Biogas production from cheese whey: Past, present and future

Transworld Research Network 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India Advances in Cheese Whey Utilization, 2008: 35-80 ISBN: 978-81-7895-359-5 Editors: Ma Esperanza Cerdán, Ma Isabel González-Siso and Manuel Becerra 2 Biogas production from cheese whey: Past, present and future R.T. Bachmann1, A.C. Johnson2 and J.E. Hernandez3 1 Malaysian Institute of Chemical and Bioengineering Technology Universiti Kuala Lumpur, 1988 Vendor City, 7800 Taboh Naning, Alor Gajah Melaka, Malaysia; 2School of Environmental Engineering, Universiti Malaysia Perlis, 02600 Jejawi, Perlis, Malaysia; 3Departamento de Engenharia Biológica Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal Abstract Cheese whey is a type of dairy wastewater that is either discharged directly into the environment or the local sewer, treated with the primary object to meet effluent discharge limits, or used as a resource to produce, for example, biofuels, single cell proteins, animal feed after condensation, sweetener after crystallization of lactose, xanthan gum, glycerol and 2,3 butanediol upon fermentation. This review has been dedicated to the production of biofuels, specifically biogas, from cheese whey. We aim to provide the reader with a brief account of how knowledge in the field of biogas production from cheese whey effluents Correspondence/Reprint request: Dr. R.T. Bachmann, Malaysian Institute of Chemical and Bioengineering Technology, Universiti Kuala Lumpur, 1988 Vendor City, 7800 Taboh Naning, Alor Gajah, Melaka, Malaysia E-mail: bachmann@micet.unikl.edu.my 36 R.T. Bachmann et al. has evolved, the current status quo, and identify areas that should be addressed in the near future. Based on the review of ca. 100 research papers published over the past 25 years following facts were extracted: A large body of knowledge exists at lab- and pilot-scale. It has been shown that cheese whey wastewater can be used either as a primary or co-substrate for the production of biomethane and biohydrogen. Amongst the reactors studied, high-rate anaerobic digesters can be operated at organic loading rates up to 40 g COD L-1 d-1 with relatively low hydraulic retention times (0.5 – 10 d). The 1980ies have seen the implementation of the first fully integrated systems in New Zealand and Ireland. It was also concluded that anaerobic treatment of full-strength cheese whey, despite COD removal efficiencies of up to 99 %, cannot meet the stringent discharge criteria set forth in most countries thus requiring an aerobic polishing step. As the biogas technology reaches technical maturity and enters the commercial stage in the dairy industry, more data and reliable statistics are needed to evaluate the economical and operational feasibility at the industrial scale, including how we deal with the anaerobic digester effluent. Life cycle and carbon footprint assessments of the overall treatment process are to be carried out before it can be promoted as a technology that can contribute towards sustainable development and a more environmentally friendly agro- industry. Cooperation and promotion should be encouraged among agro- industries, government and national research institutes as well as providers of technology, operators and clients of digestion products. Consequently, decision makers will be able to choose the best technologies and options to promote the digestion products. It should also be recognized that the competition between different processes exploiting cheese whey as a resource will continue due to technological advances. 1. Introduction Cheese whey is a type of dairy wastewater that originates from the manufacture of cheese. The production of 1 kg of cheese releases between 6 – 11 kg of cheese whey (Kosikowski, 1979; Switzenbaum et al., 1982; Hobman, 1984; Barford et al., 1986; FAO, 1998; Danalewich et al., 1998). Cheese production worldwide has steadily increased over time as shown in Figure 1, and with it the problem of what to do with the whey. It is either discharged directly into the environment or the local sewer, treated with the primary object to meet effluent discharge limits, or used as a resource to produce biofuels, single cell proteins, animal feed, sweetener, xanthan gum, glycerol and 2,3 butanediol to name but a few. Many factors play a role in the fate of cheese whey utilization such as existence and enforcement of environmental laws, legislative hygiene requirements, market Biogas production from cheese whey: Past, present and future 37 size and price for neutraceuticals, animal food etc., cheese factory size, geographic location and density of cheese factories in a given area, knowledge and technology available on-site to operate valorization or anaerobic digester technology, electricity and fuel price, and political agenda - government may provide grants for renewable energy project in an effort to reduce reliance on fossil fuel; earn tax credits for low CO2 emission; or a green certificate for renewable energy production. Surveys have shown that in Australia 40 % of the cheese whey is discarded on land, into water or treated at sewage works, while 18.5 % was used for pasture irrigation; in Norway, 38 % is discarded as effluent (Kosikowski, 1979). Another recent survey established that all cheese manufacturers in the Upper Midwest of the USA generate varying quantities of wastewaters which contain cheese whey in considerable amounts (Danalewich et al., 1998), and that these wastewaters are discharged into the public sewer, applied on land, treated in aerated lagoons, trickling filters or by the activated sludge process. In this chapter, we shall focus on the production of biogas from cheese whey. Biogas is a combustible mixture of methane, carbon dioxide and traces of water and hydrogen sulphide. Lactose, a disaccharide comprising of galactose and glucose, is the main carbohydrate in cheese whey (Table 1) and a readily available substrate for anaerobic acidogenic bacteria producing organic acids such as acetate, propionate, iso- and n-butyrate, iso- and n-valerate, caproate, lactate, formate and ethanol. Some of these metabolites are in turn utilized by methanogens to produce methane. 1.E+07 Annual cheese production [t] 8.E+06 6.E+06 OECD Non-OECD 4.E+06 World 2.E+06 0.E+00 1 00 01 02 03 04 05 06 07 08 -0 20 20 20 20 20 20 20 20 20 97 19 Figure 1. Production of cheese world-wide (OECD, 2008). Table 1. Composition of cheese whey (units in percent). 38 R.T. Bachmann et al. Biogas production from cheese whey: Past, present and future 39 A knowledge of the stoichiometry of the process (Equation 1) permits calculation of a theoretical mass balance between substrate composition and methane production (Symons and Buswell, 1933; Buswell, 1952; Hobman, 1984). a b ⎛n a b⎞ ⎛n a b⎞ C n H a Ob + ( n − − ) H 2 O → ⎜ − + ⎟CO 2 + ⎜ + − ⎟CH 4 Eq. (1) 4 2 ⎝2 8 4⎠ ⎝2 8 4⎠ The stoichiometric relationship for lactose is provided in Equation 2: C12 H 22O11 + H 2O → 6 CO2 + 6 CH 4 Eq. (2) It can be calculated that 1 kg of lactose (3 moles) theoretically will yield 18 moles CH4 or 0.42 m3 CH4 at 15°C and 1.013 bar. For a "typical" cheese whey with 50 g lactose L-1, the estimated yield of methane under standard conditions is approximately 21.2 m3 CH4 m-3, which has an energy equivalent of ca. 740 MJ m-3 cheese whey (equivalent to 18.6 liters of fuel oil). As will be seen later, the CH4 yield is slightly less than the theoretical because: • Some of the organic matter is used by the cell to carry out metabolism and cell growth, • Digestion of organic matter is usually incomplete, and • Methane is slightly soluble in water (e.g. 5.4 vol-% at 2°C and 1.013 bar). Another relation may be obtained based on the COD. The COD of methane is 2 moles O2 per mole CH4 or 2.857 g COD L-1 CH4. CH 4 + 2O 2 → CO 2 + 2 H 2 O Eq. (3) In other words 0.35 L of methane is theoretically produced under standard conditions for every g COD that is removed from the waste during anaerobic digestion (van den Berg, 1982). In the following sections we aim to provide a brief account of how anaerobic technology has evolved in the digestion of cheese whey along with a critical discussion of the various anaerobic processes proposed or used, and identify areas where particular research and more attention is required in the foreseeable future. 2. Historical aspects 2.1. History of cheese making The use of milk of animals as food for man goes far back of all recorded history (Holmes, 1928). Cheese has been mentioned in the earlier books of the Old Testament implying its previous use for an extended time. Crude equipment for the manufacture of cheese and butter has been found in 40 R.T. Bachmann et al. Switzerland among the remains of the "lake dwellers" of the prehistoric period (Holmes, 1982). Cattle and/or sheep were found in France, Portugal, Spain, Greece and India 4000 – 2000 B.C. During these ancient periods and the next ten to twelve centuries dairy cattle were kept to supply the milk, butter, and cheese for immediate family use (Kosikowski, 1979; Holmes, 1982). The secondary product, whey, has shown up to challenge man to put it to some use, any use initially as long as this yellowish-green, slightly sticky liquid was not there on the following day to plague him. In the middle ages, for example, whey was applied as a pharmaceutical drug, a component of soothing salves for burns, a skim balm, a potion to inspire vitality and to restore hair, but rarely was it used as a food for humans (Kosikowski, 1979). The modern cheese industry as we know it today had its beginning with the industrial revolution during the nineteenth century. An increasing volume of cheese whey accompanied large-scale production of it for which there was little demand. It became customary to dispose whey into the nearest stream or river, and many cheese factories were built over or near such waterways for this purpose (Kosikowski, 1979). Little fuss was made of the ecological damage until the age of conservation, where cheese manufacturers had to comply with strong new regulations prohibiting the dumping of the highly polluting whey into streams, rivers and municipal sewerage systems. Cheese makers who could not or would not adapt had to close their plant. New avenues for cheese whey utilization had to be explored such as the fed to farm animals, spread over fields, or locally processed, but certain limitations of the period tended to restrain such outlets (Kosikowski, 1979). 2.2. History of AD of cheese whey Early attempts to treat this highly pollutional waste were reported by Bowles (1911), who recommended the septic tank system to treat and reduce the pollutional load of creamery waste. Many such installations may be found, but most devices did not perform satisfactorily (Levine, 1929). For example, accumulations of precipitated undigested casein and very malodorous effluents were frequently observed, partly due to inappropriate operation. No pH control was applied, and rapid acidification due to anaerobic decomposition of lactose inhibited the proteolytic and acidogenic bacteria and thus the septic tank process. Based on his observations, Levine (1929) proposed to eliminate the acid-producing constituents through biological oxidation of this waste by the activated sludge process, for example. For small creamery with its fluctuating quantity and variable waste characteristics, not to mention limited personnel, trickling filters were perceived to be feasible and economical. In 1932, Buswell and co- workers reported the successful stabilization of milk waste under anaerobic conditions achieving a 95 % reduction in pollutional load. This work was a Biogas production from cheese whey: Past, present and future 41 milestone in the biomethanization of dairy waste and provided the scientific fundament for further developments in this field. Buswell et al. (1932) established that anaerobic digestion of dairy waste coupled with an aerobic polishing step was more cost-effective than aerobic processes alone, since less sludge is produced alongside the generation of combustable biogas. The following decades have seen a range of modifications and improvements of Buswell’s process with the development of high-rate anaerobic digesters being the most important step-change ever since. In order to illustrate the advances made and deeper knowledge gained, we compiled a graph illustrating the number of papers published annually over time (Figure 2). This graph contains only peer-reviewed papers found in following databases: Cambridge Scientific Abstracts, Scirus, Scopus, Web of Knowledge and PubMed. Other publications such as conference proceedings were excluded. The graph illustrates that advances were made in the 1980ies and 90ies. A more complete understanding of the process has subsequently resulted in the patenting and commercialization of various processes as will be discussed in section 5. The past two years, however, have seen a renewed increase in publications, which is attributed, at least in part, to the production of hydrogen from cheese whey (Ferchichi et al., 2005; Kapdan and Kargi, 2006; Yang et al., 2007) instead of methane. Moreover, continuous progress in experimental microbial growth studies alongside technological advances in computing provided impetus for the 8 100 7 90 80 6 Cumulative publications 70 Publications p.a. 5 60 4 50 3 40 30 2 20 1 10 0 0 1900 1920 1940 1960 1980 2000 2020 Figure 2. Number of peer-reviewed papers (© cumulative and published ♦ annually) over time, dealing with various aspects of anaerobic digestion of cheese whey. 42 R.T. Bachmann et al. simulation of anaerobic cheese whey degradation processes (Boening and Larsen, 1982; Yang et al., 1988; Yang and Guo, 1990; Kalyuzhnyi et al., 2006; Gelegenis and Samarakou, 2007). Microbial characterization of the acidogenic and methanogenic biomass digesting cheese whey was carried out using cultivation-based (de Haast and Britz, 1986; Chartrain et al., 1987) and cultivation-independent methods (Yu et al., 2006). 