脱氢酶文献

le c, H ong ng-g my e UASBr and irradiated to the granules. tion we tion en e dehyd aves po spectively, compared to the control. These increments were confirmed by spe- cific methanogenic activity test. When ultrasonication (UD 0.05 W/mL, UT 5 min) was irradiated every are as follows: (1) reduced waste volume, (2) generating energy- rich gas in the form of methane (CH4), and (3) yielding nutrient- containing final products. However, the slow growth rate of the methanogens has been identified as a disadvantage coupled with the performance fluctuation due to their highly sensitive charac- teristics (Mata-Alvarez et al., 2000; Lu et al., 2008; Speece, 1996). In order to retain a highly concentrated biomass, various types of high rate anaerobic treatment systems such as the anaerobic substrate was often limited (Jeison and Chamy, 1999). In order to solve the problem, expanded granular sludge bed reactor (EGSBr), establishing the fluidization of the granular sludge bed by recircu- lation of effluent to the conventional UASBr, was introduced (Kato et al., 1994). Accelerated mass transfer rate could lead to the up- graded digestion performance especially at higher organic loading rate (OLR) (Seghezzo et al., 1998). However, it would be much ben- eficial if we could achieve not only the accelerated mass transfer rate but also enhanced microorganism activity. Ultrasound has been widely applied to biological cell disrup- tions in order to recover intracellular materials (Harrison, 1991), ⇑ Corresponding author. Tel.: +82 42 821 1263; fax: +82 42 821 1476. Bioresource Technology 120 (2012) 84–88 Contents lists available at T els E-mail address: saeun@hanbat.ac.kr (S.-E. Oh). High rate anaerobic treatment system Upflow anaerobic sludge blanket reactor (UASBr) Low strength ultrasonication Dehydrogenase activity (DHA) Adenosine triphosphate (ATP) 8 h during the continuous operation of UASBr, it caused a gradual drop of methanogenic activity, com- plete loss after 20 days. At further operation, UT was decreased to 1 s but irradiated every 1 min, which resulted in a 43% higher specific CH4 production rate. � 2012 Elsevier Ltd. All rights reserved. 1. Introduction The steep increase in energy prices since the 1970s has reduced the attractiveness of aerobic waste treatment, contributing to the redirection of the research efforts towards energy saving alterna- tives such as anaerobic digestion (AD) (van Haandel and Lettinga, 1994). AD is the bioconversion process consisting of hydrolysis, acidogenesis, acetogenisis, and methanogenesis. Its advantages contact reactor, anaerobic filter reactor, fluidized bed reactor, ex- pended bed reactor, anaerobic membrane bioreactor, and upflow anaerobic sludge blanket reactor (UASBr) have been introduced (Lettinga et al., 1987). Among these reactors, UASBr has been con- sidered as the most powerful process with its successful operation and worldwide popularity being attributed to the dense sludge bed made of granules in the reactor bottom. However, as there is no mechanical mixing, the mass transfer between the granules and Keywords: optimal conditions, resulti tent by 257%, and 374%, re " Ultrasonicator was installed inside th " 0.05 W/L, 1 s per 1 min of ultrasonica " Forty-three percentage of CH4 produc " It was attributed to the increase of th " In addition, propagation of acoustic w a r t i c l e i n f o Article history: Received 14 April 2012 Received in revised form 13 June 2012 Accepted 15 June 2012 Available online 21 June 2012 0960-8524/$ - see front matter � 2012 Elsevier Ltd. A http://dx.doi.org/10.1016/j.biortech.2012.06.046 re applied to methanogenic granules. hancement was observed during the continuous operation. rogenase activity and ATP content. ssibly accelerated the mass transfer. a b s t r a c t In this study, low-strength ultrasonication was applied at various ultrasonication densities (UD) (0–0.1 W/mL) and ultrasonication time (UT) (0–30 min) to methanogenic granules on