Turnover of Macromolecules in Thermophiles

Early theories of thermophily suggested that high temperatures led to a rapid turnover of macromolecules, particularly proteins. This, however, has not been demonstrated in B. stearothermophilus nondiastaticus, which does not turn over its proteins and RNA (other than mRNA) faster than do mesophiles (Sundaram, 1986). Low protein turnover has also been reported in T. aquaticus (Kenkel and Trela, 1979). It has been reported that thermophile proteins are more stable than their mesophile counterparts, hence the low turnover at high temperatures (Moat and Foster, 1995). However, turnover of macromolecules in thermophiles may be significantly affected by medium composition (Sundaram, 1986). Macromolecule stability may be due to their association with high concentrations of carbohydrates and their ability to bind certain ions more strongly (Bergquist et al., 1987). Increased stability of proteins has also been attributed to strategic substitutions in their amino acid sequence. Enhanced DNA stability has been attributed to a higher G+C content in thermophiles than in mesophiles (Lindsay, 1995). Enhancement of tRNA stability with growth temperature has been reported in T. thermophilus in which the ribothymidylate, a normal component of tRNA is replaced with 5-methyl.2-thiouridylate, and the level of substitution increases with temperature. The higher growth and decay rate reported in thermophiles from the TAD of slaughterhouse waste, than mesophiles indicate that there are differences in the stability and turnover of macromolecules between thermophiles and mesophiles (Couillard et al., 1989).

Development, Adaptation and Stability: Thermophiles in TAD

Thermophiles responsible for TAD of sewage sludge develop from the proliferation of populations naturally present in the sewage (Ugwuanyi et al., 2008b; Sonnleitner and Fiechter, 1983a). As with compost (Abouelwafa et al., 2008; Raut et al., 2008; Yu et al., 2008; Cayuela et al., 2008; Adams and Frostic 2008; Li et al., 2008; Cunha-Queda et al., 2007; Mari et al., 2003; Heerden 2002; Hassen et al., 2001; Thambirajah et al., 1995), these remain dormant during startup while mesophiles, and then facultative thermophiles, build up the temperature until a thermophilic range is reached, when they begin to grow (Yun et al., 2000; Burt et al., 1990b). Although Sonnleitner and Fiechter (1983a) observed up to 5-log orders in the fluctuations of populations of thermophiles, washout was never observed. Over a two-year observation of a pilot scale continuous TAD, the viable thermophilic population remained at or greater than 105 g-1 of sludge even when hydraulic retention time (HRT) was reduced to as low as 10h, at aeration rates ranging between 0.02 and 0.3vvm. Extreme thermophiles (growth at > 60°C) were particularly well maintained. Rapid and comparable development of different thermophiles was observed during the TAD of potato process waste at aeration rates ranging from 0.25vvm to 1.0vvm and different temperatures (Ugwuanyi et al., 2008b). Similar stability was reported in the thermophilic biofilm responsible for the stabilisation of swine waste (Beaudet et al., 1990). This has led to suggestions that TAD should be operated at the highest possible temperature, since that could result in substantial reduction in HRT without compromising efficiency of waste stabilisation and pasteurisation.

Although high thermophilic populations are maintained during TAD, Beaudet et al. (1990) however, reported a drop in treatment efficiency as the temperature of the waste exceeded 60°C due to a restriction in the diversity (and population) of the digesting thermophiles. The persistence and stability of thermophiles in TAD has been attributed to the selection of populations with very rapid growth rate (Ugwuanyi et al., 2008b; Sonnleitner and Fiechter, 1983a,b). Unlike (thermophilic) anaerobic digestion where the overall pace of the process is determined by the growth rate of the slowest growing populations in a synthrophic interaction, the fastest growing populations in TAD determines the stability and pace of digestions.

