�max, 0.11 day−1; Ks, 0.44 mM) [28,29]. In addition, the majority(87%) of the total bacterial clones obtained in his study belongedto the phyla Deferribacteres, Nitrospira, and Chloroflexi [26]. Uncul-tured clades belonging to the phylum Deferribacteres represented34% of the bacterial population. This high occurrence in suchmethanogenic ecosystems indicates that they might play a role inpart of the food web for the methanogenic degradation of organiccompounds [22].

In a similar study, all archaeal clones were affiliatedwith Methanosaeta concillii, and no clones were related tohydrogenotrophic methanogens. However, electron microscopicexamination detected hydrogen-consuming Methanosarcina-likecells and Methanobrevibacter-like cells [30]. This difference wasdue to the bias of the rRNA approach when there are signifi-cant differences in the number of studied microorganisms [31].The bacterial clones in this study were mostly affiliated with anot-yet-cultured Clostridium cluster (>50%) [30]. In general, aceto-clastic methanogens in particular Methanosaeta concillii are moreabundant than hydrogenotrophic ones in methanogenic consortiaduring anaerobic digestion of brewery wastewater (Table 3).

2.2. Palm oil mill effluent (POME)

Palm oil mill effluent is unquestionably the largest waste gener-ated from the oil extraction process [32]. For every tonne of oil palmfresh fruit bunch, it is estimated that 0.5–0.75 t of POME will be dis-charged from the mill [33]. This wastewater is a viscous, brownishliquid containing about 95–96% water, 0.6–0.7% oil and 4–5% totalsolids (including 2–4% suspended solids). It is acidic (pH 4–5), witha high temperature (80–90 ◦C), high organic COD, (50,000 mg l−1),and high BOD, (25,000 mg l−1) [34]. It is 100 times more recalci-trant than domestic wastewater [35]. Therefore, due to its highlypolluting properties (high BOD and COD), the most cost-effectivetechnology is anaerobic treatment [36].

Over the past decade, several cost-effective anaerobic treatmenttechnologies have been developed for the treatment of POME suchas closed digester tank (CDT) [32], completely stirred tank reactor(CSTR) [37], the modified anaerobic baffled reactor [38], anaero-bic filter and anaerobic fluidized bed reactor [39], thermophilic

Fig. 1. Typical fluorescent in situ-hybridized cells of dominant methanogens in anaerobic treatment of the majority of various wastewaters: (A) Methanosaeta concilii; (B) acluster of Methanosaeta concilii and (C) Methanosarcina sp. hybridized with FITC-labeled methanogens probe (MSMX860) (provided by the authors).



Table 3A summary of kinetic data, main characteristics, and methanogenic population of major wastewaters.

Type of wastewater Main characteristics DominantMaethanogens

�max (day−1) Ks (mM) Other Methanogens Reference

Brewery wastewater Favorableconcentration ofacetate forMethanosaeta

Methanosaeta concilii 0.11a 0.44 Methanosarcina mazei,Methanospirillum hungatei

[26,27,28,29]

Hydrogen-consumingMethanosarcina-like cells andMethanobrevibacter-like cells

Palm oil mill effluent Highly favorableconcentration ofacetate forMethanosaeta

Methanosaeta concilii 0.11 0.44 Methanosarcina sp. [29,31]

Dairy wastes High levels of freeammonia and VFAs

Methanosarcinaceae 0.21 4 Methanomicrobiales [28,54,58,61]

Methanobacteriumthermoautotrophicum

– –

Cheese whey anddairy wastewater

Presence of LCFA inparticular oleic acid

Methanosaeta spp. 0.11 0.44 Methanobrevibacter sp. [78,79,80,81]

Methanococcus spp.(towards the end ofoperation)

– – Methanosarcina sp.