the purpose of increasing their activity, and eventually, enhancing the performance of upflow anaerobic sludge blanket reactor (UASBr). Batch test results showed that 5 min of ultrasonication at 0.05 W/mL was found to be the ng in the increase of dehydrogenase activity and adenosine triphosphate con- h i g h l i g h t s Enhanced activity of methanogenic granu Si-Kyung Cho a, Dong-Hoon Kimb, Moon-Hwan Kim aDepartment of Civil and Environmental Engineering, KAIST, 373-1 Guseong-Dong, Yuse bClean Fuel Department, Korea Institute of Energy and Research, 102 Gajeong-ro, Yuseo cDepartment of Environmental Engineering, Hanbat National University, San 16-1, Duck Bioresource journal homepage: www. ll rights reserved. s by low-strength ultrasonication ang-Sik Shin a, Sae-Eun Oh c,⇑ -gu, Daejeon, Republic of Korea u, Daejeon, Republic of Korea oung-dong, Yuseong-gu, Daejeon, Republic of Korea SciVerse ScienceDirect echnology evier .com/locate /bior tech strength was applied to the methanogenic granules with the objec- tive of enhancing methanogenic activity and improving mass was observed, the ultrasound was irradiated every minute at 0.05 W/mL of UD and 1 s of UT (Phase II). From the 51th day, ultra- sonication was ceased for 10 days (Phase III), and then turned on again (Phase IV). The detailed ultrasonication conditions in the continuous operation of UASBr are summarized in Table 1. 2.2. SMA test SMA test was conducted in order to confirm the optimized ultrasonication conditions determined by the analysis of dehydro- genase activity and ATP content. In a 300 mL serum bottle, 30 g of methanogenic granules were placed with 150 mL of substrate con- taining 2 g glucose/L. Initially, pH was adjusted to 7.5, and then the anaerobic medium solution was added. Each liter of anaerobic medium solution contained 0.53 g of NH4Cl, 0.27 g of KH2PO4, 0.35 g of K2HPO4, 1.20 g of NaHCO3, 0.075 g of CaCl2�2H2O, 0.10 g of MgCl2�6H2O, 0.02 g of FeCl2�4H2O, 0.05 g of MnCl2�4H2O, 0.05 g of H3BO3, 0.05 g of ZnCl2, 0.03 g of CuCl2, 0.01 g of Na2MoO4�2H2O, 0.50 g of CoCl2�6H2O, 0.05 g of NiCl2�6H2O, and 0.05 g of Na2SeO3 (Kim et al., 2007). All bottles were purged with N2 gas in order to provide anaerobic conditions. The bottles were incubated in a shaking incubator at 35 �C, and all tests were conducted in duplicate. Biogas production and its constituents were monitored every day, and CH4 production was calculated from the headspace mea- surements of gas composition and the total volume of biogas pro- duced at each time interval using the mass balance Eq. (1). VH;i ¼ VH;i�1 þ CH;iðVG;i � VG;i�1Þ þ VHðCH;i � CH;i�1Þ ð1Þ where VH,i and VH,i�1 = cumulative biogas volumes at the current (i) Tec transfer, and finally increasing the specific CH4 production rate. In order to optimize the ultrasonication condition, the ultrasound was applied at various ultrasonication densities (UDs) and ultra- sonication times (UTs). As an indicator of the methanogenic activ- ity, the dehydrogenase activity and adenosine triphosphate (ATP) content were measured, then it was confirmed by a conventional specific methanogenic activity (SMA) test. Finally, the effect of intermittent low-strength ultrasonication was investigated during the continuous operation of UASBr. 2. Methods 2.1. System setup and operating conditions The methanogenic granules used in this study was obtained from a full scale anaerobic plant treating brewery wastewater lo- cated in Cheongwon, Korea. The pH and concentrations of volatile suspended solids (VSS) were 7.6 and 105.4 g/L, respectively. As shown in Fig. 1, 5 L of UASBr (lower part: 690 mm height � 85 mm inside diameter (i.d.); upper part: 165 mm height � 130 mm i.d.) installed with four vibrators (50 W, 20 kHz) was prepared for the tests. After placing two liters of gran- ules into the UASBr, ultrasound was applied for 10 min at various UDs, ranging from 0 to 0.1 W/mL. Next, UT was varied from 0 to 30 min at a fixed UD of 0.05 W/mL. At the end of each