3. High-rate AD for cheese whey biomethanization All modern high-rate anaerobic processes for biomethanation are based on the concept of retaining high viable biomass by some mode of bacterial sludge immobilisation to treat soluble organic wastes, and have been reviewed extensively in the literature (van den Berg, 1982; Barber et al., 1999; Rajeshwari et al., 2000; van Lier et al., 2001; Demirel et al., 2005). High biomass retention can be achieved by one of the following methods (Hulshoff and Lettinga, 1986). ƒ Formation of highly settleable sludge aggregates combined with gas separation and sludge settling, e.g. upflow anaerobic sludge blanket reactor (UASB) and anaerobic baffled reactor. ƒ Bacterial attachment to high density particulate carrier materials, e.g. fluidised bed reactors and anaerobic expanded bed reactors. ƒ Entrapment of sludge aggregates between packing material supplied to the reactor, e.g. downflow /upflow anaerobic filter. The high-rate digesters have reduced hydraulic detention times (HRT) from days to a few hours. As the industrial wastewater moves past the retained biomass, soluble constituents are converted to gas, while particulate solids have little opportunity to be hydrolyzed and degrade, unless the solids become attached to the biomass. In anaerobic wastewater treatment, OLR plays an important role. In the case of non-attached biomass reactors where the HRT is long, overloading results in biomass washout, decrease in efficiency and process failure. Fixed film, expanded and fluidized bed reactors can withstand higher organic loading rate and quick recovery and better stability from shockload. Moreover, greater COD reduction is achieved even at high loading rates at a short hydraulic retention time. The organic and hydraulic loading potential of a reactor depends on the following factors (Rajeshwari et al., 2000): • Amount of active biomass that can be retained by a reactor per unit volume. • Contact opportunity between the retained biomass and the incoming wastewater. • Diffusion of substrate within the biomass. Biogas production from cheese whey: Past, present and future 43 In order to accommodate some of the inadequacies, established high-rate technologies are being either combined or modified in laboratory scale to enhance the wastewater treating capability and methane generation from cheese whey. In the following section, some of the well established digester technologies available at present are discussed and illustrated. 3.1 Anaerobic contact process (AC) The anaerobic contact process was developed during the 1950ies. The process primarily uses a continuously stirred tank reactor (CSTR) and a sedimentation tank as shown in Figure 3. The sludge from the sedimentation tank is recycled back to the CSTR. Biogas produced in the process is vented from top of the digester to the boiler or flare. The performance of this process mainly depends on the degree of mixing of digester contents and extent to which the sludge settle out in the sedimentation tank for return to the digester. Biogas production rates of up to 1.5-3 L L-1 d-1 is achieved. Advantages of this process are: a) Digester sludge concentration can be kept high (10-15 g L-1) by recycling; b) OLR is normally high (2-10 g COD L-1 d-1); c) HRT is short (1-2 days); d) Can be used to treat wastewater with floating solids and large granules with no plugging problems. Biogas Digested liquid Influent Supernatant Sedimentation Digester tank Recycled sludge Figure 3. Schematic of the anaerobic contact process. Biogas production from cheese whey: Past, present and future 45 Biogas Biogas Influent Effluent Filter Filter Effluent Influent Downflow Upflow Figure 4 Schematic of the anaerobic filters. Limitations of this process include: a) can be plugged by floating organics and biofilm growth; b) difficult to clean. 3.3 Upflow anaerobic sludge blanket reactor (UASB) The upflow anaerobic sludge blanket reactor was developed in the Netherlands during the 1970ies after the limitations encountered with the anaerobic filter. The essential feature of the reactor is the presence of a very active sludge blanket in the bottom of the reactor. The active biomass in the form of sludge granules is retained in the reactor by direct settling for achieving high mean cell retention time. The wastewater is introduced at the bottom of the reactor into the sludge bed and the gas and effluent are collected from the top of the reactor (Figure 5). Gas production rate is up to 10 L L-1 d-1. The gas formed causes sufficient agitation to keep the sludge bed particles moving around and the volume of the bed fully mixed. Sludge granules that are lifted up above the sludge blanket settle down once the entrapped gas is released. The sludge blanket is mostly composed of bacteria, but usually contains 10 - 20 % inorganics to give them a reasonably high density to settle quickly and stay in the digester. Factors suggested to affect the performance of UASB are presence of trace metals (iron, nickel, cobalt and manganese for increase in biomass specific activity); nitrogen and calcium supplementation; types of methanogens and the type of waste (Ergüder et al. 2001). Some of the benefits of this technology are: 46 R.T. Bachmann et al. a) Comparatively less investment requirements when compared to an anaerobic filter; b) Recycling of the effluent may not be required; c) OLR can be between 1 - 30 g COD L-1 d-1; d) HRT is short (0.5 - 7 days). Limitations of this technology are: a) Requires sufficient amount of granular seed sludge for fast start-up; b) Wash-out of sludge during the initial phase of the operation is likely and therefore skilled operation is necessary; c) Requires gas-solids separator. Biogas Effluent Sedimentation area Gas collector Sludge blanket Influent Figure 5. Schematic of the upflow anaerobic sludge blanket reactor (UASB). 3.4 Anaerobic fluidized bed reactor (AFBR) The fluidized bed process was also developed during the 1970ies. The process uses small particle size, granular medium (e.g. sand, activated carbon, gravel, plastics) to support bacterial attachment and growth which is kept in the fluidized state by drag forces exerted by the upflowing wastewater as shown in Figure 6. When the wastewater passes through the fluidized bed, the organics in the wastewater come in contact with the biofilm adhered to the fluidized medium which is digested anaerobically. Recycling of the effluent may be necessary to achieve bed expansion as in the case of expanded bed Biogas production from cheese whey: Past, present and future 47 reactor. In the expanded bed design, microorganisms are attached to an inert support medium similar to the fluidized bed process. However, the diameter of the particles is slightly bigger. The principle used for the expansion bed is similar to that followed in the fluidized bed process, i.e. by a high upflow velocity and recycling. The produced biogas (up to 10 L L-1 d-1) is discharged from the top of the reactor. Benefits of this process include: a) Higher concentration of bacteria; b) OLR is between 1 - 100 g COD L-1 d-1 and greater resistance to inhibitors (Rajeshwari et al., 2000); c) HRT is short (0.25 - 5 days); d) No plugging problems in the bed because of fluidized state of the medium; e) Better contact between microorganisms and the substrate; f) Greater surface area per unit reactor volume; g) Lower capital cost because of reduced reactor volumes. Disadvantage of this process are: a) High energy requirement; b) High level of system management is required. Biogas Digested liquid Supernatant Digester Sedimentation tank Return of Biofilm coated media media Influent Fluidization pump Figure 6. Schematic of the fluidized bed reactor. 48 R.T. Bachmann et al. 3.5. Anaerobic fixed film reactor (AFFR) The anaerobic fixed film reactor relies on biofilm support structures such as activated carbon, PVC (polyvinyl chloride) supports, hard rock particles or ceramic rings for biomass immobilization (Figure 7). These supporting structures are inert with high surface to volume ratio and porosity promoting the development of biofilms (Murray et al., 1981). The degradation of the substrate is accomplished by the bacteria in 1) the liquid phase; 2) on the surface of the biofilm; 3) within the biofilm. The performance of the reactor depends on the quantity of active bacterial mass per unit surface, and how much surface area is present per volume. The product of these two factors gives the amount of microbial mass per unit volume which determines the volumetric rate of methane production. This type of reactor is capable of producing biogas between 3 to 5 L L-1 d-1. There are various types of fixed film reactors, which are generally grouped as either stationary or moving bed reactors. The advantages of this process are: a) Simplicity of construction; b) No mechanical mixing; c) Better stability at higher loading rates; d) Can recover very quickly after a period of starvation; e) Changes readily from one waste to another with relatively little loss of capacity; f) Can tolerate infrequent feeding. Biogas Biogas Effluent Influent Vertical channel support for biofilm development Packing media Influent Effluent Media packed Vertical channel fixed film reactor fixed film reactor Figure 7. Schematic of the anaerobic fixed film reactor. Table 2. Comparison of various anaerobic treatment processes for cheese whey. Biogas production from cheese whey: Past, present and future 49 50 R.T. Bachmann et al. Some of the limitations of this process are: a) Reactor volume is high because of the addition of support media; b) Clogging of the reactor due to biofilm thickness and / or solids in the wastewater; Table 2 compares the performance of various anaerobic reactor configurations used in the treatment of cheese whey, while Figure 8 illustrates how much research in terms of papers has been carried out with different anaerobic reactor systems. Based on the high COD removal efficiency, maximum achievable OLR of soluble wastewater and biogas production of established technologies, UASB and AFBR stands out distinctly. In addition, the fixed film reactors posses lower capacity for biomass retention per unit volume of the reactor because of the presence of the packing media. Fluidized bed reactors have the largest surface area for biomass attachment and better contact between the incoming wastewater and the biomass. On the other hand, the main limitation common to all non-attached biomass reactors is the floatation of sludge granules and shearing of these granules at high loading rates (Rajeshwari et al., 2000)[UM1]. The latter constrain is also valid to a lesser extent to fixed film and rotating biological contact reactors. 4% 1% 6% 24% 7% CS TR AFFR UA SB 9% AFB AFT AnRBC MPAR AP F 11% Sept ic t ank 21% 18% Figure 8. Percent anaerobic digester type used in lab- and pilot-scale studies of biomethanation of cheese whey. Abbrev.: CSTR – Continuously stirred tank reactors; AFFR – Anaerobic fixed film reactor; UASB – Upflow anaerobic sludge bed reactor; AFB – Anaerobic fluidized bed reactor ; AFT – Anaerobic fermentation tank (similar to CSTR but without stirring); AnRBC – Anaerobic rotating biological contact reactor; MPAR – Multiplate anaerobic reactor; APF – Anaerobic percolating filter. Biogas production from cheese whey: Past, present and future 51 4. Factors influencing biomethanation of cheese whey The anaerobic digestion process is affected significantly by a number of operational, environmental and technical conditions. As the process involves the formation of organic acids from a range of agro-industrial wastes, it is important that the bacteria are acclimatized to the substrate composition and concentration. In addition, the rate of reaction should be such that there is no accumulation of acids, which would result in the failure of the digester. This, in turn, is governed by the loading rate and the strength of the influent. Temperature and pH are other important variables as the methane producing bacteria are sensitive to these as well Rajeshwari et al. (2000). 