Preface Preface In November 1776,Alessandro Volta performed his classic experiment disturb- ing the sediment ofa shallow lake,collecting the gas and demonstrating that this gas was flammable.The science ofBiomethanation was born and,ever since,sci- entists and engineers have worked at understanding this complex anaerobic bio- logical process and harvesting the valuable methane gas produced during anaer- obic decomposition.Two lines ofexploitation have developed mainly during the last century:the use of anaerobic digestion for stabilization of sewage sludge, and biogas production from animal manure and/or household waste.Lately,the emphasis has been on the hygienic benefit ofanaerobic treatment and its effect on pathogens or other infectious elements.The importance ofproducing a safe effluent suitable for recirculation to agricultural land has become a task just as important as producing the maximum yield of biogas from a given type of waste.Therefore,anaerobic digestion at elevated temperatures has become the main area ofinterest and has been growing during the last few years. Anaerobic digestion demands the concerted action of many groups of microbes each performing their special role in the overall degradation process. Both Bacteria and Archaea are involved in the anaerobic process while the importance,if any,of eukaryotic microorganisms outside the rumen environ- ment is still unknown.The basic understanding ofthe dynamics ofthe complex microflora was elucidated during the latter part of the last century where the concept ofinter-species hydrogen transfer was introduced and tested.The isola- tion of syntrophic bacteria specialized in oxidation of intermediates such as volatile fatty acids gave strength to the theories.Lately the use ofmolecular tech- niques has provided tools for studying the microflora during the biomethana- tion process in situ.However,until now the main focus has been on probing the dynamic changes ofspecific groups ofmicroorganisms in anaerobic bioreactors and less emphasis has been devoted to evaluating the specific activities of the different groups ofmicrobes during biomethanation.In the future we can expect that the molecular techniques will be developed to allow more dynamic studies of the action of specific microbes in the over-all process. From the present studies we know that many unknown microbes are found in anaerobic bio- reactors. Especially within the domain of Archaea, there are whole phyla of microbes such as the Crenarchaeota, which make up significant fractions of microbes in a reactor but without cultured representatives.Improving the tech- niques for the isolation of presently unculturable microbes is a major task for the future. X Preface Anaerobic digestion ofwaste has been implemented throughout the world for treatment of wastewater,manure and solid waste and most countries have sci- entists,engineers and companies engaged in various aspects ofthis technology. Even though the implementation of anaerobic digestion has moved out of the experimental phase,there is still plenty of room for improvements.The basic understanding of the granulation process,the basis for the immobilization of anaerobic microbes to each other without support material in UASB reactors,is still lacking.Like any other bioprocess,anaerobic digestion needs further con- trol and regulation for optimization. However, until now suitable sensors for direct evaluation of the biological process have been lacking and anaerobic bioreactors have generally been controlled by indirect measurements ofbiogas or methane production along with measurements of pH and temperature.The newly development ofan on-line monitoring system for volatile fatty acids could be a major step in the right direction and the use of infra-red monitoring sys- tems could bring the price down to a reasonable level.A better performance of large-scale anaerobic bioreactor systems for treatment of complex mixtures of waste can be expected to be based on on-line monitoring of the process in the future along with controlling software for qualified management ofthese plants. Besides treatment of waste,anaerobic digestion possesses a major potential for adding value to other biomass converting processes such as gasification, bioethanol or hydrogen from ligno-cellulosic materials.Conversion ofligno-cel- lulosic biomass will often leave a large fraction of the raw material untouched which will be a burden for the over-all economy ofthe process and will demand further treatment.Anaerobic digestion will on the other hand be capable ofcon- verting the residues from the primary conversion into valuable methane,which will decrease the cost and the environmental burden ofthe primary production. Biomethanation is an area in which both basic and applied research is involved. Major new developments will demand that both disciplines work together closely and take advantage