Development of Hydrolytic Enzymes During TAD

The development of enzyme activities during TAD has not been actively investigated, even though it is believed that this may aid the development of control parameters for the process (Ugwuanyi 1999; Bomio et al., 1989). This is unlike the case with composting in which hydrolytic enzyme development has been studied in considerable detail and has been used variously to characterise and monitor process development (Abouelwafa et al., 2008; Raut et al., 2008; Yu et al., 2008; Cayuela et al., 2008; Adams and Frostic 2008; Li et al., 2008; Mari et al., 2003; Heerden et al., 2002; Hassen et al., 2001; Thambirajah et al., 1995). Protease was been reported as the major activity present in sewage sludge during TAD (Ponti et al., 1995a,b; Bomio et al., 1989). Bomio et al. (1989) also detected low activity of lysozyme, and concluded that other macromolecules whose corresponding enzymes were not detected were either not present (hence did not induce activities) or passed out of the system with only minor modifications.

However, Burt et al. (1990b) recorded considerable degradation of lipids in sludge during TAD, and suggested that lipases are probably produced during the process but may be easily degraded by proteases in the digesting waste (and so not readily measured). Grueninger et al. (1984) also suggested that amylases produced by the Bacillus spp. in TAD may be degraded by protease. During TAD of potato process waste, Ugwuanyi et al. (2004ab) demonstrated rapid development of hydrolytic enzymes including amylases, cellulose, proteases and xylanases. These enzymes were constitutive but their activities also responded to the dynamics of substrate concentration and population development in the digesting waste.

Microorganisms associated with TAD have shown considerable capacity to degrade various organics in the treated wastes, and have been considered as a possible reservoir of organisms capable of producing thermostable, industrially useful enzymes (Ugwuanyi et al., 2004ab; Malladi and Ingham, 1993). The elaboration of a variety of the hydrolytic enzymes is probably repressed in the presence of easily metabolisable waste components (Ugwuanyi 1999; Sonnleitner and Fiechter, 1983b). It is reasonable to expect that enzymatic activities associated with TAD will vary with the composition of waste, and knowledge of these may be employed to improve the digestion efficiency in the process (Kim et al., 2002).

Efficiency and Performance of TAD

Standards for comparing the efficiency of TAD processes are non-existent or at best discordant, apparently due to the variety in the type of wastes to which TAD may be applied as well as the potential products of the digestion processes. Performance of processes designed to achieve protein enrichment of waste for reuse in animal nutrition will need to be assessed differently from those intended to pasteurise waste prior to treatment by another method. These may also be assessed differently from those in which the process is employed to achieve waste stabilization, prior to disposal. Where TAD is used for waste treatment, prior to disposal, the desired performance depends on whether the process is used alone for complete treatment, or as a pre/ post treatment step (Frost et al., 1990; Loll, 1989).

Matsch and Drnevich (1977) recorded 30-40% volatile suspended solids (VSS) removal at 50°C and 4.2d retention time, while treating waste secondary sludge. Similar degradation was obtained by Jewell and Kabrick, (1980), while Burt et al. (1990a,b) achieved over 60% COD removal and 45% destruction of soluble solids during continuous operation of a pilot scale sewage sludge digestion at just under 50°C. Loll (1989), reported that TAD at over 50°C, and HRT of 0.5-3d when used in conjunction with a mesophilic anaerobic process led to a reduction in overall treatment time required in the anaerobic process by up to 10 days, in addition to pasteurisation of the sludge. TAD results in enhanced solids destruction, because of the high maintenance energy requirements of thermophiles (Brock, 1986). It has been reported that the settleability of sludge increases with increase in treatment temperature due to reduction in viscosity (Kambhu and Andrews, 1969). TAD also achieves odour reduction (resulting in sludge with acceptable odour) in sewage works at various retention times and loading rate (Murray et al., 1990).

