Biotechnology Advances 29 (2011) 972–982 Contents lists available at SciVerse ScienceDirect Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv Research review paper From wastewater to bioenergy and biochemicals via two-stage bioconversion processes: A future paradigm Wen-Wei Li, Han-Qing Yu ⁎ Department of Chemistry, University of Science & Technology of China, Hefei, 230026, China a r t i c l e i n f o Article history: Received 20 December 2010 Received in revised form August 2011 Accepted 14 August 2011 Available online 22 August 2011 Keywords: Acidogenesis Anaerobic Biochemical Bioenergy Optimization Two-stage bioconversion process (TSBP) Volatile fatty acids (VFAs) Wastewater a b s t r a c t Recovery of bioenergy and biochemicals from wastewater has attracted growing and widespread interests In this respect, two-stage bioconversion process (TSBP) offers an appealing avenue to achieve stepwise and directional substrate conversion in separated stages Such a biosystem not only enables enhanced degradation of organics, but also favors a high product yield and quality Various TSBRs have been developed for the production of methane, hydrogen, electricity, bioplastics, bioflocculants, biopesticides, biosurfactants and other value-added products, demonstrating marked advantages over the conventional one-stage processes It represents a promising, and likely the sole viable, paradigm for future application However, there are also many remaining challenges This paper provides an overview of the various TSBPs, introduces the recent advances, and discusses the major challenges and the future perspectives for practical application © 2011 Elsevier Inc All rights reserved Contents Introduction Characteristics of TSBPs TSBPs for bioenergy production from wastewaters 3.1 Methane production 3.2 Hydrogen production 3.3 Bioelectricity production TSBPs for biochemicals production from wastewaters 4.1 PHA production 4.2 Biopesticide production 4.3 Bioflocculant production 4.4 Biosurfactant production 4.5 Other value-added products generation Challenges and future perspectives 5.1 How to make a choice among the different processes and end-products? 5.2 How to connect the two stages? 5.3 Process optimization 5.4 Product separation and purification 5.5 Other technological and economical considerations 5.6 Outlook ⁎ Corresponding author at: Department of Chemistry, University of Science & Technology of China, China Tel.: + 86 551 3607592; fax: + 86 551 3601592 E-mail address: hqyu@ustc.edu.cn (H.-Q Yu) 0734-9750/$ – see front matter © 2011 Elsevier Inc All rights reserved doi:10.1016/j.biotechadv.2011.08.012 973 973 973 973 974 974 975 975 976 976 976 976 976 977 977 977 978 979 979 W.-W Li, H.-Q Yu / Biotechnology Advances 29 (2011) 972–982 Conclusions Acknowledgements References 973 979 979 980 Introduction Characteristics of TSBPs Environmental pollution and fossil fuel depletion are driving worldwide intense research into alternative energy sources exploration and pollution control One of the most attractive avenues to achieve both goals is to recover energy and resources from waste streams through bioconversion processes (Angenent et al., 2004; Cantrell et al., 2008) In this respect, intensive studies have been conducted in the past few decades, and the various “green technologies” have been extensively reviewed (Angenent et al., 2004; Hahn-Hägerdal et al., 2006; Hallenbeck and Ghosh, 2009; Kleerebezem and Loosdrecht, 2007; Rozendal et al., 2008) For many years, one-stage anaerobic digestion has been a prevailing technology for methane production, in which substrate is converted to methane and other products under a joint effort of several microbial groups in one single reactor (Sterling et al., 2001) Such one-stage bioconversion processes (OSBPs), attributed to the simple reactor configuration and easy operation, have also been widely employed for production of biohydrogen, electricity, PHA and other biochemicals However, they are inherently problematic when dealing with complex wastewater that contains recalcitrant and toxic substances These compounds are generally inhibitive to biodegradation by the specific energy/biochemical-producing microorganisms and thus cause low bioconversion efficiency (Neves et al., 2009; Salminen and Rintala, 2002) Moreover, a mixed culture of hydrolysis/acidification bacteria and the functional microorganisms would increase the difficulty of bio-product separation and raise the operation costs (Brar et al., 2009; Salehizadeh and Loosdrecht, 2004) These drawbacks severely limit their application In fact, no large-scale application of OSBPs has been truly achieved in wastewater treatment so far except for methane production (Safley and Westerman, 1989) Even for the methane fermentation that has seen considerable applications due to the high competitive ability of the methanogens, there are still remaining constraints (Neves et al., 2009) Because the acid- and methane-forming microorganisms prefer different culturing conditions, a suboptimal-condition operation generally lead to low methanogen activity Furthermore, the fast-growing acidogens and prevailing acidification usually lead to organic acid accumulation, which further suppress the activities of acetogens and methanogens (Cohen et al., 1980; Fox and Pohland, 1994; Ghosh et al., 1995; Zoetemeyer et al., 1982) Although great previous efforts have been directed towards enhancing the bioconversion efficiency of OSBPs, so far the progresses achieved are rather limited There seems to be limitations inherently insurmountable by such one-stage processes As a likely solution, twostage bioconversion processes (TSBPs) are recently gaining increasing popularity, and opening up the possibility for a fundamental breakthrough of the above barriers Through a properly designed TSBP, the organic compounds that are not directly utilizable by the functional microorganisms can be stepwisely converted to valuable products, usually at higher conversion efficiency than OSBP (Demirel and Yenigun, 2002) However, TSBPs also face ubiquitous challenges arisen from the separation of conversion reactions, such as stage connection and complicated process control Notwithstanding the many remaining limitations, TSBP undoubtedly presents a promising and versatile strategy that is readily applicable to almost all bioprocesses, thus embracing numerous possibilities for future application This paper offers an overview of the various TSBPs for bioenergy and biochemicals recovery from wastewater in respect of recent advances, existing challenges and future perspectives A typical TSBP consists of two separated stages (Fig 1): hydrolysis and acidogenesis of organic matters in wastewater to volatile fatty acids (VFAs) and other intermediates at the first stage (primary processing) by mixed cultures (Yang et