4.1. Inoculum The definition of an optimal microbial inoculum and starting procedure is an important step in the optimization of an anaerobic digestion process and the modelling of microbial ecosystems (Fournier et al., 1993). In the field of biomethanation of cheese whey, however, only a few studies have been carried out to specifically examine the effect of the inoculum on digester performance, while most provided a brief description of the start-up process (Table 3). Chartrain et al. (1987) compared the performance of a designed starter culture consisting of Leuconostoc mesenteroides, Desulfovibrio vulgaris, Methanosarcina barkeri, and Methanobacterium formicicum, with that of a diverse adapted mixed-culture inoculum, in a continuous contact digestor system to which 10 g of dry whey per liter was added. Both inocula displayed a similar biomethanation performance. In another study, Fournier et al. (1993) investigated the effect of different heterogeneous mixed bacterial inocula (calfpat, cowpat and digester sludge) on the acidogenic fermentation of cheese whey permeate. The authors found that after a few months of continuous cultivation at pH 6 and a HRT of 40 h, all mixed bacterial populations produced large amounts of lactate and butyrate. When the HRT was modified, it appeared to be the most important factor to control the dynamic equilibrium between the bacterial species and/or the metabolic pathways leading to butyrate and lactate, whereas the production of ethanol and acetate was largely depending on the source of the inoculum under these conditions. Denac (1988) compared the degradation of whey in cultures that had or had not been acclimated to this substrate. The authors found that the performance of the FBR with whey was comparable to its performance with molasses, and switching of molasses to whey feed resulted in immediate good performance without adaptation. Approximately 14 % of the COD was not degradable. In summary, most inocula for lab or field based studies were taken from industrial AD treating a variety of wastes. The relative amount added and Table 3. Experimental conditions for the start-up of anaerobic digesters treating cheese whey. 52 R.T. Bachmann et al. Biogas production from cheese whey: Past, present and future 53 wastewater history affected the duration of the start-up period but not biomethanation. A large inoculum from digesters treating similar waste with similar operating conditions (OLR, reactor type) can reduce the start-up time significantly, perhaps with one exception, where biofilm formation processes are required. 4.2 Trace elements Twenty five years ago, Speece (1983) argued that the majority of industrial wastewaters that appear to be good candidates for anaerobic treatment require new phases of inquiry. In order to accomplish faster COD degradation and enhanced methane production rates particularly in high-rate anaerobic digesters, the study of trace nutrient requirements of methanogens was identified to be an important factor in anaerobic biotechnology. Methanogens were found to have relatively high internal concentrations of Fe, Ni and Co. For example, iron is present in cytochromes, siderophores and other enzymes and plays a role in redox catalytic reactions, while Ni and Co are present in several enzyme-catalysed gas metabolism reactions and vitamin B12 or derivatives, respectively (Bachmann and Edyvean, 2005). Rajeshwari (2001) pointed out that these elements may not be present in sufficient concentrations in wastewater streams from the processing of one single agro- industrial product or the wastewater derived from condensates. In such cases, the wastewater may have to be supplemented with the trace elements prior to treatment. A number of research groups have studied the trace nutrient requirements of anaerobic cheese whey digestion in a more or less systematic fashion. A brief account is provided in the following. Callander and Barford (1983) carried out a theoretical and experimental assessment of soluble, complexed (chelated) and precipitated alkali earth and transition metals in defined and complex wastewater (e.g. whey and pig manure). The pH was fixed at pH 7.3, and the effect of OH-, PO43-, CO32- and S2- anions discussed. Based on equilibrium calculations it was hypothesized that: 1) Hydroxide precipitates should not form; 2) Mg and Ca in excess of 3 x 10-4 M (7 mg L-1) and 5 x 10-5 M (2 mg L-1) will precipitate as carbonates or, at high PO43- levels, as phosphates or MgNH4PO4; 3) Mn will be precipitated as sulfide in high-sulfide digesters, and otherwise as carbonate; 4) Fe, Co, Ni, Cu, and Zn precipitate as sulfides or, if the total concentration of these metals exceeded the total sulfide level, as carbonates, with the exception of Ni (as phosphate). 54 R.T. Bachmann et al. Comparison of theoretically predicted and experimentally observed metal concentrations in whey showed that measured Mg concentration (96 mg L-1) exceeded predicted metal concentration by a factor of 6, Ca (280 mg L-1) by a factor of 50, Fe (0.56 mg L-1) by a factor of 300, and Co (0.114 mg L-1) by a factor of a million. The large discrepancies were primarily attributed to inaccurate equilibrium constants, the possibility that equilibria in the wastewater have not been reached because the kinetics of interspecies equilibria or precipitation are slow compared to process kinetics, the excretion of chelators by bacteria (e.g. siderophores), and the presence of metal chelators cysteine, NTA, citric acid etc. Owing to the limited range of anions considered, the formation of bidentate complexes was also not taken into account. Moreover, more powerful speciation modeling softwares such as PHREEQc and VisualMinteq are available now, which should improve the accuracy of theoretical predictions further. Based on the assumption that Fe-amino acid chelates are significant in methanogen Fe uptake, Barford and Callander (1983) recommended that any steps which reduce the amino acid content of digester feeds should be avoided. Streicher (1991) performed anaerobic digestion of diluted cheese whey in UASB reactors under different operating conditions. Without nutrient addition the specific maximum COD removal rate was about 6 g COD degraded per litre packed bed volume per day. Nutrient additions, consisting of iron, nickel and cobalt salts of dried yeasts, allowed an almost seven-fold increase, mainly due to higher biomass accumulation inside the reactor. In an effort to improve the anaerobic digestion process of mixture of cheese whey, poultry waste and cattle dung, Patel et al. (1994) studied the effect of various doses of FeCl3, NiCl2, CoCl2, CuCl2 and ZnCl2. Among the trace nutrients used, CoCl2 was found to increase the gas production by more than 80 % with enriched methane content. Ergüder et al. (2001) conducted biochemical methane potential (BMP) tests to establish the combined effect of trace elements, nutrients and buffering on biogas production rate and quantity from medium-strength cheese whey. The authors added a mix of nutrients and inorganic salts to cheese whey (final concentration per litre: 1200 mg NH4Cl, 400 mg MgSO4 7H2O, 400 mg KCl, 300 mg Na2S 9H2O, 50 mg CaCl2 2H2O, 80 mg (NH4)2HPO4, 40 mg FeCl2 4H2O, 10 mg CoCl2 2H2O, 10 mg CoCl2 6H2O, 10 KI, 0.5 mg MnCl2 4H2O, 0.5 mg CuCl2 2H2O, 0.5 mg ZnCl2, 0.5 mg AlCl3 6H2O, 0.5 g NaMoO4 2H2O, 0.5 mg HBO3, 0.5 mg NiCl2 6H2O, 0.5 mg NaWO4 2H2O, 0.5 mg Na2SeO3, 10 mg cysteine, 6000 mg NaHCO3), and established a five fold increase in cumulative biogas production with basal medium addition after a 70 d incubation period. Owing to the nature of the experiments, cause-and-effect relationships of particular ingredients could not be established. Biogas production from cheese whey: Past, present and future 55 Zayed and Winter (2000) carried out toxicity tests with either CuCl2, ZnCl2 or NiCl2 on whey effluents from an anaerobic fixed-bed reactor. Fifty per cent inhibition of methanogenesis was noted in the presence of >10 mg CuCl2 L-1, > 40 mg ZnCl2 L-1 and > 60 mg NiCl2 L-1, respectively. After a 12 day exposure to Cu2+, Zn2+ or Ni2+, a complete recovery of methanogenesis by equimolar sulphide addition was possible upon prolonged incubation. Recovery failed, however, for CuCl2 concentrations > 40 mg L-1. Simultaneous addition of sulfide and the three heavy metal salts resulted only in slight delay of methanogenesis, and the same amount of methane as in non-inhibited controls was reached either 1 day (40 mg ZnCl2 L-1) or 2 days later (10 mg CuCl2 L-1). Up to 60 mg NiCl2 L-1 had no effect if sulfide was present. Sulfide presumably precipitated the heavy metals as metal sulfides and by this means prevented heavy metal toxicity. From the data presented it can be concluded that addition of trace elements such as Co to cheese whey can have positive effects on the COD reduction as well as biogas production rate. However, more comprehensive studies are needed to establish the background levels of trace elements present in cheese whey and to identify which elements are growth-limiting or inhibiting, secondly to address the bioavailability of these metals and their metal uptake in axenic and mixed cultures of methanogens. Not much has been done to identify economically effective metal chelators, which could be added to the digester feed since Callander and Barford’s (1983) work. A thorough understanding of the quantitative trace element requirements will ultimatively enable the practitioner to apply the correct dose and mixture to the anaerobic reactor. 4.3 Macronutrients The required optimum C : N : P ratio for enhanced yield of methane were reported to range between 100 : 2.5 : 0.5 (Rajeshwari, 2000) and 600 : 15 : 5 : 1 (C : N : P : S; Weiland, 2001). The minimum concentration of macro- and micronutrients can be calculated based on the biodegradable COD concentration of the wastewater, cell yield and nutrient concentration in bacterial cells. It is recommended that the nutrient concentration in the influent should be adjusted to a value twice the minimal nutrient concentration required in order to ensure that there is a small excess in the nutrients needed. The rate of methanogenesis also depends on the type and content of the organic matter undergoing digestion. For example, poultry manure is difficult to handle by anaerobic digestion systems due to heavy ammonia toxification (Desai et al., 1994). When co-digesting whey and poultry waste, however, a favourable C/N ratio is maintained in the digester, and fermentation of the mixed waste material has proved to be more effective for methane generation than the materials treated individually (Desai et al., 1994). 56 R.T. Bachmann et al. Most authors report the COD : N: P ratio used in their study either directly or indirectly (Table 4). In a few cases, the C:N:P ratio was predicted, based on typical chemical formulas for carbohydrates (C6H10O5)n, proteins (C5H7NO2) and lipids C57H104O6) (Gelegenis et al., 2007). Attempts to explicitly test the effect of different ratios of C/N or C/P on methane production from cheese whey are rare (Backus et al., 1986). In this study the authors used sixteen one- liter digesters to test four C/N ratios of 8.4, 13.9, 22.2, and 27.6 and four HRTs of 12, 18, 24, and 30 days. The authors observed a correlation between methane content and methane production and C/N and HRT, respectively. As can be seen from the ratios provided in Table 4, the variation between COD, N and P is relatively small, and C : N : P ratios are similar to the ones reported by Rajeshwari (2000) and Weiland (2001). Table 4. COD : N : P ratios reported in literature for various types of cheese whey wastewater Ratio type Ratio Wastewater type Reference COD : TKN : P 100 : 1.8 : 0.8 Full-strength cheese whey Barford et al., 1986 C:N:P 100 : 3.3 : 0.7 Synthetic cheese whey Kissalita et al., 1989 COD : N : P 100 : 2.1 : 0.4 Cheese whey permeate Hwang et al., 1992 COD : N : P : S 100 : 1.1 : 0.5 : 0.1 Cheese whey permeate and Guiot et al., 1995 cheese factory effluent (1 : 1 v/v) CODt : TKN : P 100 : 1.7 : 0.6 Dairy wastewater Gavala et al., 1996 CODt : TKN : P 100 : 3.5 : 0.8 Full-strength cheese whey Malaspina, 1996 COD : N : P 100 : 2 : 0.4 Cheese whey powder Yilmazer and Yenigün, dissolved in distilled water 1999 COD : N : P : S 100 : 2.4 : 0.5 : 0.2 low-strength reconstituted an Frigon et al., 2007 NaHCO3 buffered cheese whey COD : N : P 100 : 1.4 : 0.4 Full-strength cheese whey Gelegenis et al., 2007 C:N:P 100 : 4.1 : 1.2 COD : TKN : P 100 : 1.6 : 0.7 Full-strength cheese whey Saddoud et al., 2007 BOD5 : TKN : P 100 : 3 : 1.3 4.4. pH and alkalinity Anaerobic reactions are highly pH dependent, owing to the fact that the optimal pH range for slow-growing, methane producing bacteria is 6.8 - 7.2, while fast-growing, acid-forming bacteria strive at lower pHs. Consequently, pH can be used as a means to control the population of acidogenic bacteria in an anaerobic, single-stage system by keeping it within the methanogenic optima (Rajeshwari et al., 2000). Failure to do so may cause VFA accumulation, inhibition or killing of methanogens and subsequently reactor failure. Maintenance of a steady pH in the presence of VFAs can be achieved through the addition Biogas production from cheese whey: Past, present and future 57 of, for example, sodium bicarbonate (NaHCO3) to raise alkalinity, or by means of co-digestion (refer to section 4.8 for further details). Switzenbaum and Danskin (1982) utilized an anaerobic attached film expanded bed (AAFEB), a type of UASB seeded with aluminium oxide particles for bacterial attachment, to treat full-strength, reconstituted sweet cheese whey supplemented with (NH4)3PO4 and a buffer. Despite the addition of 5 g of NaHCO3 per litre whey feed, the pH decreased at higher loading conditions (OLR 60 g COD L-1 d-1) indicating overloading and