ofeach other’s field ofcompetence.The two volumes on Biomethanation within the series ofAdvances in Biochemical Engi- neering and Biotechnology have been constructed with this basic idea in mind and,therefore,both angles have been combined to give a full picture ofthe area. The first volume is devoted to giving an overview of the more fundamental aspects of anaerobic digestion while the second volume concentrates on some major applications and the potential ofusing anaerobic processes.The two vol- umes will therefore be of value for both scientists and practitioners within the field of environmental microbiology, anaerobic biotechnology, and environ- mental engineering.The general nature of most of the chapters along with the unique combination ofnew basic knowledge and practical experiences should, in addition,make the books valuable for teaching purposes. The volume editor is indebted to all the authors for their excellent contribu- tions and their devotion and cooperation in preparing these two volumes on Biomethanation. Lyngby,January 2003 Birgitte K.Ahring CHAPTER 6 Perspectives for Anaerobic Digestion Birgitte K.Ahring University ofCalifornia,Los Angeles (UCLA),School ofEngineering and Applied Science, Civil and Environmental Engineering Dept.,5732 Boelter Hall,Box 951593,Los Angeles, California 90095-1593,USA Present address:Biocentrum,The Technical University ofDenmark,DTU,Block 227, 2800 Lyngby,Denmark.E-mail:[email protected] The modern society generates large amounts ofwaste that represent a tremendous threat to the environment and human and animal health.To prevent and control this,a range ofdiffer- ent waste treatment and disposal methods are used.The choice of method must always be based on maximum safety,minimum environmental impact and,as far as possible,on val- orization of the waste and final recycling of the end products.One of the main trends of today’s waste management policies is to reduce the stream ofwaste going to landfills and to recycle the organic material and the plant nutrients back to the soil.Anaerobic digestion (AD) is one way ofachieving this goal and it will,furthermore,reduce energy consumption or may even be net energy producing.This chapter aims at provide a basic understanding ofthe world in which anaerobic digestion is operating today.The newest process developments as well as future perspectives will be discussed. Keywords. Anaerobic digestion,Carbon-flow,Microbiology,Antimization,Gas yild,Effluent quality 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 Microbiology ofAnaerobic Digestion . . . . . . . . . . . . . . . . 3 2.1 General Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.2 Syntrophic Acetate Conversion . . . . . . . . . . . . . . . . . . . . 5 2.3 Microbiology ofThermophilic Digestion . . . . . . . . . . . . . . 7 2.4 Establishing a Stable Microflora in Thermophilic Reactors . . . . 8 3 Anaerobic Digestion Plants . . . . . . . . . . . . . . . . . . . . . 12 4 Anaerobic Digestion as a Way to Add Extra Value . . . . . . . . . 14 5 Optimization ofAnaerobic Digestion . . . . . . . . . . . . . . . . 15 5.1 Increasing the Digestibility ofthe Waste . . . . . . . . . . . . . . 15 5.2 Optimization ofReactor Configuration . . . . . . . . . . . . . . . 17 5.3 Optimizing Process Control and Stability . . . . . . . . . . . . . . 19 5.4 Improving the Microbial Process and its Efficiency . . . . . . . . . 22 Advances in Biochemical Engineering/ Biotechnology,Vol.81 Series Editor:T.Scheper © Springer-Verlag Berlin Heidelberg 2003 2 B.K.Ahring 6 Optimization ofEffluent Quality . . . . . . . . . . . . . . . . . . 23 6.1 Inactivation ofPathogens and Other Biological Hazards . . . . . . 23 6.2 Control ofChemical Pollutants . . . . . . . . . . . . . . . . . . . 25 7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 8 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 1 Introduction The modern society generates large amounts ofwaste that represent a tremen- dous threat to the environment and human and animal health.To prevent and control this,a range ofdifferent waste treatment and disposal methods is used. The choice of method must always be based on maximum safety, minimum environmental impact and,as far as possible,on valorization of the waste and final recycling ofthe end products.One ofthe main trends oftoday’s waste man- agement policies is to reduce the stream ofwaste going to landfills and to recy- cle the organic material and the plant nutrients back to soil.Waste is increasing- ly becoming a problem and secure recirculation is gaining more and more atten- tion.Anaerobic digestion (AD) is one way ofachieving this goal and it will,fur- thermore,reduce energy consumption,or may even be energy producing,which is ofmajor importance to the global environment.Anaerobic digestion has been implemented for years as a means for the stabilization of sewage sludge;how- ever,during the