al., 2003), then a subsequent conversion of the VFAs to bioenergy or biochemicals by specific-functional bacteria at the second stage (fine processing) (Pohland and Ghosh, 1971) Such a strategy of stage separation not only ensures a considerable degradation of organics but also warrants a high yield and purity of target products First, a selective degradation and directional conversion of complex wastes into favorite intermediates can be attained at the first stage by mixed cultures, which are more stable and adaptive to a wider range of substrates compared with pure cultures and need no sterilization (Kleerebezem and Loosdrecht, 2007) This not only alleviates the negative impacts of inhibitive compounds and complex microbial consortia to the functional bacteria, but also favors a further conversion of the intermediates at the subsequent stage Secondly, the recovery of energy or biochemicals can be maximized by adopting appropriate fine processing approaches and specific functional microorganisms that utilizing the produced VFAs as substrate Thirdly, it allows a separated optimization of the microbial community and the operation conditions in individual stages Finally, it shows a great potential for widespread application and standardized operation, as almost all organic materials in wastewater, regardless of their origins, can be uniformly degraded to relatively simple VFAs and then selectively converted to valuable end-products in separated stages TSBPs for bioenergy production from wastewaters 3.1 Methane production Methanogenic anaerobic digestion is a classical anaerobic bioconversion process that has been practiced for over a century and used in full-scale facilities worldwide (Alvarez et al., 2000; Bouallagui et al., 2005; Pavan et al., 2000; Schober et al., 1999; Zhang et al., 2005) This is a complicated process that involves a mixture of population of microorganisms and several gasses and liquid products, thus strict process control and product purification are required (Harper and Pohland, 1986; Schnurer et al., 1999) In comparison, a two-stage anaerobic digestion process, through a separation of hydrolysis/acidogenesis and methanogenesis steps, enables a more flexible operation and a higher product yield and purity (Blonskaja et al., 2003; Demirer and Chen, 2005) This TSBP favors the enrichment of specific microorganisms in individual stages (Fezzani and Cheikh, 2009; Rubia et al., 2009) and increases the process stability (Held et al., 2002) A short HRT is generally adopted for the first stage to wash out the methanogens, while a relatively long HRT can be maintained for the second stage to ensure sufficient retention of the slow-growing methanogens (Ponsá et al., 2008) and to prevent overloading and accumulation of toxic substances or acids (Bouallagui et al., 2004; Demirer and Chen, 2005) Other attractive attributes of such TSBPs include better decomposition of solid contents in wastewater (Neves et al., 2009) and more effective pathogenic destruction in the hydrolysis/acidification stage (Bendixen, 1994) The concept of two-stage anaerobic digestion has now been widely accepted and successfully demonstrated, and TSBP for methane production from wastewater has been a common practice worldwide (Borja et al., 1996; Göblös et al., 2008; Ince, 1998; Kim et al., 2004; Shin et al., 2010) Nevertheless, the existing TSBP systems mostly 974 W.-W Li, H.-Q Yu / Biotechnology Advances 29 (2011) 972–982 Specific functional microbial group Hydrolysis/ acid-forming microbial group Methane Hydrogen Organic wastewater VFAs Electricity PHA Alcohol ··· ··· Intermediates Endproduct Bio-pesticide Bioflocculant Biosurfactant Other value-added prdocut 1st Stage (Primary processing) 2nd Stage (Fine processing) Fig Schematics of TSBP for energy and biochemical recovery from wastewater still suffer from low conversion efficiency for wastewater treatment due to several remaining limitations First of all, the presence of refractory organics in wastewater may constitute a big challenge for the conversion, and this barrier is actually encountered in all bioconversion processes (Angenent et al., 2004; Gavrilescua and Chisti, 2005) Furthermore, the separation of hydrolysis/acidogenesis and methanogenesis could also negatively affect the syntrophic association and interspecies hydrogen transfer between acidogenens/acetogens and methanogens (Reith et al., 2003) These factors should be comprehensively considered when designing a TSBP for methane production 3.2 Hydrogen production Hydrogen has been widely recognized as an ideal alternative energy source to fossil fuel (Hallenbeck and Ghosh, 2009), and is considered a more desirable energy carrier than methane (Lee et al., 2010) A variety of biohydrogen production processes have been developed, such as dark fermentation, photofermentation and biophotolysis to directly recover hydrogen from the wastewater (Angenent et al., 2004; Benemann, 1996; Das and Veziroglu, 2008; Fang and Yu, 2002; Yang et al., 2006; Yu et al., 2002) However, such one-stage hydrogen production process encounters many similar challenges as methanogic anaerobic digestion (Angenent et al., 2004; Mohan 2008; Vazquez and Varaldo, 2009) For example, many organics in wastewater are not directly useable by the hydrogen-fermentative bacteria, and the process performances are highly sensitive to the operating conditions like solid retention time (SRT), organic loading rate and gas partial pressure (Antonopoulou et al., 2008; Mu et al., 2006; Zhao et al., 2008) Particularly, hydrogen fermentation requires inhibition of hydrogenconsuming species (Mohan 2008; Oh et al., 2003; Yu and Mu, 2006) This can be very difficult in practice as the presence of methanogens, sulfate-reducing bacteria and nitrate-reducing bacteria is usually unavoidable in a mixed-culturing anaerobic system (Chang et al., 2008) Although many countermeasures, such as inoculum pretreatment and process control are available to inhibit these competitive microorganisms, they also complicates the process and increase the operation cost (Chang and Lin, 2004; Oh et al., 2003; Okamoto et al., 2000; Shin et al., 2004) Therefore, direct hydrogen production from wastewater through OSBP appears to be restricted, while TSBPs demonstrate a promising solution In the past decade, various hydrogen TSBPs have emerged to decouple the hydrogen production process from the hydrolysis/ acidification step, by employing photofermentation, biophotolysis or bioelectrolysis as a second stage specifically for hydrogen production (Call and Logan, 2008; Chen et al., 2008; Liu et al., 2006; Rozendal et al., 2006; Shi and Yu, 2006a, 2006b; Tao et al., 2007) It should be pointed out that, notwithstanding the many drawbacks of mixed culturing hydrogen-producing and acid-forming bacteria, an incorporation of dark-fermentation