unbalanced treatment. Boening et al. (1982) added an unspecified amount of NaHCO3 to a UASB reactor treating lactic casein whey permeate at an OLR of up to 30 g COD L-1 d-1, and managed to maintain effluent alkalinity above 2 g L-1. Yang et al. (1989) added 2 g NaHCO3 L-1 and 6.6 g K2HPO4 L-1 to full-strength cheese whey as a buffer and nutrient supplement, respectively. Sodium hydroxide solution was used to adjust the pH value of the acidic whey to pH 7.0. The so-prepared cheese whey was digested in an UASB, and stable operation could be maintained up to an OLR of 7.76 g COD L-1 d-1, with a COD removal efficiency of 98 % and an average methane yield of 0.32 L CH4 g-1 COD removed. Acetic and propionic acid concentration in the effluent (pH 7.2) were 80 and 64 mg L-1, respectively. Hwang et al. (1992) used continuous UASB reactors to digest medium-strength whey permeate from reconstituted whey powder buffered with up to 2.5 g NaHCO3 L-1. The reactor could be operated at OLRs up to 13 g COD L-1 d-1 with COD removal efficiencies > 92 % and biogas production of 4.24 L L-1 d-1 (CH4 content 63 %). pH remained stable at around 7. Kato et al. (1994) provided alkalinity to low-strength cheese whey wastewater in the form of NaHCO3 (0.5 – 1.0 g NaHCO3 for each g COD). COD removal efficiencies of > 91 % at OLRs of 2.0 – 2.4 g COD L-1 d-1 could be achieved in the UASB reactors deployed, while the pH remained at pH 7. The authors also reported that 15 % of the total CH4 dissolved in the effluent and therefore lost. McHugh et al. (2006) used two modified UASB reactors R1 and R2 to treat low- (1 g L-1) and medium-strength (10 g L-1) reconstituted whey buffered with NaHCO3 (R1: 1 g L-1; R2: 10 g L-1). The pH of both reactors remained between 7 and 8, while COD removals of 80 - 90 % at OLRs up to 13.3 g COD L-1 d-1 could be obtained. Wildenauer and Winter (1985) adjusted the pH of full-strength cottage cheese whey to pH 6.7 by means of a pH titrator prior to anaerobic treatment in an upflow, packed-bed (using porous clay beads) column digester (5 x 200 cm). The digester operation was stable at an OLR of 14.1 g COD L-1 d-1 (HRT 5 d). In a similar study carried out by the same group, Zellner et al. (1987) slightly modified the design of the upflow pack-bed reactor (dimensions: 8 x 45 cm), and achieved stable operation up to an OLR of 36 g COD L-1 d-1with pH control of incoming full-strength cheese whey (pH 6.5). When whey permeate was treated instead, satisfactory operation was achieved up to 7.7 g 58 R.T. Bachmann et al. COD L-1 d-1 (HRT 4.3 d) at a pH of 5.5. Denac and Dunn (1988) also controlled the pH of reconstituted whey treated in a fluidized bed by automatic pH titration. Stable operation at an OLR of 25 g COD L-1 d-1 with a biogas production of 10 L L-1 d-1 was reported (COD reduction 63 %). A general characteristic of low-residence time operation was a relatively low CO2 composition of the biogas due to the high solubility of CO2 at pH 7. In addition, NaOH titration also caused the precipitation of carbonates. Elmamouni et al. (1995) assessed the impact of lime (Ca(OH)2) on the anaerobic treatment of deproteinated whey permeate effluents in a multiplate anaerobic reactor (MPAR). The amount of Ca(OH)2 required to maintain a pH of 5 ranged between 3.0 and 4.5 g L-1. Dissolved COD removal efficiency was > 92 % with a methane production rate of 6.7 L CH4 L-1 d-1 at an OLR up to 20 g COD L-1 d-1. Control experiments without liming or the addition of a different buffer agent such as sodium bicarbonate where not carried out. After three months of operation the MPAR experienced accumulation of calcium precipitates in the sludge bed, which reached 0.19, 0.25 and 0.33 g Ca2+ g-1 SS in the lower, the middle and the upper compartment of the MPAR, respectively. The greater concentration of Ca2+ precipitates in the upper compartments of the MPAR may be due to elevated pHs as a result of VFA removal by methanogens. Callander et al. (1983) adjusted the pH of reconstituted acidic cheese whey to 6.5 prior to anaerobic CSTRs by the addition of NaOH. No further pH control was necessary up to the maximum stable OLR of 8.5 g COD L-1 d-1. Lebrato et al. (1990) digested a mix of cheese factory wastewater and whey (80 % : 20 %) in a CSTR without pH adjustments. The maximum OLR under these configurations was 1.7 g COD L-1 d-1 (HRT 10 d) with a COD reduction efficiency of 78 %. The authors noted a self-regulation of pH at 7 - 7.4 due to the formation of HCO3NH4 from CO2 and NH3, which increased the alkalinity. Thus, any variation of volatile acids did not affect the pH. In another study, Fang (1991) digested whey processing plant wastewater (pH 7) in an anaerobic CSTR without pH control or buffering. Anaerobic sludge was recycled. At an OLR of 0.812 g COD L-1 d-1 (HRT 2 d) a BOD5 reduction of 87 % was achieved. Malaspina et al. (1996) coupled two CSTR reactors to treat full- strength cheese whey in effluent recycle mode, and was able to operate the 2-stage system at OLRs of up to 3 g COD L-1 d-1 (COD removal efficiencies > 90 %) without external buffering. The pH in the acidogenic reactor fluctuated around pH 6.5 with VFA concentrations measured between 4 – 8 g L-1 (acetic acid prevalent), while the pH in the methanogenic reactor was pH 7.5. No external alkalinity was added. Ratusznei et al. (2003) aimed to establish the minimum amount of NaHCO3 required at which process stability and efficiency could be accomplished in a stirred sequencing batch reactor containing immobilised biomass. The reactor was fed with low-strength Biogas production from cheese whey: Past, present and future 59 reconstituted cheese whey at OLRs up to 5.7 g COD L-1 d-1 (COD reduction 96 %), while 1 mg NaHCO3 was sufficient per 10 mg COD under steady-state conditions. In a similar, later study, Mockaitis et al. (2006) reported mg NaHCO3 to mg COD ratios of 25 – 50 %. Lo and Liao (1986) successfully treated full-strength cheese whey in an AnRBC without pH control down to an HRT of 5 days (OLR 10 g L-1 d-1). Attempts were made to further reduce the HRT to 4 days. However, extremely high VFA concentrations accumulated in the reactors and the pH fell to 5.1 within 2 days. Desai et al. (1994) co-digested full-strength cattle : poultry : cheese whey mix (2 : 1 : 3) without pH control or buffering. The pH remained almost unaffected by the variation in temperatures and retention. In another study, Lo et al. (1988) treated a mixture of dairy manure and cheddar cheese whey (1 : 2 v/v) in an AnRBC without pH control or buffer addition. OLRs of up to 20.9 g COD L-1 d-1 could be obtained with a COD removal efficiency of 50 % and methane production of 3.74 g CH4 L-1 d-1. When OLR was increased to 32 g COD L-1 d-1, steady-state operation could not be maintained. After initial buffering with NaHCO3, COD removal of 83 % for a mix of OMW and cheese whey (75 % : 25 %) in an anaerobic fixed bed digester could be achieved without further pH control up to an OLR of 3 g COD L-1 d-1 (Martinez-Garcia et al., 2007). Alkalinity at the end of the experiment was 4.5 g L-1 CaCO3. Most two-stage reactor configurations for the treatment of cheese whey as discussed in section 4.7 did not require buffer addition provided they are operated within a certain OLR range, which results in considerable cost savings for chemicals. In summary, pH control during anaerobic digestion of cheese whey was implemented in most studies by means of a chemical buffering agent (NaHCO3, Ca(OH)2), automated NaOH addition or co-digestion of whey with waste of sufficient buffering capacity. The degree of control was directly influenced by the OLR and reactor configuration. A general characteristic of high OLR operation was reported to be a relatively low CO2 composition of the biogas due to the high solubility of CO2 at pH 7 and the precipitation of carbonates due to NaOH titration. 4.5. Temperature Anaerobic digestion is strongly influenced by temperature and the process is classified as psychrophilic (0 - 20°C), mesophilic (20 - 42°C) and thermophilic (42 - 75°C). It is commonly reported that the bacterial activity and growth decreases by 50 % for each 10°C drop in the mesophilic range (van den Berg et al., 1985; Rajeshwari, 2000; Ke et al., 2005). Thus, for a given degradation degree, the lower the temperature the longer the degradation time. 60 R.T. Bachmann et al. Nonetheless, the psychrophilic range is of particular interest to AD operators in moderate climates treating pathogen-free organic waste, where the energy required to heat the meso- or thermophilic reactors utilises a significant proportion of the produced biogas energy with considerable implications on process economics (McHugh et al., 2006). However, the major constraints to a high rate anaerobic treatment system at low temperatures are the very low bacterial growth rates and the low specific sludge activities. To enable an efficient and stable anaerobic treatment system under psychrophilic conditions, the following conditions should be met (van Lier et al., 2001): • an extremely high biomass retention time under high hydraulic loading conditions, since only very little viable biomass can be allowed to wash out from the reactor; • an excellent contact between the retained biomass and the wastewater in order to utilise all the available capacity in the digester. An important characteristic of anaerobic bacteria is that their decay rate is very low at temperatures below 15°C (Ke et al., 2005). Thus, it is possible to preserve the anaerobic sludge for long periods without losing much of its activity. This is especially useful in the anaerobic treatment of wastewater from seasonal industries, which may be inactive during winter. Thermophilic conditions, on the other hand, are of interest where high conversion rates of organic substances coupled with hygienic requirements are needed. The upper temperature limit for conventionally designed, thermophilic, single-stage sludge bed reactors, for example, is defined by poor biomass retention if COD removal rates exceed 50 – 60 g L-1 day-1 (van Lier et al., 2001), poor sludge separation due to the lower liquid viscosity and the occasional occurrence of less stable thermophilic aggregates. In order to circumvent the problems, van Lier et al. (2001) suggested the use of staged reactor systems in which the produced biogas is evenly withdrawn from the reactor. The following section discusses the literature on the effect of temperature on anaerobic treatment of cheese whey. From the literature survey it was established, that reactors were operated at a wide temperature range (10 – 60°C), with the majority focusing on the mesophilic range (> 85 % of published research papers). Only a few papers were found to examine the effect of temperature on biogas production and COD reduction from low and medium-strength cheese whey (Boening et al., 1982; McHugh et al., 2006) or full-strength cheese whey and manure (Desai, 1994). Boening et al. (1982) investigated the effect of 15°C, 25°C and 35°C on the performance of an UASB reactor treating low-strength lactic casein whey permeate (3 g L-1), and found that COD reduction (from 35 % to 70 %) and methane production (0.232 – 0.349 L CH4 g-1 COD removed) increased, while Biogas production from cheese whey: Past, present and future 61 VFA concentration, biomass concentration and optimum HRT (from 0.58 days to 0.10 days) decreased when the temperature was raised. Solubility of methane was suggested to increase at lower temperatures, which makes biogas collection more inefficient thus partially explaining the drop in methane production rate at low temperatures. It was reported that pH control was not required (varied between pH 6.9 and 8.3). In another study, McHugh et al. (2006) used two modified UASB reactors R1 and R2 to treat low- (1 g L-1) and medium-strength (10 g L-1) reconstituted whey buffered with NaHCO3 (1 g L-1 R1; 10 g L-1 for R2), supplemented with macro- and micro-nutrients over a period of 500 days. Temperatures tested varied between 12 and 20°C. At steady-state, COD removal (80 %), methane content (70 %) and VFA concentrations (< 25 mg L-1) in R1 remained unaffected by temperature, while COD removal (90 %), methane content (55 %) and VFA concentration (< 200 mg L-1) in R2 remained unchanged at steady state until 14°C. When the temperature in R2 was lowered to 12°C, the reactor became unstable, and granule disintegration was observed. A reduction in OLR from 13.3 to 6.6 g COD L-1 d-1, however, has resulted in a gradual recovery of the system. At the end of the experiment, COD removal efficiencies in excess of 75 %, a methane content of 57 % and relatively low effluent VFA concentrations (< 700 mg L-1) were reported. EPS content based on the uronic acid method was monitored in both reactors. No noticeable changes in EPS composition were found for R1 and R2 prior to failure. The pH range of both R1 and R2 mixed liquor and effluent samples remained between 7 and 8 throughout the study. Desai et al. (1994) studied the anaerobic degradation of a full-strength cattle : poultry : cheese whey mix (2 : 1 : 3) under mesophilic and thermophilic conditions (20 – 60°C at 5°C increments) in a sludge-bed reactor at HRTs ranging from 3 to 15 days. The authors noted two temperature optima, 40°C and 60°C, for COD removal and biogas production, and a further two optima, 20°C and 50°C, for VFA production. The pH remained almost unaffected by the variation in temperatures and retention. In general, the pH was slightly lowered with increased retention at all temperatures. From the limited amount of information available it can be concluded that digestion of low-strength cheese whey in UASB reactors under psychrophilic conditions is possible. Buffering may be required to stabilize or enhance reactor performance. Other high-rate anaerobic digester configurations need to be evaluated under psychrophilic conditions, and biogas collection efficiency at low temperatures should be given further attention. The study carried out by Desai et al. (1994) agrees with Mudrak and Kunst (1986), who reported that the effect of temperature on acidogenic bacteria is not very significant, as among the mixed population there are always some bacteria which have their optimum within the range concerned, whereas acetogenic and methanogenic bacteria are much more sensitive towards temperature change. 