Pulp and paperwastewaters

Toxic and resistant tobiodegradationcompounds (i.e.lignins, resins, tanninsand highly chlorinatedorganics such aschlorophenoliccompounds)

Methanosarcina sp.(Methanosarcinabarkeri)

0.023 (h−1) 320 (as mgCOD/l−1) Methanobacterium sp. [12,94,95,100,101,110,111,112,113]

Methanosaeta sp.(Methanosaeta concilii)

0.11 0.44 Methanobrevibacterarboriphilus

Olive oil millwastewaters

Acidic pH, presence ofinhibitory/toxiccompounds such ashigh sodiumconce-ntration, highcontent of polyphenols,tannins, and lipids

Methanobacteriaceae(Methanobacteriumformicicum)

0.053 (h−1) – Methanosaeta sp. [12,118,131,133,134]

Methanomicrobiaceae

a Growth on acetate.

upflow anaerobic filter [40], membrane anaerobic system (MAS)[41], UASB reactor [42,43], and rotating biological contactors [44].To date, only a few studies have been conducted on the micro-bial aspects of POME anaerobic treatment [31,35,45,46]. Tabatabaeiet al. conducted a comprehensive study on the methanogenicdiversity during the anaerobic treatment of POME in a CDT. Themajority (>99%) of the total methanogens counted by using flu-orescent in situ hybridization (FISH) in their study belonged tothe genus Methanosaeta (Table 3). However, 16S rRNA cloningalong with denaturing gradient gel electrophoresis (DGGE) analysisshowed that M. concilii was the only member of the genus present.Methanosarcina accounted for <1% of the whole methanogenic pop-ulation [31] (Fig. 1). The high number of M. concilii was attributedto the highly favorable concentration of acetate in POME.

2.3. Dairy waste

An average dairy cow (450 kg) produces approximately 37 kgof waste (manure and urine) d−1; thus, a 1000-cow dairy pro-duces approximately 13,500,000 kg of waste annually [47]. Thiswaste is usually stored in lagoons until it can be applied to agricul-tural fields as a soil fertilizer for crops. However, there are seriousdrawbacks for the current procedure. First, cow manure may con-tain pathogenic bacteria to both humans and animals, such asEscherichia coli O157:H7 [48], Campylobacter spp. [49], Salmonellaspp. [50], and Mycobacterium spp. [51,52,53]. Therefore, crops fer-tilized with dairy waste may transmit these pathogens to livestockor humans who consume them. Second, the release of odorous com-

pounds into the air severely affects the air quality [47]. Veterinarystudies indicate that anaerobic treatment at 60 ◦C with a guaran-teed holding period of 4–6 h before waste is pumped out of thereactor is acceptable in order to treat potentially dangerous wastes[54]. Hence, the use of high-rate anaerobic treatment technologiesfor dairy waste before it enters the lagoons is an advantageous solu-tion in order to reduce organic matters [55], and pathogens [56] aswell as methane production.

Demirer and Chen reported that two-phase anaerobic digestionfor unscreened dairy manure at a ratio of sludge retention time(SRT) to hydraulic retention time (HRT) of 10 days (2 days aci-dogenic and 8 days methanogenic) resulted in 50 and 67% higherbiogas production at OLRs of 5 and 6 g VS l−1 d−1, respectively, rel-ative to a conventional one-phase configuration with SRT/HRT of20 days. Moreover, it made an elevated OLR of 12.6 g VS l−1 d−1

possible which was not achievable for conventional one-phase con-figuration and therefore, was made significant cost savings due toboth superior performance and reduced volume requirements [57].

The dominant methanogens in manure digesters have neverbeen well documented. Karakashev et al. reported that organismsassumed to be acetoclastic (Methanosarcinaceae and Methanosae-taceae) are more abundant than organisms assumed to behydrogenotrophic (Methanobacteriales, Methanomicrobiales, andMethanococcales) in anaerobic treatment of dairy waste. However,as manure contains high levels of ammonia and of volatile fattyacids (VFA), its anaerobic digestion leads to the domination ofmembers of the Methanosarcinaceae (Fig. 1C) [58] due to the intol-erance of members of the Methanosaetaceae for high ammonia