Monitoring and Control of Aerobic Thermophilic Digestion

In spite of the advantages of TAD, comparatively little has been done to achieve good understanding of the process, particularly in the area of process monitoring and control (Maloney et al., 1997; Sonnleitner and Fiechter, 1983b). This is due, both to the relative newness of the process, and the diversity of possible applications, resulting in poorly defined process objectives and end points. Consequently, control of the process is still subject of trial and error, with no TAD specific control parameters in use. As a waste treatment process, control parameters have largely been based on those applied to activated sludge (from which it has evolved). Besides, much of the work carried out on the process have been limited to sewage sludge treatment (and hygeinisation), where classical control parameters have been conveniently, though inadequately applied (Kelly et al., 1993).

Based on the "degree-day" control process for activated sludge process (Koer and Marvinic (1977), Vismara (1985) recommended that treatment may be considered satisfactory if the product of operation temperature and time equal or exceed 250. As a complete waste treatment process applied to sewage sludge, EPA suggests that 40% of volatile suspended solids of sludge should be removed, while maintaining a minimum dissolved oxygen content of 2.0mg l-1 (Matsch and Drnevich, 1977). Additionally, sludge to be disposed of to agricultural land should have been held at 55°C for at least three days. The UK Department of the Environment (DoE) recommends that sludge to be disposed of to agricultural land should have been held at 55°C for a minimum of four hours at a retention time of 7 days (Frost et al., 1990).

Matsch and Drnevich (1977) suggested criteria based on the odour producing potential, provided that the parameters can be related to an easily measurable variable such as Oxygen Uptake Rate (OUR) or redox potential. Other control parameters that have been applied to TAD include, total/ viable microbial count and count of thermophiles at 65 °C, ATP level and dehydrogenase activity, as well as lipid content, and changes in pH (Messenger et al., 1990; Burt et al., 1990a; Droste and Sanchez, 1983). These are in addition to classical waste quality parameters such as those related to profile of COD, BOD, TSS, and VSS. Although the latter parameters are acceptable where the target is the safe disposal of waste such as in sewage sludge, they are inadequate where TAD is part of an integrated process, and in particular during waste upgrading and recycling reactions.

The development of hydrolases has been studied in a number of waste treatment processes as monitoring parameters, including in landfill (Barlaz, 1997; Yamaguchi et al., 1991; Barlaz et al., 1989; Jones and Grainger, 1983a,b), anaerobic digestion (Sarada and Joseph, 1993a,b; Palmisano et al., 1993a,b; Godden et al., 1983) as well as in composting (Raut et al., 2008; Cayuela et al., 2008; Poulsen et al., 2008; Garcia et al., 1993). In these cases, different activities were considered reliable for following process performance. This is understandable, as the degradation of polymers is known to be a rate controlling step in waste digestion (Rivard et al., 1994; 1993; Mason et al., 1987, 1986; Eastman and Ferguson, 1981).

Though protease has been reported as the predominant activity during TAD of sewage sludge (Mason et al., 1992; Bomio et al., 1989), it has not been used as a monitoring tool nor has the profile of any other hydrolytic activities been used. Amylase, xylanase, cm-cellulase and proteases have also been shown to be related with the degradation and stabilization of waste and thermophilic population development during TAD of potato process waste

(Ugwuanyi et al., 2004ab). It was suggested that the profile of some of these enzymes may be used as process monitoring tools during TAD. This should be the case, since the degradation of the polymers determines the pace of waste stabilization in TAD processes. There is sparse information that describes hydrolytic activities or even polymer breakdown in TAD, although Bomio et al. (1989) suggested that up to 66% of the metabolic activities in TAD may be due to degradation of insoluble polymers.

Measurements of hydrolytic activities are considered economical, easy to execute and interpret, and may be process and waste type specific and adaptable. Whenever hydrolytic activities may be correlated with the degradation of polymers, it can become handy for monitoring the progress of waste stabilization. Ultimately, the monitoring and control of TAD will need to take into consideration the source and type of waste, and the aim of the treatment process, as a waste stream that is being reprocessed for use in animal nutrition will require different control parameters from wastes meant for land application and disposal. Similarly, a stand alone TAD process will require different control parameters from one in which it is part of an integrated process, as either pre- or post-treatment, pasteurisation or hydrolytic process. Ultimately, a process specific control fingerprint will need to be developed for any and each particular process.