62 R.T. Bachmann et al. 4.6. Seasonality of milk production Various industries processing agricultural products are, at least to some extent, affected by seasonal availability (Gavala et al., 1996; Ke et al., 2005). For example, the seasonality of the US dairy industry some sixty years ago has been described by Trebler and Harding (1947) as follows: “Considerable effort is being made to obtain a more even milk supply by better feeding during the winter and by better distribution of the calving season, but the industry is still highly seasonal, with 50 % higher average daily milk production during the late spring and early summer months when pastures are affordable for cheap feed.” As a consequence, the wastewater generated throughout the year varies in terms of quantity and composition, which makes the operation of individual anaerobic treatment plants harder. One solution to this problem is co-digestion, a topic discussed later in section 4.8. The problem of seasonal milk production and consequently processing has not been fully resolved yet. In Greece, for example, sheep milk is processed only from October to June (Gelegenis et al., 2007). In another publication concerning South-Western Greece (Gavala et al., 1996), it was reported that dairy wastewater is produced from January to June only. Danalewich et al. (1998) conducted a survey of fifteen milk processing plants (twelve produced one or more types of cheese), chosen to be representative for the dairy industry in the Upper Midwest of the U.S. Most plants reported large hourly, daily, and seasonal fluctuations in wastewater flow rates. Minimum wastewater flow rates ranged from 4 to 1703 m3 day-1 and maximum wastewater flow rates varied from 257 to 2650 m3 d-1. High seasonal variations correlated with the volume of milk received for processing, which typically is high during summer months and low during winter months (Danalewich et al., 1998). In New Zealand, waste load to the treatment process reflects the seasonal production pattern of the Southern Hemisphere, which commences in August and peaks rapidly by late September, declining gradually through February to tail out in April. The winter period is normally dormant (Hamilton and Archer, 2007). 4.7. Separation of acidogenic and methanogenic stages In order to accelerate and optimise the acidogenic and methanogenic processes AD may be carried out in two physically separate, individually controlled (e.g. pH, T, HRT) reactors. The following section provides a brief overview of research activities in the field of anaerobic cheese whey digestion, while a more general review and discussion of two-stage anaerobic digestion is provided by van Lier et al. (2001), Demirel and Yenigün (2002) and Ke et al. (2005). Two-stage reactors as defined above are sometimes referred to as two- phase, an ambiguous term since phase may not only refer to a mix of two or more immiscible liquids or materials but also the state of matter (i.e. gaseous, liquid, solid). Consequently, stage will be preferred over phase in the present Biogas production from cheese whey: Past, present and future 63 context. Two-stage aerobic-anaerobic or anaerobic-aerobic treatment systems for cheese whey effluent, as studied by Sewards and Holden (1975), Lo and Liao (1989), Germirli et al. (1993), Malaspina et al. (1995), Monroy et al. (1995), Kosseva et al. (2003) and Frigon et al. (2007), Garcia et al. (2007), are also excluded from the following discussion as these systems deal with different aspects of the process. Staging may be done by operating various high rate anaerobic reactors in series such as AnRBC-AnRBC (Lo and Liao, 1986a,b), CSTR – AnRBC (Lo and Liao, 1988), CSTR - upflow anaerobic filter (UAF) (Yilmazer and Yenigün, 1999), septic tank – CSTR (Ghaly, 2000), CSTR – CSTR (Malaspina et al., 1996; Saddoud et al., 2007), UASB – UASB (Cohen et al., 1994; Ergüder et al., 2001), and AFBR – AFBR (Hickey and Owens, 1981). Other combinations such as CSTR – UASB, CSTR - AFBR, and anaerobic packed bed reactor (APBR) have not been reported to the best of our knowledge. Studying methane generation from full-strength, acidic cheese whey in AFBRs, Hickey and Owens (1981) found that methane yield (0.363 L CH4 kg-1 COD fed) and total COD removal efficiencies (84 %) were higher in two reactors operating in series than in one reactor at the same loading (13.4 g COD L-1 day-1), but pH control was necessary in their digestion process. Lo and Liao (1986b) studied a two-stage digestion of full-strength cheddar cheese whey using two AnRBCs. The second-stage reactor receiving partially treated effluent from the first-stage reactor (HRT 6 days; COD reduction 76 %; 2.6 L CH4 L-1 d-1) could be operated at a HRT of one day with a further COD reduction of 18 % and biogas production rate of 1 L CH4 L-1 d-1. Some kind of pH control for the second reactor was indicated but not specified by the authors. In another study, Lo et al. (1988) coupled a CSTR with an AnRBC to produce biogas under mesophilic conditions from full-strength cheddar cheese whey. Acidogenic pretreatment at a HRT of 1.1 d prior to the methanogenic stage (HRT 5 d) resulted in an overall increase in methane yield (0.469 L CH4 g VSS-1) and higher treatment efficiency in terms of COD reduction (87 %) over the one-stage control AnRBC reactor (HRT 6 d; 0.324 L CH4 VSS-1 and 78 %, respectively). Overall methane production rate, on the other hand, remained fairly constant in both systems (3.1 L CH4 L-1 d-1). During two-stage operation, the acidogenic reactor produced VFAs at following concentrations: 300 mg L-1 acetic acid (52 %), 207 mg L-1 propionic acid and 41 mg L-1 butyric acid. Another advantage of the CSTR – AnRBC system is that stable operation could be achieved without buffer addition. Ghaly et al. (2000) investigated the effect of re-seeding and sodium bicarbonate on the quality and quantity of biogas production as well as the pollution potential reduction of a mesophilic, two-stage, septic tank – CSTR configuration (HRT 15 days), treating full-strength acid cheese whey. The results indicated that operating the digester without pH control resulted in a 64 R.T. Bachmann et al. low pH (3.3) which irreversibly inhibited the methanogenic bacteria. Only when the system was reseeded and the pH controlled, the biogas production resumed (1.63 m3 m-3 d-1) with a methane content of 52 %. Total and soluble COD reduction in the order of 59 and 65 % could be achieved. The addition of sodium bicarbonate resulted in a total alkalinity of 8230 mg L-1 as CaCO3. Malaspina et al. (1996) coupled two CSTR reactors to treat full-strength cheese whey in effluent recycle mode. The system was operated at OLRs of up to 3 g COD L-1 d-1 with overall COD removal efficiencies greater 90 %. Methane production averaged 290 mL CH4 g-1 CODin. The pH in the acidogenic reactor fluctuated around pH 6.5 with VFA concentrations measured between 4 – 8 g L-1 (acetic acid prevalent), while the pH in the methanogenic reactor was pH 7.5. No external alkalinity was added. Two- stage mesophilic anaerobic digestion of full-strength acidic cheese whey was also investigated by Saddoud et al. (2007) in a CSTR – CSTR system, with the methanogenic reactor coupled to a membrane filtration system to enable removal of soluble effluent whilst retaining solids. The acidogenic reactor was operated at a HRT of one day, whilst the methanogenic reactor received an organic load up to 19.8 g COD L-1 d-1, corresponding to a HRT of 4 days. Average COD removal in the two-stage system was 98.5 %, with a daily biogas production exceeding 10 times reactor volume and biogas methane content in excess of 70 %. During two-stage operation, the acidogenic reactor produced VFAs at following concentrations: 3.2 g L-1 acetic acid (63.7 %) and 24.7 % propionic acid. 18 g L-1 lactic acid was also produced. The two-stage reactor was operated for 47 days without indications of an increase in cross membrane pressure drop. However, membrane (bio)fouling is a problem that is likely to arise during long-term operation (Cinar et al., 2006; Judd, 2008). Yilmazer and Yenigün (2007) studied the performance of a mesophilic CSTR – UAF system digesting medium-strength reconstituted cheese whey (20 g COD L-1). The cheese whey was supplemented with trace and macro- nutrients and buffered using ammonium bicarbonate and dipotassium hydrogen phosphate. The acidogenic reactor was operated at an optimum HRT of 1 day with OLRs ranging from 0.5 to 2 g COD MLSS-1 d-1. At 18 hours HRT, settling of acidogenic sludge became difficult. HRT of the UAF varied between 3 - 6 days for the optimum COD removal efficiency and biogas production. At an HRT of 4 days, a maximum 90 % soluble effluent COD removal efficiency was obtained with an outmost biogas yield of 0.55 m3 kg-1 COD removed. A two stage anaerobic substrate shuttle process (SSP), comprising of an acidogenic UASB connected to a balancing tank with filtration unit, and a methanogenic UASB receiving acidogenic whey effluent from the balancing tank via ion exchange units (IEU), was tested by Cohen et al. (1994). One IEU was operated in a load cycle, while the other was being regenerated. This was a Biogas production from cheese whey: Past, present and future 65 feasibility study, and thus performance data were not reported. While the SSP process was successfully operated over a period of 109 days, fluid flow in the IEUs and ion exchange capacity utilization were considered non-ideal requiring further optimization. Ergüder et al. (2001) operated two UASBs in series in order to treat full-strength cheese whey buffered with NaHCO3 (6 g L-1) and supplemented with nutrients. The first reactor was essentially operated as a single-stage unit, achieving COD removal efficiencies in excess of 92 % at OLRs up to 22.2 g COD L-1 d-1. pH in the effluent varied between 7 – 8, and VFA concentrations were below the detection limit (50 mg L-1). The second UASB reactor achieved a further COD reduction of up to 60 % at an OLR of 2.52 g COD L-1 d-1. From the above it is clear that two-stage reactors are amenable for anaerobic cheese whey digestion. Most configurations do not require buffer addition provided they are operated within a certain OLR range and/or in effluent return mode, which results in cost savings for chemicals. It should be noted that for some reactor set-ups such as the CSTR – UASB system (no literature data available for cheese whey digestion) experience has shown that pre-acidification of carbohydrate wastewater requires elaborate pH control prior to the methanogenic stage (granulation efficiency drops drastically in acidic wastewaters), as well as adverse effects of incoming SS on methanogenic granule formation (van Lier et al., 2001). Other reactor designs aiming at enhancing spatial biomass separation include integrated staged reactor concepts which vary from vertically oriented 3 to 5 stage reactors, to horizontally oriented baffled reactors with up to 8 stages (Van Lier et al., 1994; Guiot et al., 1995; Bachmann et al., 1985; Grobicki and Stuckey, 1991; Skiadas and Lyberatos, 1998). For more details refer to van Lier et al. (2001). 