and VFA levels [58,59,60]. In contrast, using 16S rRNA sequenceanalysis, low levels of members of the Methanosarcinaceae andhigh levels of members of the Methanomicrobiales were observedin a full-scale manure-fed reactor [61]. Ahring counted acetate-and hydrogen-utilizing methanogens in thermophilic biogas reac-tors treating a mixture of cow and pig manure and found thehydrogen-utilizing methanogens in particular Methanobacteriumthermoautotrophicum absolutely dominant [54]. In his study, allacetate-utilizing methanogens identified belonged to the genusMethanosarcina and the majority were in the form of individualsingle cells in the reactor. Hence, it could be concluded that thegenus Methanosaeta plays no or little role in acetate conversion inthe therrnophilic biogas reactors [54,62,63]. An experiment withradio-labeled acetate (14CH3COOH) [54] showed that acetate inthe thermophilic anaerobic reactor was converted by the aceto-clastic reaction at high concentrations of acetate and by a two-stepmechanism involving the microbial oxidization of acetate to hydro-gen and carbon dioxide and the transformation of these productsinto methane by hydrogen-utilizing methanogens [64] when theacetate concentration was lower than the threshold level for theMethanosarcina species and in the absence of Methanosaeta speciesin the reactor. In addition, a mixture of both types of metabolismoccurred close to the threshold level.

In general, in contrast to sludge digesters where, Methanosae-taceae are the main acetoclastic methanogens (Table 3) [26,30,31],Methanosarcinaceae are either the only or the most abundantacetate-utilizing methanogens in manure digesters [543,58,61].The predominance of Methanosarcinaceae could be indirectlyattributed to the high free ammonia levels of manure which restrictMethanosaetaceae [58]. Methanosarcinaceae particularly M. conciliiare the most ammonia-sensitive methanogen, and it is completelyinhibited at a concentration of 560 mg (total) NH4-N l−1 at a pHlevel of 7.0 [65,66]. Therefore, high free ammonia levels causethe accumulation of VFA, which then allow Methanosarcinaceaewhich have a higher threshold for acetate [28,29,67] to outcompeteand restrict Methanosaetaceae. Finally, reducing ammonia levels orits inhibitory effect such as by addition of lipid-containing waste[68] in manure digesters should change the equation in favor ofthe members of the Methanosaetaceae and consequently reduceorganic acid levels considerably [58].

2.4. Cheese whey and dairy wastewater

The liquid waste in a dairy originates from manufacturing pro-cess, utilities and service sections with a high COD ranging from 1 to10 g l−1 and a high BOD5 ranging from 0.3 to 5.9 g l−1 [69–71] rep-resenting its high organic content. Moreover, the dairy industry isone of the largest sources of industrial effluents for instance, a typ-ical European dairy generates about 180,000 m3 of waste effluentannually [72]. However, there are high seasonal variations corre-lated with the volume of milk received for processing; which is ingeneral high in summer and low in winter months [73]. The varioussources of waste generation from a dairy are spilled milk, spoiledmilk, skimmed milk, whey, wash water from milk cans, equip-ment, bottles and floor washing [69]. Among those, whey is themost difficult high-strength waste product of cheese manufactur-ing (COD of more than 60 g l−1) [71,74] which contains a proportionof the milk proteins, water-soluble vitamins and mineral salts.Therefore, high COD concentration of dairy effluents, their hightemperature, no requirement for aeration, low amount of excesssludge production and low area demand make them ideal candi-dates for anaerobic treatment [71] using various types of anaerobicreactors [75–77]. The acetoclastic methanogenic activity measuredin anaerobic treatment of dairy wastewater was found to be duemostly to Methanosaeta species whilst Methanosarcina-like speciescontributed insignificantly [78]. However, Methanococcus species

seemed to become the most dominant group towards the end ofthe operation [78,79].

A study where a polymer-amended anaerobic baffled reactor(ABR) was used revealed that partial spatial separation of anaero-bic bacteria appeared to have taken place with the predominanceof acidogenic bacteria in the initial compartments and the pre-dominance of methanogenic bacteria in the final compartments.It also showed that the dominant methanogens in the initial com-partments of the ABR were those which could consume H2/CO2and formate as substrate, i.e. Methanobrevibacter, Methanococcus,with populations shifting to acetate utilizers (i.e. Methanosaeta,Methanosarcina) in the final compartments [79].