into the hydrolysis/acidification process is still preferred for TSBPs in most previous practices, in light of the higher process simplicity and the additional hydrogen output in the first stage TSBPs for high-rate hydrogen production have been demonstrated in numerous studies with great success Through such a strategy, it is theoretically possible to completely recover the 12 mol H2/mol hexose from complex organics (Bartacek et al., 2007; Guo et al., 2010; Hallenbeck and Ghosh, 2009; Li and Fang, 2007; Redwood et al., 2009) In practice, high hydrogen yield of up to 1.34 m 3-H2/kg-COD has been achieved in such TSPBs (Lu et al., 2009), which incorporate an acidogenic fermentation stage (with or without dark fermentation) and a subsequent optional photo-fermentation or electrohydrogenesis stage (Kim et al., 2006: Levin et al., 2004; Sun et al., 2008) (Table 1) Despite of the high performances and great potential of TSBPs, however, there are remaining hurdles (Lee et al., 2010) For the hydrolysis/acidification and dark fermentation as a hybrid first stage, it remains a problem in respect to strengthening the competitivity of hydrogen-producing bacteria and improving VFA properties (Brentner et al., 2010) For a subsequent photo-fermentation process, the low light penetration and conversion efficiencies and the high costs of gastight and transparent photobioreactors present two major challenges (Chen et al., 2008; Tao et al., 2007) While, for the electrohydrogenesis, an additional electricity input is required to drive the hydrogen production in microbial electrolysis cells (MECs), and the expensive platinum catalyst for cathode leads to high reactor cost (Cheng and Logan, 2007) Moreover, specific bacteria of photosynthetic bacteria (PSB) and anode-respiring bacteria (ABR) are required in such systems (Hallenbeck and Ghosh, 2009; Logan, 2009), which are likely to be affected by wastewaters that contain various toxics and competitive microbial consortia if the process is not properly controlled 3.3 Bioelectricity production Being capable of extracting energy from a wide range of complex organics and renewable biomass by the form of electricity, microbial fuel cells (MFCs) have also received significant attention and intensively studied in recent years (Logan, 2009; Rozendal et al., 2008) MFCs possess several unique advantages over the conventional bioenergy technologies (Rabaey and Verstraete, 2005) First, electricity is more convenient in transmission and utilization compared to other energy carriers Secondly, it enables high conversion efficiency by directly W.-W Li, H.-Q Yu / Biotechnology Advances 29 (2011) 972–982 975 Table Main TSBPs for energy and biochemical recovery from wastewater Products Wastewater type Inoculums Productivity Reference Methane Dairy wastewater Palm oil mill effluent Fat-containing wastewater Instant-coffee-production wastewater Dairy wastewater Acid whey wastewater Food waste-recycling wastewater Sucrose-containing wastewater Glucose-containing wastewater Olive mill wastewater Cellulose-containing wastewater Molasses wastewater Coffee processing wastewater Food processing wastewater Citrate and n-octane wastewater Starch-containing wastewater Starch-containing wastewater Glucose containing wastewater Municipal wastewater Raw rice grain-based distillery spentwash Starchy wastewater Paper mill wastewater Anaerobic sludge Anaerobic sludge Anaerobic sludge Anaerobic sludge Anaerobic sludge Anaerobic sludge + Kluyveromyces lactis Anaerobic sludge Anaerobic sludge + PSB Anaerobic sludge + PSB Anaerobic sludge + PSB Anaerobic sludge + ARB Anaerobic sludge + ARB Anaerobic sludge + acclimated activated sludge Anaerobic sludge + acclimated activated sludge P oleovorans Anaerobic sludge + R eutropha Anaerobic sludge + R eutropha Anaerobic sludge + A eutrophus Activated sludge Activated sludge Anaerobic sludge Activated sludge 0.15 m3 CH4/kg COD 0.33 m3/kg-COD 0.15 m3/kg-COD 0.32 m3/kg-COD 0.40 m3/kg-volatile solid 0.20 m3/kg-COD 0.46 m3/kg-COD 0.39 m3-H2/kg-COD 0.53 m3-H2/kg-COD 0.66 m3-H2/kg-COD 0.32 m3-H2/kg-cellulose 1.34 m3-H2/kg-COD 88.18 kJ/kg-COD 14.20 kJ/kg-COD 0.63 g-PHA/g-VSS 0.18 g-PHA/g-VSS 0.73 g-PHA/g-VSS 0.63 g-PHA/g-VSS 0.30 g-PHA/g-VSS 0.67 g-PHA/g-VSS 0.34 g-PHA/g-VSS 0.48 g-PHA/ g-VSS Ince (1998) Borja et al (1996) Kim et al (2004) Dinsdale et al (1997) Dugba and Zhang (1999) Göblös et al (2008) Shin et al (2010) Tao et al (2007) Shi and Yu (2006a) Eroglu et al (2006) Wang et al (2010a) Lu et al (2009) Nam et al (2010) Oh and Logan (2005) Jung et al (2001) Yu and Si (2001) Du and Yu (2002) Jin et al (1999) Chua et al (2003) Khardenavis et al (2007) Yu (2001) Bengtsson et al (2008b) Hydrogen Electricity PHA converting the substrate energy to electricity Thirdly, MFCs can operate efficiently at ambient and even low temperatures, thus they require no extra energy input and offer opportunities for application in remote locations lacking electrical infrastructures A number of studies have shown that the electricigens can efficiently utilize VFAs such as acetate (Lee et al., 2008a, 2008b; Rabaey et al., 2005), propionate (Bond and Lovley, 2005) and butyrate (Liu et al., 2005) as well as mixture VFAs from fermentation broth (Freguia et al., 2010; Teng et al., 2010) as carbon sources for current generation Among these, acetate and propionate are preferentially converted in mixed VFA feeds (Jeong et al., 2008) In addition, it has been found that a moderate acidophilic environment favors electricity production in MFCs (Mohanakrishna et al., 2010; Raghavulu et al., 2009) Therefore, MFCs may serve as an ideal process for energy production following a primary-processing step of hydrolysis/ acidification So far, a maximum power density of 2.98 W/m3 has been achieved in a granular activated carbon augmented MFC that was fed with acidified effluent from hydrogen fermentation process (Nam et al., 2010) While this technology may sound like an answer to our energy crisis and significant progresses have been achieved in recent years, it is not yet viable for practical applications (Li et al., 2011; Logan, 2010) The current available power output is still too low for practical use, the cost of the component materials and operations at the present stage far exceeds the value of the energy generated, and the options of microorganism for such processes are still very limited Furthermore, although many researches have been reported on the utilization of acetate and mixed VFAs for electricity generation at laboratory scale, the performances of such TSBP systems for practical wastewater treatment are yet to be ascertained at pilot-scale applications (Rozendal et al., 2008) TSBPs for biochemicals production from wastewaters 4.1 PHA production Apart from bioenergy that can be recovered from wastewater in the form of gaseous