past years anaerobic digestion technologies have been expand- ed to emphasize treatment and energy recovery from many other types of wastes including animal wastes,source-sorted household wastes,organic indus- trial wastes and industrial wastewater. Compared to incineration, anaerobic digestion creates more energy during the treatment of wastes,which normally have high water content.During incineration the nutrients are lost.Following the increasing interest in implementation of anaerobic digestion,optimization ofthis process is becoming increasingly more important.Despite the increased efforts spent on waste reduction,the amounts ofwaste are increasing through- out the world.This has led to ideas for a total removal ofwaste through injection into the deep underground (below 2 km) into old oil wells far below any the groundwater level [1].The recovery ofmethane will,however,be ofimportance for the feasibility and economy of this technique and methane development at these high temperatures,pressures and salinity is now under investigation. This chapter focuses on the perspectives for optimization ofanaerobic diges- tion after a briefintroduction to the microbiology ofanaerobic digestion.Opti- mization is a double-sided task:it involves both an increase ofthe biogas yield, which again implies an increased removal ofthe organic material in the waste, as well as ensuring an effluent with a sufficiently high quality to allow for recy- cling ofthe material as a fertilizer.A number ofareas for improving the biogas yield will be discussed such as,for example,increasing the digestibility of the Perspectives for Anaerobic Digestion 3 waste,optimizing process control,and improving the microbial process.With respect to effluent quality,emphasis will be on inactivation of pathogens and control ofchemical pollutants. 2 Microbiology of Anaerobic Digestion 2.1 General Scheme A major value of anaerobic digestion is linked to the production of biogas (methane and carbon dioxide) formed as the end product during degradation of organic material without oxygen.This energy is renewable and CO neutral and 2 can be used for production of electricity and heat.Many different consortia of microorganisms with different roles in the overall process scheme are needed for the AD process,which occurs naturally in anaerobic ecosystems such as sed- iments,paddy fields,water-logged soils and in the rumen [2]. Three major groups of microorganisms have been identified with different functions in the overall degradation process [3] (Fig.1): 1. The hydrolyzing and fermenting microorganisms are responsible for the ini- tial attack on polymers and monomers found in the waste material and pro- duce mainly acetate and hydrogen,but also varying amounts ofvolatile fatty acids (VFA) such as propionate and butyrate as well as some alcohols. Fig.1. The anaerobic degradation process 4 B.K.Ahring Fig.2. Carbon flow in anaerobic environments with active methanogens Fig.3. Carbon flow in anaerobic environments without active methanogens 2. The obligate hydrogen-producing acetogenic bacteria convert propionate and butyrate into acetate and hydrogen. 3. Two groups of methanogenic Archaea produce methane from acetate or hydrogen,respectively. The major part ofthe carbon flow in a well-operating anaerobic reactor occurs between the fermentative microorganisms and the methanogens.Only between 20 and 30% of the carbon is transformed into intermediary products before these are metabolized to methane and carbon dioxide (Fig.2) [4]. A balanced anaerobic digestion process demands that the products from the first two groups of microbes responsible for hydrolyzing and fermenting the material to hydrogen and acetate,simultaneously are used by the third group of microbes for the production ofmethane and carbon dioxide.The first group of microorganisms can survive without the presence of methanogens but will, under these conditions,form an increased amount ofreduced products such as VFA (Fig.3). The second group does,however,rely on the activity ofthe methanogens for removing hydrogen to make their metabolism thermodynamically possible as their reactions are endergonic under standard conditions and only occur when hydrogen is kept below a certain concentration.The relationship between the VFA-degrading bacteria and the hydrogen-utilizing methanogens is defined as syntrophic due to the dependent nature of this relationship and the process is Perspectives for Anaerobic Digestion 5 Fig.4. Interspecies hydrogen transfer called interspecies hydrogen transfer (Fig.4) [3].The lower the hydrogen con- centration the better are the thermodynamics ofthe VFA degradation.The dis- tance between the VFA degrader and the hydrogen utilizer does,therefore,affect the concentration ofhydrogen in the liquid phase,which again affects the ther- modynamics of the process.Therefore,the conversion is improved in granules and flocks compared to a situation where the microbes are distributed freely in a liquid solution [5].The two partners have to share a very small amount of energy and the conditions for ensuring energy for both microbes is very strict and can only be met within a narrow range ofhydrogen concentrations [6]. 