Milk fat was found to have an immediate influence on reducingmethane gas production rate in reactors to which it was added [80].Similar observations were reported by Uyanik et al and Demirel andYenigun indicating that the densities of total bacteria and autofluo-rescent methanogens both decreased during start-up operation ofdairy wastewater anaerobic treatment [79,81]. This was explaineddue to the presence of long chain fatty acids and in particu-lar oleic acid, which is a major derivative of milk fat hydrolysis[80]. Oleic acid was found to have inhibitory effects on methaneproduction and on ATP concentration in the sludge which is anindicator of sludge’s total physiological activity [80] particularlythrough acetoclastic methanogenesis. Oleic acid at a concentra-tion of 4.4 mM (300–1500 mg l−1) resulted in 50% inhibition inmethanogenesis from acetate at 30 ◦C [82]. Under thermophilicconditions (55 ◦C), 100–1000 mg l−1 oleic acid inhibited acetic acidremoval [83]. Lalman and Bagley also reported that oleic acid atconcentration above 30 mg l−1 inhibited acetoclastic methanogen-esis at 20 ◦C [84]. They also pointed out that slight inhibition ofhydrogenotrophic methanogenesis occurred. Furthermore, milk fatalso contributes to the sludge flotation problems [80,85] which con-sequently plays a role in biomass wash-out from the reactor [48].About 70% of milk lipids are adsorbed by the granular sludge [86]which reduced the adhered fraction of biomass [87]. Rinzema etal. reported sludge flotation and a total sludge wash-out in a UASBreactor with a lipid loading rate more than 2–3 gCOD l−1 d−1 [88].Taking all into account, Perle et al advised to treat dairy effluentsby anaerobic digestion only after reduction of the milk fat concen-tration below 100 mg l−1, and after careful acclimatization of thedigester culture to casein in order to develop proteolytic enzy-matic system [80]. To the contrary, some studies reported thatthe intermediates of fat degradation (mainly oleic acid) seem notto reach concentrations high enough to affect the anaerobic pro-cess or hardly affected the overall performance [87,89]. It was alsoreported that the anaerobic biodegradation rate of fat-rich wastew-aters is slower than that of fat-poor wastewaters, due to the slowerrate of the fat hydrolysis step [89]. Having considered various fac-tors, Vidal et al. recommended reactor operation for anaerobictreatment of dairy wastewater at COD concentrations between 3and 5 kg COD m−3 to ensure the highest levels of biodegradabilityand biomethanation of both wastewaters and eliminate flotationproblems [89]. It was documented that anaerobic treatment of afat-rich dairy wastewater is enhanced when repeated pulse feedingis applied by promoting the accumulation of long chain fatty acids(LCFA) into the biomass and allowing them to be biodegraded after-wards. This is attributed to the fact that LCFA degradation processincreased the tolerance of the acetoclastic methanogens to LCFAeffect, by significantly dropping the lag phases observed before thebeginning of methane production [90].

2.5. Pulp and paper wastewater

The pulp and paper industry is a very water-demanding indus-try and can consume as high as 35 m3 of freshwater t−1 of paperproduced [91]. This results in the generation of various types of



wastewater such as papermaking effluent, de-inking process efflu-ent and pulping process effluent with an average COD value as highas 11,000 mg l−1 [89]. For each tonne of manufactured pulp, thewastewater discharge volume will be a minimum of 30 m3 [92].The characteristics of the pulp and paper-effluent are highly depen-dent on raw materials and manufacturing process adopted [92,93].Moreover, these effluents are strongly polluting and toxic owingto the presence of lignins, resins, tannins and chlorophenolic com-pounds that are resistant to biodegradation [94,95].

Application of a “zero liquid effluent” process was reported asa feasible option for the paper mills and found to be profitable[96,97]. The wastewater is generated at a temperature ranging from50 to 60 ◦C and therefore, thermophilic anaerobic treatment com-plemented with appropriate post-treatment is considered as themost cost-effective solution to meet re-use criteria of the processwater as well as maintaining its temperature [98]. Anaerobic treat-ment is well feasible for effluents generated by recycle paper mills,mechanical pulping (peroxide bleached), semi-chemical pulpingand sulfite and kraft evaporator condensates. [99]. This is due tothe tolerance to toxicity of anaerobic microorganisms [100]. Inthe proposed closed-cycle, the anaerobic treatment step removesthe largest fraction of the biodegradable COD and sulfur as H2Sfrom the effluent, without the use of additional chemicals, and isregarded as the only possible location to eliminate sulfur from theprocess water cycle [98]. Buzzini and Pires studied the treatmentof diluted black liquor from a kraft pulp plant by using a UASB reac-tor and obtained a COD removal efficiency of 80% [101]. The blackliquor comprises only 10–15% of the total wastewater, however,contributes approximately 95% of the total pollution load of pulpand paper mill effluents [102]. Therefore, due to its higher contentsof chemicals and organic substances and consequent high pollu-tion strength, low influent concentration was found essential forgranulation when UASB reactors are applied [103]. Van Lier et al.compared H2S stripping efficiency of UASB and gas-supplied UASBreactors treating paper mill effluent and showed 3–4 times highervalues in the gas-supplied UASB [98]. In a study where an anaerobicbaffled reactor was used for continuous anaerobic digestion of pulpand paper mill black liquors, OLRs higher than 5 kg COD m−3 d−1