ultrasonica- tion, the granules were taken in order to measure the dehydroge- nase activity and ATP content, and new granules were prepared for the next experiment. All tests were performed in duplicate and the results were averaged. In the continuous operation of UASBr, 5 g chemical oxygen de- mand (COD)/L of acidified mixture (food waste and livestock waste; V:V = 6:4) was fed at an organic loading rate (OLR) of 2.5 g COD/L/day. The produced gas was collected by a gas collector and sampled using a 1 ml syringe to analyze CH4 content. During the continuous operation, the dehydrogenase activity and ATP con- and its applications have spread to various research fields such as enzyme extraction, pollutant removal, and coal cleaning (Bougrier et al., 2005; Tiehm et al., 2001). During ultrasonication, an acoustic wave propagates in the liquid media, and then cavitation bubbles are generated in the rarefaction region. The hydro-mechanical shear stress localizes the temperature increase up to 5000 K, and the OH-radicals generated via the cavitation rupture lead to the destruction of microorganism cell walls and membranes (Riesz and Kondo, 1992). While ultrasound was applied at high strength levels in the above cases, it was sometimes applied at low strength levels that does not cause cell disruption but increases the enzyme activity and cell membrane permeability (Liu et al., 2003; Pitt and Ross, 2003). By applying ultrasonication at 0.2 W/cm2 for 10 min, the activity of anaerobic digester sludge was enhanced with organic removal increase by 30% (Xie et al., 2009). When ultrasound was irradiated (less than 113.9 mW/cm3) for 1–8 min, the biosynthesis of the shikonin was stimulated (Lin and Wu, 2002). In nitrogen re- moval process using anaerobic ammonium oxidation bacteria, 25.5% of performance enhancement was observed by applying ultrasonication at 0.3 W/cm2 for 4 min (Duan et al., 2011). As men- tioned, low-strength ultrasound has been applied to many types of microbial consortiums; however, there has been no attempt to methanogenic granules. With this research background, in the present work, low S.-K. Cho et al. / Bioresource tent of the granules were measured every other day. For the first 20 days, the ultrasound was irradiated every 8 h at 0.05 W/mL of UD and 5 min of UT (Phase I). However, as the performance failure Fig. 1. Schematic diagram of ultrasonicator attached UASBr. Table 1 Ultrasonication conditions of ultrasonicator attached UASBr. Phase (operation day) Ultrasound density (W/mL) Ultrasound time Ultrasound interval I (Day 0–20) 0.05 5 min 8 h II (Day 21–50) 0.05 1 s 59 s III (Day 51–61) – – – IV (Day 62–70) 0.05 1 s 59 s hnology 120 (2012) 84–88 85 and previous time (i � 1) time intervals; VG,i and VG,i-1 = total biogas volumes in the current and previous time intervals; CH,i and CH,i�1 = the fractions of methane gas in the headspace of the bottle immediately. All sample tubes were shaken slightly for reaction Fig. 2 shows the effect of UD on dehydrogenase activity and ATP reported that microbial granules withstand compression and high shear owing to DLVO (named after Derjaguin, Landau, Verwey, and Overbeek)-type interaction, extracellular polymeric substances bridging effect, and hydrophobic interaction (Liu et al., 2009). The ATP content also showed a similar trend to that of dehydroge- nase activity: a significant increase at 0.05 W/mL, from 2.86 to 19.34 � 104, and a negligible increase with further UD increase. In the next test, UD was maintained at 0.05 W/mL while the ultrasound was irradiated for 0 min to 30 min. Unlike the effect of the UD level, significant decreases in both dehydrogenase activ- ity and ATP content, from 3.01 to 1.24 and from 18.94 to 7.32 � 104, respectively, were observed when ultrasound irradi- ated longer than 10 min (Fig. 3). Loosen and partially broken gran- ules were observed when the ultrasound was irradiated for a long period (figure not shown). From the above experiments, 0.05 W/ mL of UD and 10 min of UT were selected as the optimum ultrason- ication conditions. 