2.2 Syntrophic Acetate Conversion Syntrophic relationships have also been found to be of importance for conver- sion of acetate when the acetate-degrading methanogens are inhibited by high concentrations of ammonia [7,8] or sulfite (unpublished).Under these condi- tions the acetate-utilizing methanogens are inhibited and other groups of microbes replace them to obtain energy from the oxidation ofacetate to hydro- gen and carbon dioxide (Fig.5). Due to thermodynamic constrains this reaction proceeds much better at increased temperatures and is the way ofacetate transformation when the tem- perature is higher than 60°C, close to the upper temperature limit of ther- mophilic acetate-utilizing methanogens [9,10].In accordance with this,the pop- ulation of Methanosarcina species disappeared more or less instantaneously from a biogas reactor operated on manure,when the temperature was increased from 55 to 65°C [11].Concurrently,the acetate concentration first increased and 6 B.K.Ahring Fig.5. Modified anaerobic degradation process with syntrophic acetate conversion then stabilized at a level somewhat higher than that found at 60°C [12].This coincided with a significant increase in the population of hydrogen-utilizing methanogens [11] indicating that this group had become dominant in the over- all conversion. Both syntrophic acetate oxidation and methanogenesis from acetate can be simultaneously active in a reactor system as indicated by several isotope studies often showing that less than 95% ofthe methane produced from acetate is derived from the methyl group. Isotope experiments with biomass from thermophilic reactors have further shown that the concentration ofacetate affects the competition between the two processes.When the concentration of acetate is low, syntrophic acetate conversion is the major process for acetate transformation [13,14].However,when the concentration ofacetate is above the threshold level [15] for the specific population ofacetate-utilizing methanogens in the reactor,these will be the major group active in the system.These findings further explain why the numbers ofhydrogen-utilizing methanogens are high in thermophilic granules,which have exclusively been fed with acetate for a long period [16]. Furthermore, the numbers of acetate-utilizing methanogens are highest close to the surface ofthe granules,where the concentration ofacetate is highest, while the populations of hydrogen-utilizing methanogens increased towards the center ofthe granules [16]. The first microbe found to perform acetate oxidation was a thermophilic bac- terium belonging to the group ofhomo-acetogenic bacteria capable ofreversing the acetate-forming reaction from hydrogen and carbon dioxide [17].This bac- terium used a very limited range ofsubstrates all related to its homo-acetogenic nature [17].Over time more microbes have been identified as being capable of Perspectives for Anaerobic Digestion 7 carrying out this reaction.Some of these microbes have been found to use a large variety ofsubstrates [18] and,furthermore,to be normal members ofthe populations of fermentative microbes in thermophilic reactors.This indicates that,at least in thermophilic reactors,syntrophilic acetate oxidation could be performed by a variety of the fermentative bacteria in the reactor when no other substrates are available.This needs,however,further verification. 2.3 Microbiology of Thermophilic Digestion Microbes thriving at high temperatures have been known for years [19].The reaction rate of many chemical reactions will double by an increase of 10°C according to the Arrhenius equation.The same is,however,not always the case for microbial reactions where the temperature response is specific for the par- ticular microbe.Different groups of microbes have been identified where the ones ofinterest for anaerobic digestion are mesophilic strains with an optimum between 30 and 40°C,and thermophilic strains with an optimum between 50 and 60°C[20].The mixed microflora found in an anaerobic bioreactor general- ly shows an increasing rate from a temperature of20 to 60°C and the theoreti- cal temperature gap between mesophilic and thermophilic strains is not appar- ent when viewing the process as a whole [21].Anaerobic digestion at a temper- ature below 20°C, or at a temperature above 60°C, generally shows a lower methane yield than within these limits.However,anaerobic digestion has been shown to be possible even at extreme thermophilic conditions of70°Cand more [28–30].Experiments with high temperature digestion ofmanure showed that major changes occurred in