resulted in loss of reactor’s stability which was apparent by thedecrease in biogas production rate and its methane content [104].This was attributed to the toxic effect of the high concentrationof tannin and lignin present in black liquor on methanogens [105].However, in a similar study in an anaerobic baffled reactor by usingan immobilized cell system, the reactor maintained its stabilitywith higher OLRs (7 kg COD m−3 d−1)[106]. This was due to theadvantages of immobilization technologies such as the retentionof catalytic activity, a high ratio of sludge retention time (SRT) tohydraulic retention time (HRT) and in particular, the protection ofcells from the effects of inhibitory/toxic substances [107,108].

Several studies have demonstrated the capacity of the micro-bial consortium e.g. methanogenic archaea to adapt to potentiallytoxic effluents present in the effluents of pulp and paper mills[101,109]. The adaptation depends on the concentration of thetoxicants and the operating conditions and the acclimation of thesludge substantially reduces the degree of inhibition [109]. Thepredomination of Methanosarcina spp. and Methanosaeta spp. wasreported during the anaerobic treatment of paper and pulp milleffluent using USAB reactors [100,101,110]. In a study, Roest etal. monitored microbial populations in a UASB reactor for treat-ing paper mill wastewater over 3 years with a combination ofdifferent molecular techniques and conventional microbiologicalmethodology. The authors were able to confirm that Methanosaetawas the most abundant archaeal genus throughout the operation[111]. They also reported the domination of sulfate-reducing bacte-ria and syntrophic fatty acid-oxidizing microorganisms during theanaerobic treatment of paper mill wastewater [111]. Methanogenic

consortia (Methanosaeta sp., Methanosarcina sp., and Methanobac-terium sp.) were found to have an important role in the degradationof highly chlorinated compounds such as chlorophenols presentin paper mill wastewaters (Table 3) [112]. This was supported bythe findings of Buzzini et al. who reported the capability of anaer-obic treatment using UASB reactors dominated by Methanosaetasp. and Methanosarcina sp. to treat this kind of wastewater withchlorinated organics removal efficiency ranging from 71 to 99.7%[110]. Using a high-rate fixed-bed loop (FBL) reactor, Ney et al.successfully treated sulfite evaporator condensate (SEC) which isa wastewater from pulp and paper [113]. He showed that witha consortium consisting of Methanosarcina barkeri, Methanobre-vibacter arboriphilus, M. concilii and Desulfovibrio furfuralis, all theconstituents of a synthetic SEC including furfural, which is a toxiccompound to anaerobic bacteria [114], were degraded at an effi-ciency of almost 90% [113].

2.6. Olive oil mill wastewater

Olive mill wastewater (OMW) generated by the olive oil extrac-tion process is the main waste product of this industry. The worldannual production of olives is approximately 10 million tonneswhere the majority of olives is produced in the Mediterraneancountries and 90% is processed for oil production [115]. The aver-age amount of olive mill wastewater produced during the millingprocess is 1.2–1.8 m3 t−1 of olives [116]. Therefore, the OMW result-ing from the production process exceeds 13.5 million m3 annually.Treatment of OMW is becoming a serious environmental problem,due to its high BOD and COD concentration (100–220 g l−1; whichis on average 100 times greater than those of common munic-ipal wastewater [114,117]), high sodium concentration [118] asconcentrations exceeding 10 g l−1 strongly inhibits methanogen-esis [119], low pH (∼5), low alkalinity (∼0.6 g CaCO3 l−1) [120]and finally because of its resistance to biodegradation due to itshigh content of polyphenols, tannins, and lipids and consequentnegatively impacts on methanogenic cells. Despite the presenceof inhibitory/toxic compounds, the high organic content of OMW,makes anaerobic treatment processes with biogas production aconsiderable option. Besides the previously mentioned advantagesof anaerobic treatment, easy restart after several months of shut-down before seasonal production campaigns as it is the case forOMW anaerobic treatment should be stressed.