Some Applications of Thermophilic Aerobic Digestion

TAD arose as a response to the need to find safe, economical and environmentally friendly means of disposing excess sewage sludge to land. This followed legislative restrictions on the disposal of sludge to sea, or of raw (unpasteurised) sludge to agricultural land (Kelly et al., 1993). It is understandable therefore, that virtually all early studies on TAD as well as more recent ones have focused on the use of this process in sewage sludge treatment and pasteurisation (Hawash et al., 1994; Chu et al., 1994; 1996; 1997). Notwithstanding, TAD has also been used for the treatment of a variety of other waste types, particularly those of agricultural and food industry origin intended for disposal. It has been deployed as the sole treatment method for different high strength wastes such as swine slurry (Beaudet et al., 1990), slaughter house effluent (Couillard et al., 1989), potato process waste (Ugwuanyi et al., 2005ab; Malladi and Ingham, 1993), and dairy, brewery/ distillery and other food industry wastes (Zvauya et al., 1994; Gumson and Morgan, 1982; Loll, 1976; Popel and Ohmnacht, 1972) spent liquor of pulp mills (Zentgraf et al., 1993), olive mill waste (Becker et al., 1999) and a wide variety of other waste, particularly those loaded with toxic organic and inorganic chemical (Adav et al., 2008). Sonnleitner and Fiechter (1983b,c) proposed that it may be used to treat virtually any kind of organic waste, provided that for particularly recalcitrant wastes enough time is allowed for the adaptation of the process, preferably in continuous processes. It has also been proposed that the metabolic versatility of TAD associated thermophiles may be exploited in the use of this process for the reprocessing and protein enrichment of a variety of wastes for use in animal nutrition, and in the production of high value bichemicals (Ugwuanyi, 1999).

Application of Thermophilic Aerobic Digestion in Waste Pasteurisation

Conventional waste treatment processes are accepted to be inefficient from a hygienic and epidemiological point of view (Pagilla et al., 1996; Plachy et al., 1995; 1993; Juris et al., 1993; 1992; EPA, 1992, 1990; De Bertoldi et al., 1988). Consequently, one of the major attractions of TAD has been the potential for exploiting its high temperatures to achieve the destruction of pathogenic microorganisms as well as protozoa, viruses and parasite eggs in treated wastes. These can all theoretically be destroyed at the temperatures typical of TAD (Wagner et al., 2008; Ugwuanyi et al., 1999; Mason et al., 1992; Kabrick and Jewell 1982).

Although various studies have been carried out on the inactivation of pathogens during waste treatment particularly in mesophilic processes and in composts (Gerba et al., 1995; Kearney et al., 1993ab; Carrington et al., 1991; Olsen and Larsen, 1987; Abdul and Lloyd, 1985), few have described the behaviour of pathogens and indicators in TAD (Borowski and Szopa 2007). As a result, various, often conflicting projections have been made on the capacity of this process to achieve pathogen destruction, based on the known sensitivity of indicator organisms to thermophilic temperatures (Ponti et al., 1995 a; Messenger et al., 1993b). Jewell and Kabrick (1980) and Jewell (1991), reported that maintenance of the temperature of digesting sludge at 50°C for 24h destroys most pathogens including bacteria, viruses, protozoa and parasite (Ascaris) eggs, as does 60°C for 1h. Carlson (1982) reported that 24h was required to completely inactivate enteric bacteria in sewage sludge at 57°C, while Burt et al. (1990b) reported a reduction in populations of E. coli in sludge from 106-107 per gram to undetectable levels after digestion at 50°C, and a HRT of 24h. Efficient destruction of helminth eggs and ova as well as protozoan parasites, at different temperatures in TAD has also been reported (Whitmore and Robertson, 1995; Plachy et al., 1995,1993; Juris et al., 1992).