methane, hydrogen or electricity, valuable biochemicals can also be harvested from the liquid or solid phase through bioconversion processes Particularly, the production of PHA from wastewater biological treatment systems has recently aroused widespread interests (Bengtsson et al., 2008b; Chua et al., 2003; Dionisi et al., 2005; Kleerebezem and Loosdrecht, 2007; Sagastume et al., 2010; Serafim et al., 2008; Wang and Yu, 2006) PHAs are biodegradable and biocompatible polyesters with interesting characteristics for a significant number of industrial applications (Serafim et al., 2008) They are stored as granules in the cell cytoplasm by microorganisms under stress conditions (Anderson and Dawes, 1990) During conventional OSBPs, the organic matters in wastewater are converted to short-chain VFAs by acid-producing bacteria and accumulated in PHA-producing bacteria cells in the same mixed-culturing system (Serafim et al., 2008) Such OSBPs face two major restrictions Firstly, the PHA-producing microorganisms have difficulties in sustaining in such a complex system, and the overall conversion efficiency is limited by the suboptimal operation conditions (Castilho et al., 2009) Second, the PHA content of the harvested solid biomass is usually not high enough for a cost-effective polymer recovery, because of the coexistence of considerable amount of nonbiodegradable content and the many non-PHA-producing cells (Yu et al., 1999) Thus, in order to achieve higher volumetric productivity and product purity, it is apparently more desirable to separate the PHA-producing process from the hydrolysis/acidification TSBPs that combine anaerobic acidogenic fermentation and aerobic VFA conversion have been demonstrated to significantly enhance the PHA production efficiency (Dionisi et al., 2005; Du and Yu, 2002) In such a process, relatively simple VFAs were formed in the first stage (usually using mixed culture), which favors the growth of PHAproducing bacteria and allows a more simple PHA content (Bengtsson et al., 2008a; Wang et al., 2010b) Meanwhile, a high concentration of PHA-producing bacteria with strong PHA storage capacity (pure cultures or enriched sludge) can be retained in the secondary stage (Dionisi et al., 2005; Fang et al., 2009; Lemos et al., 2006), thus enabling a higher PHA yield (Ruan et al., 2003) Moreover, TSBPs allow a flexible operation of aerobic dynamic fermentation (Dionisi et al., 2004; Majone et al., 1996) or anaerobic/aerobic mode (Dai et al., 2007) for culture selection, which can be further differentiated from the PHA accumulation step (Dionisi et al., 2005), and favor the predominance of PHAproducing microorganisms However, the requirement for a strict process control presents a potential challenge for such processes The physical and mechanical properties of PHAs depend largely on the composition of the produced VFAs (Lemos et al., 2006) In practical processes, however, the acidogenic fermentation products can vary significantly with wastewater type and operation conditions (Bengtsson et al., 2008a; Horiuchi et al., 2002; Salehizadeh and Loosdrecht, 2004; Yu and 976 W.-W Li, H.-Q Yu / Biotechnology Advances 29 (2011) 972–982 Fang, 2002) Thus, a strict control of the process conditions is of critical importance for a VFA production TSBP to ensure a relatively stable product yield and quality (Albuquerque et al., 2007; Chen et al., 2005; Yu and Fang, 2000) Furthermore, despite of the employment of pureculture in the second stage, the product extraction and purification still represent a considerable percentage of the manufacturing cost because of the coexistence of many other microbial metabolites and recalcitrant matters bioflocculant production from various wastewaters is technologically viable and expectable Most of all, unlike other biochemicals that usually need high product purity for application, bioflocculant can be directly used in biological wastewater treatment systems with not purity requirement As thus, this process shows great promise for large-scale application However, like other biochemical recovery processes, the similar barrier of low production yield and high recovery cost need to be overcome to make such processes commercially viable 4.2 Biopesticide production 4.4 Biosurfactant production Biopesticide is another obtainable value-added product from microorganisms during wastewater fermentation Biopesticides are generally highly target specific, leave no toxic residues, and produce a lesser overall impact on the environment than chemical pesticides One of the most well-known and dominant biopesticide is derived from Bacillus thuringiensis (Bt), and mainly consists crystal deltaendotoxins and other pesticidal substances (Brar et al., 2006; Sanchis and Bourguet, 2008) The use of agro-industrial wastewater or sludge for Bt cultivation and biopesticide production has been extensively investigated (Khuzhamshukurov et al., 2001; Sachdeva et al., 2000; Tirado-Montiel et al., 2001; Vidyarthi et al., 2002; Yezza et al., 2006) Wastewater can provide the necessary nutritional elements to sustain growth, sporulation and crystal formation by Bt Nevertheless, these processes generally suffer from low product yield and complicated composition, attributed to the complex nature of wastewater and the coexistence of other competitive bacteria Moreover, most of the nutrients in the wastewater are unavailable to Bt This weakens its ability to produce pores, crystals and other insecticidal metabolites and leads to low entomotoxicity Therefore, similar to PHA production OSBPs, problems also occur in one-stage pesticide fermentation in terms of product harvesting and separation (Brar et al., 2009) And likewise, a TSBP strategy seems to provide a viable solution Tirado-Montiel et al (2001) found that acid hydrolysis of wastewater sludge can improve the entomotoxicity of Bt by 24% attributed to the increasing bioavailability of substrate and nutrients Meanwhile, a relatively simplified and optimized environment in the subsequent step can favor the predominance of Bt and lead to higher biopesticide yield and quality Thus, although no TSBP has been experimentally demonstrated for biopesticide production from wastewater so far, it exhibits a high potential to recover biopesticides from wastewaters with enhanced product quality and productivity Nevertheless, the challenges of process control and product separation/ purification, which seem to be encountered in all biochemicals recovery process, are yet to be addressed 4.3 Bioflocculant production Bioflocculant, as another valuable microbe-derived metabolite with high biodegradability and ecological safety, has also recently attracted considerable attention (Salehizadeh and Shojaosadati, 2001) Bioflocculant is extracellular biopolymers excreted by microorganisms, including