To date due to the characteristics of OMW, various anaerobictreatment approaches have been applied such as high dilution ofOMW with tap water [121,122], combined treatment (co-digestion)of OMW together with other waste such as manure, householdwaste (HHW) or sewage sludge to compensate for its low alka-linity and nitrogen [123,124], the use of pretreatment systemsbefore anaerobic treatment such as sand filtration and subsequenttreatment with powdered activated carbon [116], using biolog-ical agents such as Aspergillus strains, Azotobacter chroococcum,Geotrichum candidum [125,126] and pretreatments with Ca(OH)2and bentonite [127].

Dalis et al. found the employment of the upflow type digestersuch as UASB as an economical and effective treatment for signif-icantly reducing the organic load of total raw olive oil wastewater(83% COD removal and 75% reduction of phenolic compounds).More satisfactory results were obtained when a fixed-bed-typedigester was connected in series with a previous one as a secondtreatment stage [128]. COD reductions of 70–80% using laboratory-scale UASB reactors were reported by other researcher [122,129].

Anaerobic biofilm reactors packed with granular activated car-bon (GAC) and ‘Manville’ silica beads showed approximately 60,250, and 100% improvement in COD removal, phenol reductionand methane yield, respectively, when compared with treatment inconventional anaerobic contact bioreactors [130]. In a similar study,



Table 4The classification of anaerobic reactors and typical examples by Fannin and Biljetina [137].

Category Retention characteristics Examples

A Microorganisms retention time is equal to that of the solid and liquid (RTm = RTs = RTl) CSTR, CDTB Microorganisms and solid retention time is higher than that of the liquid (RTm and RTs > RTl) CSTR or CDT with solid recycle,

UASB, Baffled flow reactorC Microorganisms retention time is higher than that of the solid and liquid (RTm > RTs and RTl) Membrane bioreactor, UASFF

RTm = Retention time of microorganism; RTs = Retention time of solid; RTl = Retention time of liquid; CDT = Closed digester tank; CSTR = Continuously stirred tank reactor;UASB = Upflow anaerobic sludge blanket; UASFF = Upflow anaerobic sludge fixed film reactor.

Bertin et al. [131] used a GAC-bioreactor to treat OMW and reportedabout 100 and 300% improved efficiency in terms of removal of CODand phenolic compounds, respectively, and by 70% in terms of CH4production [131]. GAC provides the microorganisms with a placeto grow and allow them to live stably in the reactor by minimizingthe inhibitory/toxic effect of the present compounds. Hence, it pro-vides the bioreactor with increased tolerance to high and variableorganic loads along with a volumetric productivity in terms of CODand phenolic compound removal. Taking all things into account,results of single anaerobic treatment are not always satisfactoryand some form of pretreatment, apart from simple dilution andnutrient/alkalinity adjustment, is usually necessary [132].

Bertin et al. (2006) analyzed the microbial diversity of a GACreactor during anaerobic digestion of OMW and found a mem-ber of Methanobacteriaceae as the sole dominant species, i.e.,hydrogenotrophic Methanobacterium formicicum representing thewhole archaeal community [131]. This methanogen was also dom-inant and persistent in a UASB pilot plant treating OMW [133]. Theabsence of acetoclastic methanogens which are highly pH-sensitiveas well [134] was due to the acidic pH environments occurred inthe reactors. In contrast to these studies, Methanobacteriaceae andMethanosaeta were both the main methanogens in a laboratory-scale upflow anaerobic digester treating olive mill effluent [134].In the latter study, at a volumetric organic loading (VOL) of 6 gCOD l−1 day−1, the hydrogenotrophic Methanobacterium predomi-nated in the reactor but decreased from 1011 to 108 cells g−1 sludgewhen the VOL was increased to 10 g COD l−1 day−1. By increasingthe VOL, the non-dominant methanogenic family i.e. Methanomi-crobiaceae increased from 104 to 106 cells g−1 sludge. On theother hand, hylotypes belonging to the acetoclastic Methanosaetawere stable throughout VOL variation and at 10 g COD l−1 day−1

dominated in the biofilm (109 cells g−1 sludge) [135]. With theabove results, we may suggest, Methanosaeta as the most tolerantmethanogen to the inhibitory/toxic substances present in wastew-aters such as OMW. This could be attributed to its high affinity foracetate enabling it to occupy the deepest or in other words, moreprotected niches in the granule or biofilm with low concentrationof substrate [136].