4.8. Co-digestion Co-digestion of cheese whey with other agro-industrial wastes may be motivated by economical and/or operational aspects. For example, the waste generated by a single cheese factory may not be sufficient or continuous throughout the year to ensure economic operation of the anaerobic digester (Gavala et al., 1996). In such cases, a centralized anaerobic waste treatment system co-digesting such seasonally produced wastewaters is believed to be economically more favorable, ensures stable year-round operation of a treatment plant, with the additional benefits of smaller capital costs and the exploitation of complementarity in waste characteristics (Trebler and Harding, 1947; Gavala et al., 1999) (refer to section 4.3). Nonetheless, anaerobic reactors digesting certain agro-industrial wastes such as manure from livestock are subject to various legislations, which can have a significant impact on the economics of the AD plant. For example, European legislation (EEC 1774/2002) requires that animal by-products from healthy animals approved for human 66 R.T. Bachmann et al. consumption (category 3) have to be treated at 70°C for 60 min before digestion (Bagge et al., 2005). A review on pathogen survival in biogas plants for in-depth information is available (Sahlström, 2003). The following section provides a brief summary of co-digestion experiments published so far. Desai et al. (1994) studied different ratios of cheese whey, poultry manure and cattle dung in a mesophilic, single stage anaerobic reactor at a HRT of 10 days, and achieved more stable operation and efficient COD reduction and biogas production compared to control experiments with individual wastes due to maintaining an optimum C/N ratio in the reactor. Best results in terms of gas production and methane content were obtained when the three wastes were used in a ratio of 3 : 1 : 2 (w/w) on a dry weight basis. Gelegenis et al. (2007) studied the co-digestion of full-strength cheese whey with poultry manure at ratios varying from 1 : 5.6 to 1 : 1 (v/v) in a mesophilic CSTR. The HRT was fixed at 20 d, while the OLR varied slightly (4.5 to 5.0 g COD L-1 d-1) due to the higher COD value of poultry manure. The authors established that optimum reactor performance in terms of biogas production without external buffering could be achieved at a whey to poultry ratio of 1 : 1.86 (v/v). Whey-to-poultry ratios of 1 : 1 resulted in a drastic decline in biogas production, and was attributed to the lower alkalinity and NH4 content. Lo et al. (1988) established that co-digestion of cheddar cheese whey with dairy manure in a mesophilic, single stage AnRBC reactor provides the necessary nutrients and buffer capacity to operate the AnBCR at an HRT as low as 2 days and an influent concentration as high as 32 g COD L-1. Martinez-Garcia et al. (2007) studied the mesophilic, co-digestion of heat-sterilized, pretreated olive mill waste with cheese whey (ratio 3 : 1) in an anaerobic fixed bed reactor. The two-stage system operated satisfactorily up to an OLR of 3.0 kg COD L-1 day-1 with a biogas production rate of 1.25 L L-1 day-1, a methane content between 68 and 75 %, and a total COD reduction in excess of 93 % (62 % COD reduction in aerobic pretreatment and 83 % COD reduction in anaerobic digestion). In summary, co-digestion of cheese whey with various agro-industrial wastes has not been studied extensively. Based on a limited number of studies available, single and two-stage reactors were found to be technically feasible. The buffer capacity provided by the complimentary feed substrate was sufficient to operate single stage reactors. So far, no study has been conducted to study the effect of heat-sterilisation, as required by EEC17174/2002, on the chemical composition and thus digestibility of cheese whey and co-substrate, and should therefore be given further attention. 5. Case studies A summary of anaerobic digesters working at industrial scale all over the world is provided in Table 5 and briefly discussed in the following sections. Table 5. Summary of biogas digesters treating cheese whey at industrial scale (all under mesophilic conditions). Biogas production from cheese whey: Past, present and future 67 68 R.T. Bachmann et al. 5.1. Canada Guiot et al. (1995) reported that a 450 m3 MPAR reactor was set-up at the Nutrinor (Lactel Group) cheese factory (Chambord, Quebec, Canada) to treat whey permeate and dairy wastewater in a 1: 1 volume ratio. Construction started in August 1991 and, after some further modifications and improvements, start-up took place in April 1992 and by June continuous operation was underway. It is not known whether the plant is still in operation. 5.2. Germany After an extension of production at the Naabtaler Milchwerke Bechtel OHG, (Schwarzenfeld, Germany), the factory produced up to 1100 m³ wastewater daily. The existing WWTP was extended with an anaerobic Biomar® AFB reactor featuring a small footprint, low operation cost as well as gain of biogas and excess sludge reduction. Wastewater is collected in a flow equalization tank, sieved and pumped to the preacidification and buffering unit. Before the preacidified wastewater enters the Biomar® AFB reactor the flotation separates fats and cheese particles. After biomethanization, effluent is polished in the existing aerobic waste water treatment unit. The biogas is separated and used in a high-speed steam generator. (EnvironChemie, 2008). 5.3. Ireland Carbery Milk Products, based in Ballineen, Co. (Cork, Ireland) processes and manufactures a wide range of food products, including cheese, whey protein, yeast extracts, flavour enhancers and, uniquely ethanol, produced by the fermentation of lactose (McHugh et al., 2003). Currently, annual milk volumes processed are in excess of 320,000 m3. The wastewater produced at Carbery Milk Products is treated using a Biopaq® – IC reactor consisting of a vertical 653 m3 reactor (6.5 m x 20 m), and a recirculation tank. The digester is operated at an average OLR of 23 g L-1 d-1. 5.4. Italy In Jesi WWTP (60,000 PE), activated sludge is gravitationally thickened and sent to a 1500 m3 anaerobic digester, where it is co-digested with liquid organic wastes such as cheese whey and residuals from olive oil making factories. Afterwards, the digested sludge is post-thickened in a gravitational tank, dewatered by belt-press and disposed of in landfills (Bolzonella et al., 2006). 5.5. Mexico The plant of "El Sauz" at Cortazar in the state of Guanajuato produces an average of 1500 tons of milk derivatives per month and 500 m3 of wastewater Biogas production from cheese whey: Past, present and future 69 per day. Several parameters (BOD5, COD, fat and grease, settleable solids, nitrogen and phosphorus) were not within the consents or particular discharge conditions. The new system consisted of: a grease trap, an anaerobic UASB type pond, an aerated pond and an effluent polishing pond with water hyacinth. The project was done at a low capital investment (US-$ 330,000) and operational costs (US-$ 0.55 m-3) which were competitive with current market prices as well as within the official environmental consents. 5.6. New Zealand The Fonterra Tirau Casein Complex is situated in the Waikato district of the North Island of New Zealand. Wholemilk is processed to produce casein, whey proteins and ethanol. The biogas plant has been in operation since 1983 features a floating XR5 membrane cover to a 26,000 m3 anaerobic lagoon and a lamella plate clarifier to separate and return anaerobic biomass, followed by an aerobic lagoon and secondary clarifiers. Two tonnes ferrous sulphate heptahydrate are added every 3 days as trace element supplement along with up to 7 tonnes of lime per day for pH control. The relatively high dosage of lime also removes most phosphorus, which is required for the discharge into the freshwater stream. In excess of 90 % BOD removal is achieved by the anaerobic contact process. Biogas is recovered for use in a boiler producing steam used in the factory (Hamilton and Archer, 2007). 5.7. Sweden Disposal and utilisation of cheese whey has long been a problem for Norrmejerier, a Swedish dairy association. The whey has been concentrated into whey powder by evaporation or sold as pig food to local farmers with the latter causing considerable handling and transportation costs. In addition, pig farming is not very common, and there are simply not enough pig farmers in Norrmejerier’s region of activities to find an outlet for the whey produced. This is not a problem for other dairies in Sweden and has therefore been a competitive disadvantage for Norrmejerier (Asplund, 2005). At the same time, consumption of low-refined dairy products such as milk, cream, soured milk etc. is decreasing in Sweden. To make use of the surplus of raw milk produced by milk farmers the cheese production at Norrmejerier was increased leading to the production of even more whey. It was considered to produce whey powder as an additive at dairies and other food industries. However, the evaporation into powder is a highly energy demanding process consuming up to 40 % of the total energy requirements of the Umeå Dairy. Further investments in evaporation equipment have therefore not been an option. In order to enhance the environmental-friendly profile of the association and to reduce the problems with whey and waste disposal, an anaerobic wastewater treatment plant has been planned and installed. In this plant 70 R.T. Bachmann et al. wastewater and residual milk from Umeå Dairy is treated together with whey permeate from both Umeå and Burträsk Dairy. The biogas produced in this plant is used to produce steam. The project (Biotrans) was partially funded by the County Administration of Västerbotten and EU’s LIFE program. 5.8. USA Fairview Swiss Cheese, Rochester The US-$ 2.2 million project is a collaborative effort between the local county government, Fairview Swiss Cheese Plant, Joy Cone Co., and Penn State Cooperative Extension of Mercer County. It involves constructing an anaerobic digester that will use cheese whey from the plant and cone batter waste to generate 1 million m3 of biogas p.a.. The biogas will be used in a boiler to produce steam and electricity for processing milk into cheese that in turn will offset the purchase of fuel oil and electricity produced from fossil fuels. The wastewater from the digester will flow to a treatment facility where the solids will be removed and clean water discharged. The joint project is funding through the Department of Agriculture’s Machinery and Equipment Loan Fund, the Department of Environmental Protection’s Energy Harvest Grant and USDA’s 9006 Renewable Energy Grant, combined with private financing from John Koller & Son Inc. The Renewable Energy Systems and Energy Efficiency Improvements program was set up to create economic opportunities and reduce energy costs for farmers and agribusinesses in rural communities. The plant is still under construction. Kraft Foods - Campbell, New York Kraft Foods manufacture cheese since 2003 at their Campbell, NY cheese making facility. The installation and operation of Ecovation’s patented Mobilized Film Technology (MFT) was completed under a design-build- finance-operate (DBFO) contract. Ecovation’s treatment solution eliminated previous discharge compliance issues, odor complaints, and trucking of waste byproducts. The system also provided pretreatment for process wastewater that significantly reduced the loading to existing on-site aeration lagoons. Recently, the MFT treatment facility has undergone a five-fold expansion to treat high-strength whey permeate. The expanded MFT system was designed to convert all of the production plant’s soluble waste byproducts into biogas and treated water. The biogas is utilized in the production plant to offset 30 % of its natural gas consumption. Kraft Foods - Lowville, NY Another Kraft Foods cheese making facility in the state of New Year generates high-strength low protein whey and UF permeate, which was used to generate biogas in an MFT ® reactor. The biogas produced helps to offset the Biogas production from cheese whey: Past, present and future 71 use of natural gas in the manufacturing plant as well as eliminates the current costly disposal practices – such as animal feed and land application. Breyers Yogurt - North Lawrence, NY The manufacturing facility is located in rural northern New York and relies on No. 6 fuel oil and electricity as its main energy sources. The project was partially funded by a US-$ 500,000 USDA rural development grant. The facility was designed, built, and is currently operated by Ecovation under a long-term agreement. The MFT ® technology generates biogas from acidic cheese whey, yogurt slop and wastes from other dairies in the area, providing 25 % of the plant’s total steam requirement. 6. Future of cheese whey biomethanation Cheese whey is a potential resource for the production of biofuels such as biogas, rich in methane or hydrogen (Ergüder et al., 2001; Ferchichi et al., 2005; Krich et al., 2005; Kapdan and Kargi, 2006; Yang et al., 2007). Nevertheless, this practice is still not common in the dairy industries. Small- to medium-size cheese manufacturers are the most-likely benefactors of this technology and should be convinced of the financial advantages of producing biogas as an alternative disposal mean. The advent of ongoing industrial applications will deliver valuable data (DEP, 2007), which in turn will be utilized by decision makers and practitioners to deliver convincing facts and figures. Producers will therefore abandon the a priori solution of utilizing this substrate as animal feed or paying a fee for land application (Fehrs, 2000). However, more data are required to demonstrate that cheese whey is a financially attractive substrate for biogas production in different geographic locations. A holistic approach could help to understand the interactions of this practice on the environment. 