3.2. Confirmation via SMA test Fig. 3. Effect of ultrasonication time on dehydrogenase activity and ATP content (dehydrogenase activity and ATP content were expressed by O.D. and R.L.U, respectively). Tec content, which were expressed via the optical density (OD) and rel- ative light unit (RLU), respectively. A significant increase in the dehydrogenase activity from 0.85 to 2.62 was observed at 0.025 W/mL, but there was a negligible increase with further UD increases. Unlike the results in this study, the dehydrogenase activ- for 30 min. A drop of vitriol oil was added to finish the reaction, and 5 mL of ethyl acetate was supplemented to sample tubes. All sample tubes were mixed thoroughly and extracted for 6 min at 90 �C, then centrifuged at 4000 rpm for 10 min. The supernatants of the samples were colormetrically measured at 485 nm, and absorbency was obtained. ATP is a multifunctional nucleotide used in cells as a coenzyme. It is often called the ‘‘molecular unit of currency’’ of intracellular energy transfer, and it transports chemical energy within cells for metabolism (Knowles, 1980). ATP was measured by ATP kit (Clean-Trace Luminometer, 3 M, USA) using three particles of gran- ular sludge, it was expressed by RLU (Relative Light Unit). Average value was obtained after 10 sets measurement. The concentrations of the COD and VSS were measured accord- ing to standard methods (APHA, 1998). The measured biogas pro- duction was adjusted to a standard temperature (0 �C) and pressure (760 mm Hg) (STP). The CH4 gas content was analyzed via gas chromatography (GC, Gow Mac Series 580) equipped with a thermal conductivity detector (TCD) and a 2 m � 2 mm stainless steel column packed with a Porapak Q mesh 80/100 with helium as the carrier gas. The temperatures of the injector, detector, and col- umn were maintained at 80, 90, and 50 �C, respectively. 3. Results and discussion 3.1. Optimization of ultrasonication conditions SMA test is an easy method for assessing the methanogenic activity, but it requires a long time to accomplish (Ince et al., 1994). In this study, instead, dehydrogenase activity and ATP con- tent were measured, which require much less time. The dehydro- genase activity is related to a group of enzymes that participate in the metabolic reactions producing energy in the form of ATP through the oxidation of organic matter (Barrena et al., 2008), and the bioconversion process of the organic matter into biogas is catalyzed through the oxidation and reduction of hydrogen. measured using gas chromatography in the current and previous intervals; VH = the total volume of headspace in the reactor (Oh et al., 2003). The accumulated methane was divided by the volatile suspended solids content of the inoculum, which serves as a proxy for active biomass in the batch reactors. 2.3. Analytical methods The mechanism of measuring DHA is that TTC (2,3,5-trip- henyltetrazoluimchloride) is used as hydrogen receiver in cell res- piration, and reduced TTC forms a reddish color substance called TF (Triphenyl Formazan) which is proportional to its concentration and can be measured colormetrically (Xie et al., 2009). The following materials and reagents were added to centrifuge tubes (50 mL): 0.5 mL of 0.36% Na2SO3, 0.5 mL of 0.00577% CoCl2, 1.5 mL of tri-buffer (pH 8), 2 mL of granular sludge, 0.5 mL of 0.4% TTC and 1 mL of synthetic substrate (4 g COD/L adjusted by glucose). After shaking, the sample tubes were placed in a water- bath at a constant temperature (37 �C) under without any light 86 S.