3. Anaerobic reactors: designs and operation

Various types of anaerobic reactors have been successfullydesigned, studied and applied to a wide range of organic richwastewaters such as UASB [23,42,43], GRABBR [24], AFB [25],CDT [32], CSTR [37], the modified anaerobic baffled reactor [38],anaerobic filter and anaerobic fluidized bed reactor [39], ther-mophilic upflow anaerobic filter [40], MAS [41], rotating biologicalcontactors [44], polymer-amended ABR [79], anaerobic biofilmreactors packed with GAC and ‘Manville’ silica beads [130,131].This section shall briefly review the common anaerobic reactordesigns used in the treatment of organic rich wastewaters and fur-ther discuss their classification and operation. The classificationof anaerobic reactors was best described by Fannin and Biljetinaaccording to the retention time characteristics of microorganisms,solid and liquid in the reactor system and is simplified in Table 4[137].

The simplest reactor design is class A, such as CDT and CSTR,where the retention time of microorganisms, solid and liquid isequal. This type of digesters is characterized by lowest construc-tion cost and simplest operation among all types of reactors. Theschematic diagrams of various anaerobic reactors are presented inFigs. 2 and 3. In the CDT reactor [32], the substrate is fed throughthe bottom inlet and displaces the treated effluent inside the tankout through the top outlet of the CDT (Fig. 2A). The mixing isachieved using a centrifugal pump which circulates the effluentintermittently inside the digester. Alternatively, an agitator couldalso be used for the mixing purpose. The mixing will release theentrapped biogas at the bottom of the tank and provides a goodcontact between microorganisms and substrate inside the digester.The biogas produced will flow out of the digester through the topoutlet for further processing. In the CSTR reactor [138], the influentis pumped into the CSTR through the bottom inlet and the efflu-ent will flow out from the top outlet (Fig. 2B). Mixing is achievedusing an agitator mounted at the top of the CSTR. The biogas isproduced inside the CSTR and released through the top outlet. Themixing could be continuous or intermittent but must be achievedcompletely in the digester.

Fig. 2. Schematic diagrams of closed digester tank (CDT) (A), continuous stirred tank reactor (CSTR) (B), and CDT with solid recycle system (C).



Fig. 3. Schematic diagrams of upflow anaerobic sludge blanket bioreactor (UASB) (A), membrane filter bioreactor (B) and upflow anaerobic sludge fixed film bioreactor(UASFF) (C).

For class B anaerobic reactors such as CSTR or CDT with solidrecycling system, UASB and baffled flow reactor, the retention timeof microorganisms and solid is higher than that of the liquid inorder to accomplish higher process efficiency. The basic schematicdiagram of a CDT reactor with solid recycle system is shown inFig. 2C [45]. The basic operation of such reactor is identical to thatof the normal CDT, however, a settling tank is added to the sys-tem to recycle the biomass and consequently increase the retentiontime of the reactor. The recycling of biomass would also help tosupplement nutrients and alkalinity to the reactor. Recently, Busuet al. reported that sludge recycling improved the overall processperformance of the reactor [139]. The schematic diagram of theUASB reactor is shown in Fig. 3A [140]. In this reactor, the sub-strate is fed through the bottom inlet and flows upward throughthe sludge blanket phase. The treated effluent will overflow at thetop of the reactor. The most important feature of this reactor isthe sludge blanket situated at the bottom part of the reactor whichaids to maintain a high amount of microorganisms including bothacidogens and methanogens in the system.