the microbial populations of the anaerobic reactor when the temperature was increased from 55 to 65°C[12].Besides a significant increase in the population of Archaea compared to Bacteria,also the popula- tions of methanogens underwent large changes over time.The population of hydrogen-utilizing methanogens did,for example,change from a major popula- tion belonging to the genus Methanobacterium to another belonging to Methanococcus over a 3-month period [11]. Such results clearly demonstrate that reactors operated at extreme conditions can take months before a stable microflora has established. With this in mind it is difficult to guess if the methane yield actually will be lower after an extended period ofmany months. Within the normal temperature range the general carbon flow of ther- mophilic reactors was found to be very similar to that of mesophilic reactors [31].A slightly higher amount ofthe carbon was channeled directly into acetate and a slightly smaller amount ofcarbon was turned over via the pool ofVFA [4]. Many extreme thermophilic Bacteria or Archaea have been found to produce mainly acetate and hydrogen as their end products [32].Therefore,less butyrate and propionate can be expected at these high temperatures.Different maximum temperatures were found for the different microbial groups in a thermophilic anaerobic reactor treating manure [33].For instance,among the methanogens, the acetate-utilizing methanogens have a much lower temperature maximum (ca. 62°C) compared to the hydrogen-utilizing methanogens (ca. 75°C) [33]. However,the actual temperature of the reactor affects the specific populations 8 B.K.Ahring which are active in the reactor.Therefore,a higher temperature optimum and maximum is found for the main metabolic groups in extreme thermophilic reac- tors compared to thermophilic reactors [28].Methane production was found in microbial mat samples taken from a slightly alkaline hot spring at 80°C [34]. This demonstrates that methanogenesis is possible even at this very high tem- perature. 2.4 Establishing a Stable Microflora in Thermophilic Reactors Waste such as sewage sludge,manure or household waste contains many differ- ent populations ofanaerobic or facultative anaerobic microorganisms.Most of these microbes are mesophilic and only a very small number of true ther- mophiles is present.The number ofmicrobes in raw sewage sludge utilizing sub- strates such as acetate or cellulose at 60°Cis extremely low (ca.100per g) [35]. The numbers are somewhat higher at 55°Cbut still much lower than the num- bers at 37°C [35].These facts clearly show the problems of establishing stable reactors at higher temperatures.Where the microflora of mesophilic reactors can be established directly based on the raw material fed to the reactor, the microflora ofthe thermophilic reactor has to be propagated from small minor- ity populations found in the raw materials [36].Many thermophilic full-scale reactors have failed through history,especially within the area ofsewage sludge treatment.The reason is basically a lack ofunderstanding ofthe principles for establishing a stable thermophilic microflora in the reactor. The same also applies to the literature,which is full ofexperiments with unstable thermophilic laboratory reactors often performing poorly compared to mesophilic reactors. When reviewing the literature describing these experiments,Wiegant [37] con- cluded that process stability is lower in thermophilic reactors and that ther- mophilic reactors generally have higher concentrations ofvolatile fatty acids in the effluent compared to mesophilic reactors.During recent years where more thermophilic reactors have been implemented,it has been shown that this con- clusion it not correct and that stable thermophilic reactors with a balanced thermophilic microflora perform just as well as stable mesophilic reactors [33, 38,39]. The key to obtain a balanced thermophilic microflora is to give optimal growth conditions to the small numbers of thermophilic populations found in the raw material during start-up ofthe bioreactor [36].Ifsufficient thermophilic seed material is available,it is possible to carry out a rapid start-up of a ther- mophilic reactor [33].The seed material should be evaluated before use with respect to the destruction ofvolatile solids in the reactor from which the seed is obtained as well as the concentration ofVFA.Ifpossible,it will be beneficial to perform a methanogenic activity testing of the seed material to establish the potential of this seed for transforming extra loads of methanogenic substrates (acetate and hydrogen) [40,41].After addition ofthe seed to an empty reactor it should be allowed to equilibrate for 1 day before feeding is initiated at the desired thermophilic temperature.A slow and graduate change ofthe tempera- ture only prolongs the start-up phase and does not select for true thermophiles