-K. Cho et al. / Bioresource ity of anaerobic flocs suddenly dropped when UD exceeded a spe- cific level (Xie et al., 2009). It appeared that this was resulted from the more rigid structure of the granules than that of flocs. It was Fig. 2. Effect of ultrasonication density on dehydrogenase activity and ATP content (dehydrogenase activity and ATP content were expressed by O.D. and R.L.U, respectively). hnology 120 (2012) 84–88 In order to confirm that the dehydrogenase activity and ATP content could represent the methanogenic activity, SMA test was conducted using the granules ultrasonicated for 0–30 min at Tec 0.05 W/mL. Cumulative CH4 production was divided by an initial biomass concentration to obtain specific methanogenic activity. As shown in Fig. 4, the activity for the control sample, 5, 10, 15, 20, and 30 min of ultrasonication were 23.5, 32.0, 32.4, 27.1, 22.3, and 20.6 mL CH4/g VSS/day, respectively, which was coin- cided with the dehydrogenase activity and ATP content results. De- creases in the methanogenic activity after 10 min of ultrasonication in the granules could be explained by the decrease of dehydrogenase activity and ATP content. In particular, 30 min of UT led to an even lower activity than the control. The maximum methanogenic activity was observed at 10 min of UT, but 0.05 W/ mL of UD with 5 min of UT were chosen as the optimum conditions because 10 min of UT resulted in only a slight enhancement of the methanogenic activity of 0.4 CH4/g VSS/day despite having double the energy consumption compared with 5 min of UT. The positive effects of ultrasonication on AD performance have been widely reported in previous researches: 64% of specific CH4 production rate enhancement through 30 min of UT (20W, 9 kHz) and 51% of specific CH4 production rate enhancement through ultrasonication by 9350 kJ/kg TS have been reported by Wang et al. (1999) and Bougrier et al. (2006), respectively. Kim et al. (2010) reported a significant increase in specific CH4 produc- tion rate from 82 to 127 ml CH4/g CODadded through combining the alkaline pretreatment (pH 9) with 7000 kJ/kg TS of ultrasonication. However, different mechanisms need to be applied in order to ex- plain the performance enhancement between low-strength and Fig. 4. Effect of low strength ultrasonication on specific methanogenic activity. S.-K. Cho et al. / Bioresource high-strength ultrasonications. While the enhancement of AD per- formance by low-strength ultrasonication was attributed to the in- crease of methanogenic activity through the stimulation of the biological enzymes, the latter was explained by the increase of the substrate availability in the solubilizing inert and slowly biode- gradable sections. Accordingly, the understanding of the decrease in CH4 production under excessive ultrasonication should be ap- proached differently. For low-strength, it was attributed to the damage or lost viability of the methanogens; however, for high- strength, it was explained by the generation of inhibitors such as melanoidines, furfural, and hydroxymethylfurfural (HMF), which are known as barely biodegradable substances (Dwyer et al., 2008; Palmqvist and Hahn-Hagerdal, 2000). 3.3. Effect of ultrasonication during the continuous operation of the UASBr In order to see the effect of low-strength ultrasonication in the continuous operation of UASBr, two UASBr (control and ultrasoni- cator attached UASBr) were operated for 70 days and the daily CH4 production is shown in Fig. 5. Unlike our expectation, although a slight higher CH4 production was observed until day 14, a drastic decrease in the CH4 production was observed at further operation coupled with a lower COD removal and the deterioration of efflu- ent turbidity probably due to the disaggregation of granules and cell rupture (Phase I). It seemed that the enhanced microbial activ- ity by low-strength ultrasonication resulted in the improvement of CH4 production in the initial period; however, a continuous ultra- sonication caused a loss of methanogenic activity. Acoustic waves could break granule formation and also make the cell wall thinner. The enhancement of the nitrogen removal performance and de- crease of the cell wall thickness from 5.6 to 4.5 nm have been re- ported by Duan et al. (2011) after 4 min of ultrasonication in the seed sludge at