To achieve a higher biomass concentration in the reactor forhigher reactor efficiency and biogas performance, Fannin and Bil-jetina suggested class C of the reactor designs [137]. The typicalexamples of such design are MAS and upflow anaerobic sludgefixed film reactor (UASFF)[141]. In the membrane anaerobic reac-tor, the influent is pumped through the top inlet to be utilizedby the microorganisms inside the reactor (Fig. 3B). The mixtureof substrate and microorganisms is then pumped into the mem-brane filtration unit for separation. The treated effluent is allowedto leave the reactor while the microorganism fraction is returnedto the reactor. The key and the most important part of this systemis the membrane unit designed to efficiently capture the microbialmass responsible for acidogenesis and methanogenesis processes.The schematic diagram of the UASFF reactor is presented in Fig. 3C[34]. The reactor column is divided into three different compart-ments. The bottom part is designed like a UASB section while themiddle and top portions are designed similar to a fixed film reactorand a gas–liquid separator, respectively. The influent is fed to thereactor from the base using a pump and flows through the reac-tor. The incoming influent will displace an equal volume of thetreated effluent flowing out through the top outlet of the reactor.The combination of sludge blanket at the bottom and fixed film atthe middle ensures high biomass retention in the system for highorganic removal efficiency and biogas production. The comparisonof performance of various reactor designs utilized for the anaerobictreatment of different types of wastewaters and biogas generationis summarized in Table 5.

4. Molecular methods for microbial ecosystem studiesduring anaerobic digestion

Although anaerobic digestion has been applied in wastewatertreatment successfully over the last 100 years, however, molecularmethods have only been applied to the analysis of communitiesin anaerobic digesters since the late 1990s [142,143]. As previ-ously mentioned, methanogenesis from complex organic matteris achieved by the microbial consortia comprising members ofboth the bacteria and the archaea. At the moment, optimizationof methane production is carried out empirically and the processis generally monitored by the determination of VFA concentrationsin the digester as described by Ahring et al. [144]. Biomonitoringdigesters using molecular methods would not only be useful toavoid failures and optimize the production of methane, but couldalso lead to the identification of new species.

Molecular methods can be generally classified into (i) analy-sis of small subunit ribosomal RNA (SSU rRNA) clone libraries,(ii) community fingerprinting techniques using SSU rRNA genesuch as denaturing gradient gel electrophoresis (DGGE), termi-nal restriction fragment length polymorphism (T-RFLP), ribosomalintergenic spacer analysis (RISA), single-strand conformation poly-morphism (SSCP), and length heterogeneity PCR (LH-PCR), and(iii) dot blotting, FISH, and stable isotope probing. Most of themolecular approaches used so far are based on the analysis of SSUrRNA but recent studies also use quantification of functional geneexpressions. Furthermore, several quantitative methods have beendeveloped such as quantitative real-time PCR assay [145,146] andquantitative FISH to investigate methanogens [31]. A recent reviewpublished by Talbot et al. highlighted the principles of culture-independent nucleic-acid-based methods for analyzing microbialcommunities in anaerobic digesters [147]. Tabatabaei et al. alsodemonstrated that by employing FISH combined with communityfingerprinting DGGE and cloning/sequencing analyses can success-fully study the methanogenic population dominating a particularsubstrate [31]. Therefore, a combination of molecular techniquesseems to be an ultimate tool in microbial ecosystem studies duringanaerobic digestion.

5. A look to the future

A look to the future of environmental biotechnology, micro-bial ecology and anaerobic digestion should focus on scientificadvances that open new possibilities that pull them towards prac-tical goals. Looking first at the science side, the capabilities ofmolecular methods to shed light on how microbial communities



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function will continue to expand and generate much larger quan-tities of throughput. A big challenge is knowing what to do withall the data. On the practical side, a chemical engineer must under-stand that we have already moved from the old-way monitoringtechniques to biomonitoring procedures of anaerobic digestionprocess using molecular techniques. For those applications, thechallenge will be to improve reliability, particularly for use at alarge scale or even any scale. This will include using a cost-effectivemonitoring of anaerobic digestion using a combination of molec-ular methods. Ultimately, if optimum process efficiency is to befully realized, then future developments in anaerobic treatmentprocesses will still require a much greater understanding of the fun-damental relationships between archeal and bacterial populationswithin the biomass.

Acknowledgements

The authors would like to thank Federal Land DevelopmentAuthority (FELDA), Ministry of Science, Technology and Innovation,Malaysia (MOSTI) and Japan Society for the Promotion of Science(JSPS).

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Importance of the methanogenic archaea populations in anaerobic wastewater treatments

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