6.1. Feasibility A cost-driven society requires that commercial industrialized processes be financially self-sustained. Biogas production from cheese whey is therefore dependent on current economical, technical, environmental and social parameters. These and other possible drivers need to be explored to consider cheese whey biomethanization as a financial strategy (Svensson et al., 2006). There are few local data sets that reflect the availability and potential energy production from anaerobic digestion of cheese whey. In Vermont (USA), 40 % of cheese whey is potentially available for energy production and represents 3 % of the total energy potential (30 MW per year) obtained from dairy industries (Fehrs, 2000). In California, the estimated potential of methane generation from cheese whey is 7 million Ft3 per year (Krich et al., 2005). More 72 R.T. Bachmann et al. data is expected from Pennsylvania (USA) where cheese whey and cone batter will be digested to produce approximately 1 million m3 of biogas per year (DEP, 2007). The results obtained in this large scale process will be helpful for optimization of the digestion process, improvements on both the post digestion processes and storage issues. There is no recent, comprehensive and accessible data on cheese whey stock. Indeed, worldwide figures are unknown. The estimates are based on the production of cheese and the cheese whey utilized by other markets (food, pharmaceuticals, etc.). Data are needed to determine if cheese whey biomethanization will allow the full utilization of both capacity of the equipment installed and energy carriers produced (Svensson et al., 2006). Consequently, it is essential to create a database for energy potential and fertilizer (compost) recovery from cheese whey biomethanization in different locations. A Geographic Information System (GIS) to assess cheese whey availability can be useful. Thus, there will be a better understanding of cheese whey production and availability of energy as biogas in different geographic locations and periods of time. The data will allow for predictions and the fulfillment of a variety of considerations regarding to characterization, finance, stakeholders’ objectives and regulations. It is also necessary to evaluate the selection of cheese whey as a substrate, or co-substrate, in combination with other well-recognized parameters like scale and utilization rate (Svensson et al., 2006). Market entry issues for the products (energy and compost) and emission credits for digesting cheese whey need to be studied. Specially, an assessment should be carried out if there is an interest in upgrading the quality of the biogas to be distributed in the low pressure national natural gas pipelines. In general, the information obtained will be helpful to determine the on-site production of biogas, cooperatives or in future centralized plants. 6.2. Environmental impact Cheese whey can be used as feedstock but the issues of its utilization and post-consumer waste should be understood even if whey is considered to be safe for humans. At present, EU guidelines on emission control (VDI, 2007) only apply for biogas facilities digesting agricultural waste. However, obviousness dictates that plants biomethanizing cheese whey will not be an exception. The issue needs therefore attention because environmental regulations are a product of political discussions. This has economical consequences on costs, which are influenced by the treatment efficiency and requirements on public or private responsibility. There is scarce knowledge about the risks and hazards, towards the environment, derived from cheese whey biomethanization. Emission control in this practice is poorly understood (odour, gaseous pollutants, dust, bioaerosols Biogas production from cheese whey: Past, present and future 73 and fermentation residues). Moreover, there is no knowledge about contamination of the digestate with pathogens similar to those found in other anaerobic processes such as Salmonella spp. and other microorganisms (Sahlstrom, 2003; Bagge et al., 2005). A management of the outputs is therefore required. Ecotoxicological consequences are unknown, e.g. biogenesis of more toxic chemicals and negative effects on microbial ecosystems. On the other hand, nutrient management of discharged digestate requires attention to prevent eutrophication and other potential disruptions in trophic chains. In addition to this, life cycle and carbon footprint assessments are needed. Removal of nitrogen and phosphorus from dairy wastewaters is gaining increasing attention, due to more strict environmental regulations (Tilche et al., 1996; Danalewich et al., 1998; Demirel et al., 2005; Bolzonella et al., 2006). As the regulations for discharge of nutrients become stricter in time, new modifications in existing treatment plants will have to be tested and eventually implemented. 6.3. Cooperation and promotion Likewise to other anaerobic digestion plants, cooperation is required; for instance, the cooperation of agro-industries, government and national research institutes as well as providers of technology, operators and clients of digestion products. Previous experiences and cooperation will allow to produce, upgrade and utilize biogas as a source of energy; compost as fertilizer and anaerobic digestion as a link in the chain of cheese whey waste treatment. Consequently, decision makers will be able to choose the best technologies and options to promote the digestion products. Industries paying for cheese whey disposal should be convinced of the economical advantage of producing biogas from this wasted product. They also need to know that they are not limited to sell methane and fertilizer products. Industries can also be rewarded with emission credits, renewable energy certificates, and potential tax credits. For instance, it would be the case in a country or city committed to increase its renewable energy capacity (Kyoto protocol) and to satisfy tightening regulations (Grommen and Verstraete, 2002). References 1. Anon. (1990) Anaerobic treatment of dairy effluents. Bull. Int. Dairy Fed., Vol. 252, pp. 3-23. 2. Arvanitoyannis, I. and Giakoundis, A. (2006) Current strategies for dairy waste management: A review. Critical Reviews in Food Science and Nutrition, Vol. 46(5), pp. 379-390. 74 R.T. Bachmann et al. 3. Asplund, S. (2005) The Biogas Production Plant at Umeå Dairy Evaluation of Design and Start-up. Linköping University Electronic Press. Thesis. 4. Bachmann, R.T. and Edyvean, R.G.J. (2005) Biofouling: an historic and contemporary review of its causes, consequences and control in drinking water distribution systems. Biofilms J., Vol. 2(3), pp. 197–227. 5. Backus, B.D., Clanton, C.J., Goodrich, P.R., and Morris, H.A. (1988) Carbon-to- nitrogen ratio and hydraulic retention time effect on the anaerobic digestion of cheese whey. Trans. ASAE, Vol. 31(4), pp. 1274-1282. 6. Bagge, E., Sahlström, L. and Albihn, A. (2005) The effect of hygienic treatment on the microbial flora of biowaste at biogas plants. Water Research, Vol. 39(20), pp. 4879-4886. 7. Barford, J.P., Cail, R.G., Callander, I.J., and Floyd, E.J. (1986) Anaerobic digestion of high-strength cheese whey utilizing semi-continuous digesters and chemical flocculant addition. Biotech. Bioeng., Vol. 28(11), pp. 1601-1607. 8. Boening, P.H. and Larsen, V.F. (1982) Anaerobic fluidized bed whey treatment, Biotech. Bioeng., Vol. 14, pp. 2539-2556. 9. Bolzonella, D., Pavan, P., Battistoni, P., Cecchi, F. (2006) Anaerobic co-digestion of sludge with other organic wastes and phosphorus reclamation in wastewater treatment plants for biological nutrients removal. Wat. Sci. Tech., Vol. 53(12), pp. 177-186. 10. Bonastre, N. & Paris, J. M. (1989) Survey of laboratory, pilot, and industrial anaerobic filter installations. Proc. Biochem., 24, 15-20. 11. Borja, R., Martin, A., Duran, M.M., and Luque, M. (1992) Kinetic study of the anaerobic purification of a cheese factory wastewater. Rev. Esp. Ciencia y Tecnol., Vol. 32(1), pp. 19-32 12. Borja, R., Duran, M.M., Martin, A., Luque, M., and Alonso, V. (1994) Influence of immobilization supports on the kinetic constants anaerobic digestion of cheese whey. Res. Conservat. Recycl., Vol. 10(4), pp. 329-339. 13. Buswell, A.M., Boruff, S. and Wiesman, C.K. (1932) Anaerobic Stabilization of Milk Waste. Ind. Chem. Eng., Vol. 24(12), pp. 1423-1425. 14. Callander, I.J. and Barford, J.P. (1983) Cheese whey anaerobic digestion - Effect of chemical flocculant addition. Biotech. Letters, Vol. 5(3), pp. 153-158. 15. Carrieri, C., Dipinto, A.C., Rozzi, A., and Santori, M. (1993) Anaerobic co- digestion of sewage sludge and concentrated soluble wastewaters. Wat. Sci. Tech., Vol. 28(2), pp. 187-197. 16. Cheng, S. and Logan, B. (2007) Sustainable and efficient biohydrogen production via electrohydrogenesis. PNAS, Vol. 104(47), pp. 18871–18873. 17. Clark, J. N. (1988) Utilization of acid and sweet wheys in a pilot-scale upflow anaerobic sludge blanket digester. N.Z. J. Dairy Sci. Technol., Vol. 23, pp. 305-27. 18. Cohen et al. (1994) Pilot-scale anaerobic treatment of cheese whey by the substrate shuttle process. Wat. Sci. Tech., Vol. 30(12), pp. 433-442. 19. Danalewich, J.R., Papagiannis, T.G., Belyea, R.L., Tumbleson, M.E., Raskin, L. (1998) Characterization of dairy waste streams, current treatment practices, and potential for biological nutrient removal. Water Res; Vol. 32, pp. 3555–68. 20. De Haast, J., Britz, T.J., Novello, J.C., and Lategan, P.M. (1983) Anaerobic digestion of cheese whey using a stationary fixed-bed reactor. New Zealand J. Dairy Sci. Tech., Vol. 18(3), pp. 261-271. Biogas production from cheese whey: Past, present and future 75 21. De Haast, J., Britz, T.J., Novello, J.C., and Verwey, E.W. (1985) Anaerobic digestion of deproteinated cheese whey. J. Dairy Res., Vol. 52(3), pp. 457-467. 22. Demirel, B., Orhan Yenigün, Turgut T. Onay (2005) Anaerobic treatment of dairy wastewaters: a review. Process Biochemistry 40, 2583–2595. 23. Denac, M., Dunn, I.J. (1988) Packed and fluidized-bed biofilm reactor performance for anaerobic waste-water treatment, Biotech. Bioeng., Vol. 32(2), pp. 159-173. 24. DEP (2007) Mercer County Cheese Plant Breaks Ground on Biogas Facility [Data file] Retrieved 09 January 2008, from site, www.depweb.state.pa.us/news/ 25. Desai, M. and Madamwar, D. (1994) Anaerobic digestion of a mixture of cheese whey, poultry waste and cattle dung - A study of the use of adsorbents to improve digester performance. Env. Poll., Vol. 86(3), pp. 337-340. 26. Desai, M., Patel, V., and Madamwar, D. (1994) Effect of temperature and retention time on biomethanation of cheese whey, poultry waste and cattle dung. Env. Poll., Vol. 83(3), pp. 311-315. 27. Desai, M. and Madamwar, D. (1994) Surfactants in anaerobic digestion of cheese whey, poultry waste, and cattle dung for improved biomethanation. Transactions of the ASAE, Vol. 37(3), pp. 959-962. 28. Dewes, E. (1960) Experience and results with a new anaerobic biological process for treatment of certain polluted industrial wastewaters. Ber. Abwass. Techn. Verein, Vol. 121, p. 169. 29. Ecovation (2008) http://www.ecovation.com/. Accessed on 12/1/2008 30. Elmamouni, R., Guiot, S.R., Mercier, P., Safi, B., Samson, R. (1995) Liming impact on granules activity of the multiplate anaerobic reactor (MPAR) treating whey permeate. Bioproc. Eng., Vol. 12(1-2), pp. 47-53. 31. Ergüder, T.H., Tezel, U., Guven, E., Demirer, G.N. (2001) Anaerobic biotransformation and methane generation potential of cheese whey in batch and UASB reactors; Waste Management, Vol. 21(7), pp. 643-650. 32. EnvironChemie (2008), accessed on 23/01/2008: ttp://www.envirochemie.com/Milk-and-cheese-Naabtaler.332.0.html?&L=1 33. FAO. (1998) Cheese production. In Food and Agriculture Organization Yearbook 51. Rome, Italy: FAO, UN. 34. Fehrs, J. E. (2000) Vermont methane pilot project: Resource assessment. Vermont Department of Public Service and Vermont Department of Agriculture, Food and Markets. Report number: 35. Ferchichi, M., Crabbe, E., Gil, G.-H., Hintz, W. and Almadidy, A. (2005) Influence of initial pH on hydrogen production from cheese whey. J. Biotechnology, 120(4), 402-409. 36. Fitzmaurice, J.R., Archer, H.E. & Fullerton, R.W. (1987) Anaerobic contact wastewater treatment, Tirau casein complex - N.Z. Co-operative Dairy Company Ltd., Trans. Inst. Prof. Engng N.Z., Vol. 14, pp. 73-84. 37. Fournier, D., Schwitzguebel, J.P. and Peringer, P. (1993) Effect of different heterogeneous inocula in acidogenic fermentation of whey permeate. Biotech. Letters, Vol. 15(6), pp. 627-632. 38. Gannoun, H., Khelifi, E., Bouallagui, H., Touhami, Y., Hamdi, M. (2008) Ecological clarification of cheese whey prior to anaerobic digestion in upflow anaerobic filter, Bioresource Technology. doi:10.1016/j.biortech.2007.12.037. 76 R.T. Bachmann et al. 39. Gavala, H.N., Kopsinis, H., Skiadas, I.V., Stamatelatou, K., Lyberatos, G. (1999) Treatment of dairy wastewater using an upflow anaerobic sludge blanket reactor, J. Agric. Engng Res. 73, 59-63. 40. Gelegenis, J., Georgakakis, D., Angelidaki, I., and Mavris, V. (2007) Optimization of biogas production by co-digesting whey with diluted poultry manure; Renewable Energy, Vol. 32, pp. 2147–2160. 41. Ghaly, A.E. (1996) A comparative study of anaerobic digestion of acid cheese whey and dairy manure in a two-stage reactor. Biores. Tech., Vol. 58(1), pp. 61-72. 42. Ghaly, A.E. and Pyke, J.B. (1991) Amelioration of methane yield in cheese whey fermentation by controlling the pH of the methanogenic stage. Appl. Biochem. Biotech., Vol. 27(3), pp. 217-237. 43. Göblös, S., Portörö, P., Bordás, D., Kálmán, M., and Kiss, I. (2008) Comparison of the effectivities of two-phase and single-phase anaerobic sequencing batch reactors during dairy wastewater treatment; Renew. Energy, Vol. 33(5), pp. 960-965. 