proteins, glycoproteins, polysaccharides, lipids and glycolipids (Salehizadeh and Shojaosadati, 2003) Direct bioflocculants production from organic wastewater has been performed in several studies with certain success (Li et al., 2008; Wang et al., 2007; Zhang et al., 2007) However, limited by a high complexity of wastewater, the low product yield remains a major constriction to its application (Zhang et al., 2007) Again, TSBP processes may offer an attractive solution (Fujita et al., 2000; 2001) Especially, some low-molecular VFAs such as acetic and propionic acids were found to give a higher flocculant yield than other simple organic carbon source (Fujita et al., 2000), while the addition of 2-keto gluconic acid can significantly increase the flocculating activity (Nakamura et al., 1976) Therefore, although utilization of VFAs for bioflocculant production has only been demonstrated in the treatment of sludge digestion liquor by far, the application of TSBP for Among the various possibilities for biochemical production from wastewater, biosurfactant may also have a place in the future paradigm Biosurfactants are amphipathic compounds excreted by microorganism, with comparable surface activity to their synthetic equivalents but much lower toxicity and higher efficiency at extreme conditions (Makkar and Cameotra, 1999; Nitschke and Pastore, 2006) The biosurfactant family covers a variety of compounds like glycolipids, lipopeptides, fatty acids, polysaccharide-protein complexes, peptides, phospholipids and neutral lipids (Yin et al., 2009) In an attempt to lower the production cost, various waste streams such as oils, sugar-containing wastewater and even lignocellulose have been used as feedstock for biosurfactant (Haba et al., 2000; Nitschke and Pastore, 2006) Generally, the type and quantity of the biosurfactant produced varied significantly with the microorganism species and the wastewater composition (Das et al., 2009) Compared to OSBP, a TSBP offers a promising strategy to simplify the substrate composition and microbial population by converting the various organics uniformly to VFAs in a preceding hydrolysis/acidification step (de-Lima et al., 2009) This fermentation liquor with optimized substrate content and balanced nutrients favors the growth of specific biosurfactant-producing bacteria, and thus enables a high yield and good properties of the biosurfactants (Panilaitis et al., 2007) However, investigations in such TSBP process are rather scarce, the practical feasibility of such processes for wastewater treatment are yet to be demonstrated 4.5 Other value-added products generation In addition to PHAs, biopesticides, bioflucculants and biosurfactants, some other value-added products can also be produced and recovered from the nutrient-rich liquor or biomass through TSBPs, such as biofertilizer and enzymes (Angenent et al., 2004; Pham et al., 2009) For instance, polyphosphate-accumulating organisms (PAO) are one of the most widely recognized bacteria species for producing storage polymers (PHA, glycogen and polyphosphates) (Cech and Hartman, 1993) Hence, the biomass from two-stage PHA production process is also rich in phosphate, and thus can be also utilized for production of phosphate biofertilizer (de-Bashan and Bashan, 2004) Another example is the further utilization of the hydrolytes of activated sludge or marine-product processing wastewater, which can be well used to culture protease-producing species like Bacillus licheniformis and to recover protease (Chenel et al., 2008; Souissi et al., 2008) In fact, there can be so many choices of recoverable biochemicals as there are many variations of microbial metabolism pathways in biological wastewater treatment systems The major challenges are how to selectively direct the reaction toward the production of valuable products and how to separate and recover them from the fermentation effluent or the biomass in a cost-effective way Challenges and future perspectives While TSBPs for methane production has already find widespread applications, the other bioenergy and biochemical TSBPs are mostly still in their infancy Apart from the diverse restrictions occurred in W.-W Li, H.-Q Yu / Biotechnology Advances 29 (2011) 972–982 the various available TSBPs, there also exist some common challenges for their scaling up and commercialization 5.1 How to make a choice among the different processes and end-products? The conversion of wastewater to bioenergy and biochemicals via TSBP has been advancing in two fronts: enhancing substrate utilization and hydrolytes optimization in the first stage, and maximizing product yield and quality in the second stage Numerous researches have demonstrated that hydrolysis and acidification presents a critical preceding step, during which almost all feedstock, from simple carbohydrates to complex biomass residues, can be well degraded to VFAs and other intermediates The produced VFAs are subjected to further utilization in the subsequent stage for production of a variety of energy and valuable products While the first stage of acidogenic fermentation can be relatively simple and uniform, the second stage allows a wide range of choices on biotechnologies and corresponding target products, such as methane, hydrogen, electricity, PHA, biopesticides, bioflocculants, biosurfactants and other valuable-added products Thus, there comes the question: how to make a choice among the different processes and end-products, so as to maximize the product output and substrate degradation? This is a critical question to be answered, but currently there are no specific selection criteria available Generally speaking, technical feasibility, simplicity, economics, societal needs and political priorities are all key factors to be considered in choosing a bioconversion process for wastewater treatment In the technical perspective, the characteristic of wastewater and the desired form of the final end-product are two overriding factors, which determine the process configuration and the bacterial culture to be adopted, and thus affect the overall process efficiency For example, bioprocesses for the production of methane and hydrogen can all utilize carbohydrates as feedstock, but the energy yield can be different Attempts have been made to evaluate energy potential of organics for production of different end products (Levin et al., 2007) Based on a hydrogen yield of 1.3 mol H2/mol hexose in an OSBP, the potential hydrogen energy was estimated to be only 41.4% of the potential methane energy However, for most hydrogen TSBPs, the hydrogen yield can actually reach over mol H2/mol hexose (Kawaguchi et al., 2001; Kim et al., 2006) A comparison of the reported methane and hydrogen yield in Table also shows that hydrogen TSBP could be more energetically productive than a methane process (Redwood et al., 2009) It