TAD has been reported to achieve better pasteurisation of waste than does composting (Paggila et al., 1996; Droffner and Brinton, 1995; Carrington et al., 1991), probably due to the presence of high levels of solids in composts, which may protect pathogens from thermal inactivation. Ponti et al. (1995a) applied TAD to achieve rapid inactivation of E. coli in sewage sludge at 55° and 57°C, and reported that increase in waste solid content considerably protected cells from thermal destruction. Morgan and Gumson (1989) reported that temperatures in excess of 50°C were required to eliminate E. coli and Salmonella in a continuous flow TAD with retention time of 8 days at Pontir Sewage Works, UK. Murray et al. (1990) reported efficient sludge pasteurisation in a small sewage treatment works even during periods of irregular sludge feed, while Spaull and McCormack (1988) employed TAD for the inactivation of plant pathogens and weed seeds with considerable success.

Though rapid destruction of mesophiles (E. coli) occurs between 54° and 62°C, recent studies have shown that mesophiles in compost may survive for very long periods, and may even multiply in the process, at temperatures comparable to what is likely to be employed in TAD (Droffner and Brinton, 1995). Besides, various pathogens can be expected to respond differently to the effect of heat at different pH, DO and waste solid contents (Plachy et al., 1995). Ugwuanyi et al., (1999) reported various Dvalues for different human and animal pathogens during TAD of agricultural waste. However, more studies are needed to be able to make projections on the survival of various pathogens in TAD, particularly given the variety of waste and digestion conditions likely to obtain in full scale processes. Since the survival of a single pathogen is often enough to cause re-infection of treated waste, Hamer, (1989) recommended that microbial death in waste treatment should be followed by plate count.

Although differences in design and assay procedures make comparisons between inactivation data in TAD difficult, the Environmental Protection Agency (EPA) recommends that sludge intended for spreading on agricultural and pasture land should be held at 55°C for 3 days to achieve pasteurisation ('class A' status (EPA, 1992)). While in the UK, the Department of the Environment (DoE) recommends that exposure of sludge to a temperature of 55°C for at least 4h, will reduce the number of viruses and pathogenic organisms to levels acceptable for disposal to agricultural land. Reduction of pathogens levels in wastes intended for land or even sea disposal is the subject of several legislations in Europe and North America, (Droffner and Brinton, 1995; EPA, 1992). This is a major driving force in the development, and acceptability of TAD. When this process is to be employed in waste reprocessing for animal feed use, new parameters related to control of infectious agents of both humans and animals will be expected to be given prominent consideration. This is particularly important with the increasing attention on the spread of zoonotic diseases.

Detoxification of Xenobiotics in TAD

The use of TAD for the destruction of xenobiotics and noxious chemicals that may contaminate otherwise recycleable waste biomass has received little attention (Laine and Jorgensen, 1996). The potential for application of TAD in this area derives from the ability of aerobic thermophiles to rapidly degrade a variety of chemicals due to their enzymatic versatility (Hernandez-Raquet et al., 2007; Moeller and Reeh 2003; Knudsen et al., 2000; Banat et al., 2000; Jones et al., 1998; Reinscheid et al., 1997) and removal efficiency may only be constrained by the bioavailability of the anthropogenic chemical (Patureau et al., 2008). Various enzymes such as oxygenases, ligninases and peroxidases, which are powerful and essential biocatalysts in xenobiotic degradation, remain the preserve of aerobic organisms (Adav et al., 2008; Verstreate et al., 1996). In TAD, these may play significant roles in the detoxification of xenobiotics, and a variety of plant toxins, such as linamarin in cassava, and related plant cyanogenic glycosides, whose presence in plants limit their use. The application of TAD in detoxification of xenobiotics and toxins will certainly increase its appeal as a waste management and recycling process.

Detoxification of cyanogenic glycoside (linamarin) associated with cassava and cassava process wastes using thermophiles isolated from TAD of agricultural waste has been demonstrated (Ugwuanyi et al., 2007). Although, little information currently exists on the use of either this process or any other for detoxification of waste in full scale operations, it is expected that the high temperatures of TAD and the selection of suitably adapted microbial population could lead to the breakdown of the toxins and noxious chemicals in various waste streams.