44. Gonzalez Siso, M.I. (1996) The biotechnological utilization of cheese whey: a review. Bioresource Technology, Vol. 57, pp. 1-11. 45. Grommen, R. and Verstraete, W. (2002) Environmental biotechnology: the ongoing quest. J. Biotechnology, Vol. 98(1), pp. 113-123. 46. Guiot, S.R., Safi, B., Frigon, J.C., Mercier, P., Mulligan, P., Tremblay, R., and Samson, R. (1995) Performances of a full-scale novel multi-plate anaerobic reactor treating cheese whey effluent. Biotech. Bioeng., Vol. 45(5), pp. 398-405. 47. Gutierrez, J.L.R., Encina, P.A.G., and Polanco, F.F. (1991) Anaerobic treatment of cheese production wastewater using a UASB reactor. Biores. Tech., Vol. 37, pp. 271-276. 48. Hickey, R.E. and Owens, R.W. (1981). Methane generation from high-strength industrial wastes with the anaerobic biological fluidized bed. Biotechnology and Bioengineering Symp., 11, 399-413. 49. Hobman, P.G. (1984) Review of processes and products for utilization of lactose in deproteinated milk serum. J. Dairy Sci., Vol. 67, pp. 2630-53. 50. Hulshoff, P. and Lettinga, G. (1986) New technologies for anaerobic wastewater treatment, Wat. Sci. Tech., Vol. 18(12), pp. 41-53. 51. Hwang, S.H., Hansen, C.L., and Stevens, D.K. (1992) Biokinetics of an upflow anaerobic sludge blanket reactor treating whey permeate. Biores. Tech., Vol. 41(3), pp. 223-230. 52. Hwang, S.H. and Hansen, C.L. (1992) Performance of upflow anaerobic sludge blanket (UASB) reactor treating whey permeate. Transaction of the ASAE, Vol. 35(5), pp. 1665-1671. 53. IRC (2008) Recovery and valorisation as feed of cheese whey (Ref: TGE - TO TR1) [Data file] Retrieved 22 January 2008, from site, http://www.innovationrelay.net 54. Kalyuzhnyi, S.V., Perez Martinez, E., and Rodriguez Martinez, J. (1997) Anaerobic treatment of high-strength cheese-whey wastewaters in laboratory and pilot UASB-reactors; Biores. Tech., Vol. 60, pp. 59-65. 55. Kapdan, I. K. and Kargi, F. (2006) Bio-hydrogen production from waste materials. Enzyme and Microbial Technology, 38(5), 569-582. 56. Ke, S., Shi, Z., Fang, H.H.P. (2005) Applications of two-phase anaerobic degradation in industrial wastewater treatment. Internat. J. Environ. Poll., Vol. 23(1), pp. 65-80. Biogas production from cheese whey: Past, present and future 77 57. Kennedy, J.L. and Droste, R.L. (1985) Startup of anaerobic downflow stationary fixed film (DSFF) reactors. Biotech. Bioeng., Vol. 27, pp. 1152-1165. 58. Kissalita, W.S., Lo, K.V., and Pinder, K.L. (1989) Influence of dilution rate on the acidogenic phase products distribution during two-phase lactose anaerobiosis Biotechnol. Bioeng., 34, 1235. 59. Kosaric, N. and Asher, Y. J. (1985) The utilization of cheese whey and its components. Adv. Biochem. Engng, Vol. 32, pp. 25-60. 60. Krich, K., Augenstein, D., Batmale, J., Benemann, J., Rutledge, B. and Salour, D. (2005) Biomethane from dairy waste: A sourcebook for the production and use of renewable natural gas in California. Sustainable Conservation 61. Lo, K.V. and Liao, P.H. (1986) 2-stage anaerobic digestion of cheese whey. Biomass, Vol. 10(4), pp. 319-322. 62. Lo, K.V., Liao, P.H. (1986) Digestion of cheese whey with anaerobic rotating biological contact reactors, Biomass, Vol. 10, pp. 243-252. 63. Lo, K.V., Liao, P.H., and Chiu, C. (1988) Laboratory scale studies on the mesophilic anaerobic digestion of cheese whey in different digester configurations. J. Agric. Eng. Res., Vol. 39 (2), pp. 99-105. 64. Lo, K.V., Liao, P.H., and Chiu, C. (1988) Mesophilic anaerobic digestion of a mixture of cheese whey and dairy manure. Biomass, Vol. 15(1), pp. 45-53. 65. Malaspina, F., Stante, L., Cellamare, C.M., Tilche, A. (1995) Cheese whey and cheese factory wastewater treatment with a biological anaerobic-aerobic process. Wat. Sci. Tech., Vol. 32(12), pp. 59-72. 66. Malaspina, F., Cellamare, C.M., Stante, L., Tilche, A. (1996) Anaerobic treatment of cheese whey with a downflow-upflow hybrid reactor, Bioresource Technology. 55, 131-139. 67. Marshall, D. and Timbers, G.E. (1982) Development and testing of a prototype fixed film anaerobic digester. Paper No. 82-6519, Winter Meeting ASAE, St Joseph, Michigan, 14-17 December. 68. Martinez-Garcia, G., Johnson, A.C., Bachmann, R.T., Williams, C.J., Burgoyne, A., Edyvean, R.G.J. (2007) Two-stage biological treatment of olive mill wastewater with whey as co-substrate. Int. Biodet. Biodeg., Vol. 59, pp. 273–282. 69. Marwaha, P. and Kennedy, J.F. (1988) Whey - pollution problem and potential utilization. Int. J. Food Sci. Tech., Vol. 23(4), pp 323-336. 70. Mawson, A.J. (1994) Bioconversions for whey utilization and waste abatement. Biores. Technol., Vol. 47(3), pp. 195-203. 71. McHugh, S., O’Reilly, C., Mahony, T., Colleran, E., O’Flaherty, V. (2003) Anaerobic granular sludge bioreactor technology. Rev. Env. Sci. Biotech., Vol. 2(2-4), 225-245. 72. Mendez, R., Blazquez, R., Lorenzo, F., and Lema, J. M. (1989). Anaerobic treatment of cheese whey: start-up and operation. Wat Sci. Tech., Vol. 21, pp. 1857-1860. 73. Mockaitis, G., Ratusznei, S.M., Rodrigues, J.A.D., Zaiat, M. and Foresti, E. (2006) Anaerobic whey treatment by a stirred sequencing batch reactor (ASBR): effects of organic loading and supplemented alkalinity; J. Env. Managem., Vol. 79, pp. 198–206. 74. Monroy, O.H., Vazquez, F.M., Derramadero, J.C., and Guyot, J.P. (1995) Anaerobic–aerobic treatment of cheese wastewater with national technology in Mexico: the case of ‘El Sauz’. Water Sci Technol; Vol. 32, pp. 149–156. 78 R.T. Bachmann et al. 75. Mudrak, K. and Kunst, S. (1986) In: Biology of Sewage Treatment and Water Pollution Control. England: Ellis Horwood Ltd, 1986. p. 193. 76. Murray, W.D. and van den Berg, L. (1981) Effect of support material on the development of microbial fixed films converting acetic acid to methane, J. Appl. Biotech., Vol. 51, pp. 257-265. 77. OECD (2008), accessed on 20/01/2008: http://www.oecd.org/dataoecd/19/33/4621291.xls 78. Patel, C. and Madamwar, D. (1994) Effect of metals on biomethanation of mixtures of cheese whey, poultry waste and cattle dung. Fres. Env. Bull., Vol. 3(10), pp. 604-609. 79. Patel, P., Desai, M., Madamwar, D. (1995) Biomethanation of cheese whey using anaerobic upflow fixed film reactor, Journal of Fermentation and Bioengineering. 79(4), 398–399. 80. Patel, C. and Madamwar, D. (1996) Biomethanation of a mixture of salty cheese whey and poultry waste or cattle dung - A study of effect of temperature and retention time. Appl. Biochem. Biotech., Vol. 60(2), pp. 159-166. 81. Patel, C., Sastry, V., Madamwar, D. (1996) Tegoprens in anaerobic digestion of a mixture of cheese whey, poultry waste, and cattle dung for improved biomethanation. Appl. Biochem. Biotech., Vol. 56(1), pp. 89-94. 82. Patel, C. and Madamwar, D. (1997) Biomethanation of salty cheese whey using an anaerobic biological contact reactor. J. Ferment. Bioeng., Vol. 83, pp. 502–504. 83. Patel, C., and Madamwar, D. (1997) Biomethanation of salty cheese whey using an anaerobic rotating biological contact reactor, J. Ferment. Bioeng., Vol. 83(5), pp. 502-504. 84. Patel, P. and Madamwar, D. (1998) Surfactants in anaerobic digestion of salty cheese whey using upflow fixed film reactor for improved biomethanation; Process Biochemistry, Vol. 33 (2), pp. 199-203. 85. Rajeshwari, K.V., Balakrishnan, M., Kansal, A., Kusum Lata, Kishore, V.V.N. (2000) State-of-the-art of anaerobic digestion technology for industrial wastewater treatment, Renewable. Sustainable Energy Reviews, Vol. 4, pp. 135-156. 86. Rodgers, M., Zhan, X.M., and Dolan, B. (2004) Mixing characteristics and whey wastewater treatment of a novel moving anaerobic biofilm reactor. J. Env. Sci. Heal. A, Vol. 39(8), pp. 2183-2193. 87. Rogers, A.A. (2008) Director Marketing Services, Ecovation, Inc. Personal communication. 88. Ryhiner, G.B., Heinzle, E., and Dunn, I.J. (1993) Modeling and simulation of anaerobic wastewater treatment and its application to control design - case whey. Biotech. Progr., Vol. 9(3), pp. 332-343. 89. Saddoud, A., Hassairi, I., and Sayadi, S. (2007) Anaerobic membrane reactor with phase separation for the treatment of cheese whey; Bioresource Technology, Vol. 98, pp. 2102–2108. 90. Sahlström, L. (2003) A review of survival of pathogenic bacteria in organic waste used in biogas plants. Bioresource Technology, Vol. 87(2), pp. 161-166. 91. Sandbach, D. M. L. (1981) Production of potable grade alcohol from whey. Cult. Dairy Prod. J., 16(4), pp. 17-19. 92. Schroder, E.W. and De Haast, J. (1989) Anaerobic digestion of deproteinated cheese whey in an upflow sludge blanket reactor. Dairy Res., Vol. 56(1), pp. 129-139. Biogas production from cheese whey: Past, present and future 79 93. Sewards, G.J. and HOLDEN, G.A. (1975) Disposal of dairy whey by stage biological treatment. I. Kinetic studies of the aerobic stage. Water Research, Vol. 9(4), pp. 409-416. 94. Short, J.L. (1978) Prospects for the use of deproteinated whey in New Zealand - a review. N.Z.J. Dairy Sci. TechnoL, Vol. 13, pp. 181-194. 95. Speece, R.E. (1983) Anaerobic biotechnology for industrial wastewater treatment. Env. Sci. Technol., Vol. 17(9), pp. 416A-427A. 96. Streicher, C., Milande, N., Capdeville, B., Roques, H. (1992) Improvement of anaerobic digestion of diluted whey in a fluidized bed by nutrient additions. Env. Tech., Vol. 12(4), pp. 333-341. 97. Strydom, J.P., Britz, T.J., and Mostert, J.F. (1997) Two-phase anaerobic digestion of three different dairy effluents using a hybrid bioreactor. Water S A; 23(2):151-6. 98. Svensson, L., Christensson, K. and Björnsson, L. (2006) Biogas production from crop residues on a farm-scale level in Sweden: scale, choice of substrate and utilisation rate most important parameters for financial feasibility. Bioprocess and Biosystems Engineering, 29(2), 137-142. 99. Switzenbaum, M.S. and Danskin, S.C. (1982) Anaerobic expanded bed treatment of whey. Agric Waste; Vol. 4, pp. 411-426. 100. Symons, G. E. and Buswell, A. M. (1933) The methane fermentation of carbohydrates. Journal of the American Chemical Society, Vol. 55, pp. 2028-2036. 101. Tilche, A., Bortone, G., Garuti, G., and Malaspina, F. (1996) Post-treatments of anaerobic effluents. AvL Int. J. Gen. Mol. Microbiol., Vol. 69(1), pp. 47-59. 102. van den Berg, L. (1982) Anaerobic digestion of wastes. Cons. Recycl., Vol. 5(1), pp. 5-14. 103. Van den Berg, L., Kennedy, K.J., and Samson, R. (1985) Anaerobic downflow stationary fixed film reactor: performance under steady-state and non-steady conditions. Wat Sci Tech, Vol. 17(1), pp. 89-102. 104. Van Lier, J.B., Van Der Zee, F.P., Tan, N.C.G., Rebac, S., Kleerebezem, R. (2001) Advances in high-rate anaerobic treatment: Staging of reactor systems. Wat. Sci. Tech., Vol. 44(8), pp. 15-25. 105. VDI (2007) Guideline VDI 3475 Part 4 (Draft): Emission Control – Agricultural biogas facilities – Fermentation of energy crop and livestock. IN V. D. Ingenierure (Ed.), VDI and DIN. 106. Vidal, G., Carvalho, A., Mendez, J.M. Lema (2000) Influence of the content in fats and proteins on the anaerobic biodegradability of dairy wastewaters; Bioresource Technology, Vol. 74, pp. 231-239. 107. Weiland, P. (2001): Grundlagen der Methangärung - Biologie der Substrate; VDI- Berichte, Nr. 1620: Biogas als regenerative Energie - Stand und Perspektiven, pp. 19-32, VDI-Verlag. 108. Wildenauer, F.X. and Winter, J. (1985) Anaerobic digestion of high-strength acidic whey in a pH-controlled up-flow fixed-film loop reactor, Appl. Microbiol. Biotechnol., Vol. 22, pp. 367-72. 109. Yadvika, S., Sreekrishna, S.T., Kohli, S., and Ran, V. (2004) Enhancement of biogas production from solid substrates using different techniques–a review. Biores. Tech. Vol. 95, pp. 1–10. 80 R.T. Bachmann et al. 110. Yan, J.Q., Lo, K.V., and Liao, P.H. (1989) Anaerobic digestion of cheese whey using up-flow anaerobic sludge blanket reactor, Biological Wastes, Vol. 27, pp. 289-305. 111. Yan, J.Q., Lo, K.V., and Liao, P.H. (1990) Anaerobic digestion of cheese whey using an upflow anaerobic sludge blanket reactor. 3. Sludge and substrate profiles. Biomass, Vol. 21(4), pp. 257-271. 112. Yan, J.Q., Lo, K.V., and Pinder, K.L. (1993) Instability caused by high-strength of cheese whey in a UASB reactor. Biotech. Bioeng., Vol. 41(7), pp. 700-706. 113. Yang, P., Zhang, R., McGarvey, J.A., and Benemann, J.R. (2007) Biohydrogen production from cheese processing wastewater by anaerobic fermentation using mixed microbial communities. Int. J. Hydr. En., Vol. 32(18), pp. 1086-1094. 114. Yang, K., Yu, Y. and Hwang, S. (2003) Selective optimization in thermophilic acidogenesis of cheese-whey wastewater to acetic and butyric acids: partial acidification and methanation; Wat. Res., Vol. 37, pp. 2467-2477. 115. Yilmazer, G. and Yenigün, O. (1999) Two-phase anaerobic treatment of cheese whey. Wat. Sci. Tech., Vol. 40(1), pp. 289-295. 116. Young, J.C. and Yang, B.S. (1989) Design consideration for full-scale anaerobic filters. Water Pollut. Control Fed., Vol. 61, pp. 1576-1587. 117. Yu, H.Q. and Fang, H.H.P. (2001) Acidification of mid- and high-strength dairy wastewaters; Water Research 35(15), pp. 3697-3705. 118. Zellner, G., Vogel, P., Kneifel, H., and Winter, J. (1987) Anaerobic digestion of whey and whey permeate with suspended and immobilized complex and defined consortia. Appl. Microbiol. Biotech., Vol. 27(3), pp. 306-314.