seems feasible to compare the energy potential of a substrate for different products However, in practice, a variety of factors including feedstock composition and concentration, microbial cultures and operation conditions can all significantly affect the overall performance of a system, and thus complicates the comparison Furthermore, there is no uniform basis for direct comparison of the yield of energy and chemicals in diversified forms Nevertheless, an approximate comparison of the merits and limitations of the different biotechnologies and bioproducts may offer some hints for the process selection Methane fermentation is the most mature technology with practical applications, but the methane gas, as a major greenhouse gas, need to be strictly controlled in transmission and has limited application Comparatively, hydrogen and electricity are more desirable forms of energy carriers with high versatility and environmental-friendliness Sugars are preferred substrates for hydrogen dark-fermentation, while VFAs from acidogenic fermentation can serve as ideal substrate for photohydrolysis and bioelectrolysis in hydrogen TSBPs However, the application of these processes is restricted by the constrained light conversion efficiency or the need for energy input MFC presents an amazing process to directly extract energy from organics, but this technology at the present stage is still limited by a lower power density and the relatively high cost of electrode and separator materials, and a pilot-scale implementation of such technology is still to be demonstrated (Logan, 2010) 977 TSBP for biochemical production presents an attractive way to recover bioproducts from the liquid phase or the biomass However, they raise more requirements on substrate composition compared with bioenergy production processes, as the impurities and toxic substances may severely affect the end-product yield and purity and arouse difficulty in product separation Moreover, more strict process control should be ensured Therefore, methane production through TSBP seems to be the most likely technology for practical use at the present stage Nevertheless, for the selection and design of a practical TSBP, a wide range of factors should be taken into account, such as the substrate type, operating condition and requirement on product quality And the future development of other biotechnologies may also bring new opportunities Production of single bioproduct with high purity is desirable for some specialized industrial applications While in a sense of maximizing resource reclamation, multi-products recovery may appear a more attractive way (Oh and Logan, 2005) For example, in hydrogen fermentation, hydrogen can be obtained as gaseous fuel, whereas the nutrient-rich slurry-phase residual can also be recovered as useful fertilizer It need be pointed out that, although the diversity in products benefits a higher process flexibility and practicability, it may simultaneously add to complexity of the system and increase cost Therefore, it should be considered whether the products have sufficient values to justify the added complexity of the system Moreover, the separation and purification of mixture products are also important factors to be considered when evaluating the feasibility of a TSBP 5.2 How to connect the two stages? For a TSBP system, the bridge connection between the two stages is a key aspect affecting the overall system productivity Transport of fermentation products from the first reactor to the second (while retaining biomass) presents an engineering challenge for scaling-up of such two-stage systems The simplest and most common method is ‘batch-transfer’ in which spent medium is transferred between reactors in batches However, for large-scale application, continuous processes are generally more preferable One common strategy for continuous transfer of fermentation products is using filtration membranes Microfiltration membranes have been used to separate the two bioreactors in a PHA-production TSBP (Yu and Si, 2001) The fermentative acids from the acidification reactor were continuously and effectively transferred to the aerobic reactor through the membrane, meanwhile a high content of biomass were maintained in the first stage However, the convective hydraulic flow across the membrane caused washout of the slow-growing PHAproducing bacteria in the second reactor, and thus lowered PHA production To sort out this problem, Du and Yu (2002) replaced the microfiltration membrane with a dialysis membrane This membrane allowed mass transfer via molecular diffusion, and thus exerted less hydrodynamic disturbance to the system As a consequence, an enriched culture of Ralstonia eutropha was successfully maintained and dominated in the second reactor, giving a very high PHA content The rapid advances in membrane technologies are making such mass transfer more efficient and selective in TSBPs Nevertheless, the membrane fouling in long-term operation may continue to be a significant barrier Thus, an investigation into effective fouling control technologies should be warranted in further studies Meanwhile, other cost-effective and efficient coupling strategies should also be explored 5.3 Process optimization For a bioconversion process, it is important to control the reaction pathway towards the selective production of desired end-products Although TSBPs enable higher operation flexibility and better process control than OSBPs, the complex and variation nature of practical 978 W.-W Li, H.-Q Yu / Biotechnology Advances 29 (2011) 972–982 wastewater as well as the many influencing factors of operation conditions may still bring about risks in low product yield and components uncertainties The concentration and composition of VFAs, as the key intermediates from acidogenic fermentation, are of critical importance for a TSBP (Belokopytov et al., 2009) The properties of VFAs, which are highly dependent on the substrate type and process conditions such pH, HRT and temperature, can significantly affect the end-product yield and quality (Banerjee et al., 1998; Yu and Fang, 2001, 2003) Therefore, it is essential to properly control the VFAs production process via operating condition optimization (Shi and Yu, 2005; Yu et al., 2004; Zheng and Yu, 2005) However, this would rely on a timely monitoring of the reactor status and may require complicated control procedures, which may present a challenge for large-scale applications Noticeably, a different bioconversion process in the first stage may also lead to different pattern of VFA production In this regard, although an acidogenic fermentation step incorporating dark-fermentation can be selected as the first stage to enable additional hydrogen production (Kyazze et al., 2007), it doesn't necessarily lead to higher overall hydrogen yield compared to that