Protein Enrichment and Reprocessing of Waste by TAD

Various organic wastes, particularly the agro-food types, may be considered as resources insofar as their energy and mineral content are concerned. However, the recycling of these wastes is strictly tied to their microbiological and sanitary quality. Although in classical studies on the use of TAD waste upgrade and reuse were not always the intended endpoints, it has vigorously been proposed that TAD, as the vehicle for the protein enrichment of waste, may be applied to waste intended for upgrading and recycling as (components of) animal feed (Coulthard et al., 1981) given also that several agricultural wastes are currently being studied for upgrading and recycling, particularly in solid state fermentations including by silage (Villas-Boa et al., 2003; Laufenberg et al., 2003). The attraction for this application of TAD is buoyed by the fact that many agricultural and food industry wastes are already being employed in this capacity (Houmoumi et al., 1998; Krishna and Chandrasekaran, 1995; Coillard and Zhu, 1993; Barington and Cap, 1990). It is envisaged that carbohydrate-rich wastes, high-quality agricultural refuse and by-products, food industry wastes and other organic wastes may eventually be treated by this process, to achieve cost-effective pasterurisation, stabilisation and protein enrichment of zero-cost materials for animal feed use (Zentgraf et al., 1993; Coillard and Zhu, 1993; Barington and Cap, 1990). The emphasis here is on exploitation of the capacity of thermophilic populations to degrade carbohydrates and lipids (with loss of carbon as carbon dioxide) while accumulating nitrogen as high quality microbial protein (Ugwuanyi, 1999; Krahe et al., 1996; Bergquist et al., 1987), besides the selective conservation of waste protein under appropriate (elevated temperature) digestion conditions where nitrification is unlikely to occur.

The pattern of protein accumulation in TAD is influenced by the metabolism of intrinsic particulate and soluble waste proteins, the metabolism and disposition of microbial proteins, as well as the metabolism, particularly the conservation as microbial protein/biomass of intrinsic and extrinsic other nitrogenous compounds including ammonia in the waste slurry. Protein enrichment of wastes by TAD has considerable implication for global food security, especially in the tropics where, on account of scarcity, animals and humans often compete directly for the same sources of nutrition. Accumulation of protein in TAD-treated waste results from the selective degradation of carbohydrate, resulting in the loss of carbon (Ugwuanyi et al., 2006a). This leads to a relative accumulation of nitrogen as protein (possibly of waste origin but also, importantly, as microbial protein), and the relative increase in percentage of digested waste remaining as protein. If mineral nitrogen is provided, this is also converted to microbial biomass protein. The result of this nitrogen turnover is the conversion of low- quality waste biomass into higher-quality (microbial) protein-enriched digest. The protein-rich digest may then be economically applied to animal feeding. The extent of accumulation of protein is a function of waste digestion conditions, including temperature, aeration rate and pH (Ugwuanyi et al., 2006a).

Concluding Remarks and Safety Consideration

Global perception of waste in general and agricultural wastes in particular is changing rapidly in response to need for environmental conservation, sustainable agricultural productivity and global food security. Consequently, most of such wastes are currently seen more as resources in the wrong form and location that needs to be reprocessed and reused than as wastes to be disposed of. The need for appropriate technologis for the reprocessing of such resources has become more compelling as are the economic prospects arising from such efforts. Protein enrichment of waste for use in animal nutrition offers opportunities for the economic reuse of abundant agricultural wastes and refuse. Low tech, low cost biotechnological processes such as TAD offers great opportunities for the reprocessing and reuse of abundant agricultural wastes (particularly those generated as slurries or at elevated temperatures) in animal nutrition. The use of thermophilic organisms (perhaps also with GRAS status) to effect the protein enrichment, biomass production and detoxification reactions will help improve confidence in the final products derived from this process, and help drive its development and application in the valorization of agricultural wastes.

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