without dark-fermentation Fig illustrates that diverse types of fermentations (e.g., lactic acid, mixed-acid and Clostridial-type fermentation) can be applied with different hydrogen yields In fact, from this diagram we can see that a higher overall yield could be expected using lactic acid or mixed-acid fermentation in the first stage followed by a more efficient step of lactate conversion Currently, intensive researches are underway to meet a target of 10 mol-H2/mol-hexose by TSBPs (Vazquez et al., 2008) Therefore, from an overall process perspective, a simple preceding stage of hydrolysis/acidification is likely to enable a better intermediate production and higher hydrogen potential However, this is yet to be experimentally evidenced, and the low efficiency of subsequent photofermentation or bioelectrolysis process presents a bottleneck at the present stage In addition to intermediate control, the presence of toxic or inhibitive compounds in wastewater, such as endocrine disrupting substances, may also constitute hurdles These substances cannot be well degraded even in an individual hydrolysis/acidification process, thus they may not only adversely affect microbial activity but also deteriorate product quality and increase separation difficulties In this respect, the recent advances in metabolic engineering and genetic engineering are bringing new possibilities, by driving more efficient bioconversion via providing the microorganism with new and enhanced capabilities in chemical production and detoxification (Barnabé et al., 2009; Gavrilescua and Chisti, 2005; Kim, 2000) Nevertheless, these approaches of metabolic engineering or molecular level manipulation are less effective in mixture culture systems, due to the high system complexity and inter-influences between different microbial groups (Stephanopoulos, 2007) Regardless of which form of energy or bioproduct becomes the dominant fuel/resource of choice for the future, microbial conversion is at present and will continue to be the main route for energy recovery from wastes Therefore, apart from substrate and process condition control, further advances in microorganism regulating technologies for complex wastewater environment are also expected 5.4 Product separation and purification The separation and purification of desired bioproducts from the complex wastewater system present another big challenge A hybrid process of hydrogen and methane fermentation may lead to high overall energy production (Gavala et al., 2005; Ting et al., 2004; Ueno et al., 2007), however, the mixture gas product of methane and hydrogen constrains its application (Liu et al., 2006; Ueno et al., 2007) Moreover, some other undesirable gasses such as H2S and NH3 may also be produced during anaerobic fermentation, which not only leads to device corrosion but also generate harmful environmental emissions However, there are technological difficulties and A) Lactic acid fermentation lactate glucose ADP NAD+ ATP NADH LDH pyruvate B) Mixed acid fermentation 0-2 lactate glucose ADP NAD+ ATP NADH LDH pyruvate ADP 2ATP PFL 0-2 formate 0-2 acetyl-CoA PTA ACK FHL 0-2 H2 ADH 0-1 acetate 0-1 ethanol C)Conventional anaerobic fermentation 0-1 butyrate glucose ADP NAD+ 0-1 ADP ATP NADH 0-1 ATP pyruvate PFOR acetyl-CoA PTA ACK H2ase 2-4 H2 Fdred 0-2 ADP 0-2 ATP 0-2 acetate Fig Theoretical hydrogen production in different fermentation types (LDH lactate dehydrogenase; PFL: pyruvate formate lyase; ACK: acetate Kinase; FHL: formate hydrogen lyase; PTA: phosphotransacetylase; ADH: alcohol dehydrogenase; PFOR: pyruvate ferrodoxin oxidoreductase) economical barriers in separation and purification of these energy gasses (Abatzoglou and Boivin, 2009), thus it seems more practical to prevent the production of impurities during the fermentation process Comparatively, the separation and purification of biochemcials from wastewater is a more critical and challenging issue, because it usually presents the highest percentage of the manufacturing cost despite the adoption of pure culture or enriched bacteria for system simplification (Castilho et al., 2009) Moreover, complex operation procedures are required to extract the valuable products from the bulk liquid or biomass (Kleerebezem and Loosdrecht, 2007) These have significantly hindered the commercialization of bioconversion technologies Therefore, on the one hand, it is highly desirable to find applications of such biochemicals with low purity demand, for example many microorganism-derived products like bioflucculants, biosurfactants and biofertilizers can be directly used after simple treatment On the other hand, for some bioproducts of specific use W.-W Li, H.-Q Yu / Biotechnology Advances 29 (2011) 972–982 such as PHAs and amino acid, it is essential to develop efficient, economic and environmental friendly extraction processes 5.5 Other technological and economical considerations Microorganism is the key to all bioconversion processes Although pure or mixed culture (after enrichment) can both be employed in the second stage for most TSBRs, a diverse species of microorganisms may arouse risk of producing more varied microbial metabolites and impurities, while a pure culture favors higher product yield and quality (Nevin et al., 2008; Serafim et al., 2008) As is shown in Table 1, utilization of pure cultures for PHA production generally leads to a higher product yield than mixed culture system However, a pure culturing may necessitate substrate sterilization and increase system sensitivity to environmental changes Therefore, a balanced consideration of the technological and economical aspects should be given in the selection of microorganisms for TSBPs Except for methane production, most of the current waste-toresource TSBPs are still at a laboratory or even conceptional scale Therefore, an experimental demonstration and scaling up of these processes are required In light of the many existing technological and economical barriers and the numerous but rather inconsistent previous research results, several issues need to be highlighted in future works First of all, a standardization of data expression is necessary to allow reliable and accurate comparison of various investigations In addition, further advances in the reactors, operation, and microbial components of various bioconversion technologies are also needed to enhance their commercial competitiveness and practical applicability TSBPs facilitate a higher bioenergy/biochemical productivity and expand the scope of substrate utilization, thus implying a promising paradigm for future application However, it also adds to system complexity in terms of reactor configuration and operation and, as a consequence, increases investment and operational costs Therefore, it needs to be evaluated that whether the gain in productivity can offset against the increased cost arise from the stage separation Economical viability is an important factor to be considered in design and implementation of a TSBP For most of the existing TSBPs, it remains to be seen whether or not sufficiently high productivity can be achieved with appropriate capital and energy input, and whether a sophisticated process of reaction control, product separation and purification can be justified by the value of bioenergy and biochemical output From a practical perspective, upgrading the existing wastewater treatment facilities and implementing intelligent control of the bioconversion processes may provide a viable choice To achieve large-scale implementation of the TSBPs, future advances in microbes, materials and separation technologies should be warranted, and exploration in new processes and biotechnologies should be continued 5.6 Outlook Although still severely constrained at the present stage, TSBP will undoubtedly take an important role in the future market of sustainable energy and green chemistry technologies, because it seems to be the only way to rapidly complete the cycle of resource–wastes–resource Despite the numerous opportunities of TSBP, such as for production of methane, hydrogen, electricity, bioplastics, bioflocculants, biopesticides and iosurfactants, one should keep in mind that the a balanced consideration of the technical limitations, economy and social needs may finally determine its future paradigm Methane production via TSBP is by far the most practical technology for commercialization, but its application range of scope is limited In light of separation and downstream processing, bioelectricity is undoubtedly one of the most appealing forms of energy source from TSBP, because energy is directly extracted from the wastewater and no further separation is needed However, the currently achievable power output is far from enough to enable a practical application, and it is argued that the greatest future values of MFC may not be for 979 power generation but for MFC-assisted bioremediation and bioproduction (Franks and Nevin, 2010) Thus, MFC technologies in combination with other processes may be a more viable solution and may ultimately hold a position in future paradigm of TSBP applications Biosynthesis of chemical compounds such as bioplastics, bioflocculants, biopesticides are also possible, but it is yet to be seen that to how much degree we can produce and recover them from wastewater at an acceptable cost While biohydrogen production from wastewater is still at its infancy stage of development, such technologies are undergoing rapid progress in recent years and showing a promising prospect The hydrogen yield has grown dramatically from below mol-H2/mol-hexose to about mol-H2/mol-hexose through dark-photofermentation TSBP (Asada et al., 2006), which has reached 60% of the theoretical limit (Lee et al., 2008a, 2008b) While efforts are underway to further improve the conversion efficiency of individual processes, the integrated application of multiple processes is continuously bringing new possibilities for maximizing the energy and products exploitation at a higher rate (Hallenbeck and Ghosh, 2009), and the recent technological advances in lignocellulosic biomass degradation are putting it a step forward toward lowcost production (Ren et al., 2009) In addition, there is a strong need to develop sustainable clean energy, like hydrogen, to replace the depleting fossil fuels, in which respect the financial incentives are growing rapidly overall the world (IEA, 2011) Thus, although most of the studies on hydrogen production are still limited at laboratory scale, multioutput TSBPs for simultaneous recovering hydrogen and other products are likely to be the most promising technology that may ultimately see commercial-scale development in the near future Conclusions The concept of wastewater treatment is evolving from pollutant removal to resource recovery In this respect, bioconversion offers an appealing avenue to recover sustainable energy and valuable biochemicals from wastewater Especially, TSBPs seems to be the only practically viable choice By far, various TSBPs have been, or are being, developed and applied with different degrees of success While practical application of TSBPs for methane production has long been achieved, those for hydrogen, electricity, PHA, biopesticides, bioflocculants, biosurfactants and other biochemicals are yet to be demonstrated at pilot or even laboratory scale Several challenges arise from the separation of reaction processes, and many barriers remain for their scaling up and commercialization These include: how to make a choice among the wide option of combined processes so to maximize the energy/biochemical output? How to properly connect the two stages so that efficient mass transfer can be achieved without causing bacteria and product contamination to the second stage? How to optimize the individual stages in a hybrid system? How to achieve product separation and purification in efficient and costeffective way? Moreover, for each individual biotechnology, there are also specific technological and economic barriers to be overcome Notwithstanding the many remaining restrictions, the rapid advances in metabolic and biochemical engineering, process optimization, integration strategy as well as cost-effective separation and purification technologies are likely to bring the TSBPs out of the laboratory systems and to substantially promote and extend their application in the near future Furthermore, a better understanding of the microbiology and the fundamentals of TSBPs is expected to benefit the scaling up of the existing systems and the development of new technologies Therefore, TSBP represents a promising biotechnology paradigm to meet the dual ends of wastewater treatment and energy/biochemical production, and may finally make “green energy” and “bioproducts” practical and sustainable Acknowledgements The authors wish to thank the NSFC (50878203), the NSFC-JST (21021140001), and 863 Project 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acidification of glucose in a mixed culture forming part of a two-stage digestion process Water Res 1982;16:313–21 ... effluent Fat-containing wastewater Instant-coffee-production wastewater Dairy wastewater Acid whey wastewater Food waste-recycling wastewater Sucrose-containing wastewater Glucose-containing wastewater... Cellulose-containing wastewater Molasses wastewater Coffee processing wastewater Food processing wastewater Citrate and n-octane wastewater Starch-containing wastewater Starch-containing wastewater... complicates the comparison Furthermore, there is no uniform basis for direct comparison of the yield of energy and chemicals in diversified forms Nevertheless, an approximate comparison of the merits