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ABSTRACT The influence of phosphorus concentration on the biodegradation of dissolved organic matter (DOM) was discussed as a possible cause of the accumulation of chemical oxygen demand by acidic permanganate method (CODMn) in Lake Biwa. Enhancement of oxygen consumption in the biodegradation test by dissolved phosphorus (DP) was observed in Lake Biwa water with the natural bacterial community. The response-curve of the oxygen consumption on DP was expressed by a modified Michaelis-Menten type equation with a threshold. The threshold of DP for oxygen consumption was in the range of 0.0031 to 0.0040 mgP/L. The pseudo first-order biodegradation rate constant of DOM was estimated to be 0.0022 d-1 in 1980s and 0.0014 d-1 in 2000s based on DP in the northern basin of Lake Biwa. Consequently, DOM in 2000s was expected to be 1.6 times higher than that in 1980s from mass balance analysis. Since the ratio of CODMn observed in 2000s to that in 1980s in the northern basin of Lake Biwa was 1.3, it was likely that the deterioration of bioactivity for DOM degradation contributed to the accumulation of CODMn in Lake Biwa.
Journal of Water and Environment Technology, Vol. 9, No.2, 2011 Address correspondence to Naoyuki Kishimoto, Department of Environmental Solution Technology, Faculty of Science and Technology, Ryukoku University, Email: naoyuki@rins.ryukoku.ac.jp Received November 19, 2010, Accepted April 1, 2011. - 215 - Influence of Phosphorus Concentration on the Biodegradation of Dissolved Organic Matter in Lake Biwa, Japan Naoyuki KISHIMOTO*, Kazunori UENO** *Faculty of Science and Technology, Ryukoku University, Otsu 520-2194, Japan **Lake Biwa-Yodo River Water Quality Preservation Organization, Osaka 540-6591, Japan ABSTRACT The influence of phosphorus concentration on the biodegradation of dissolved organic matter (DOM) was discussed as a possible cause of the accumulation of chemical oxygen demand by acidic permanganate method (COD Mn ) in Lake Biwa. Enhancement of oxygen consumption in the biodegradation test by dissolved phosphorus (DP) was observed in Lake Biwa water with the natural bacterial community. The response-curve of the oxygen consumption on DP was expressed by a modified Michaelis-Menten type equation with a threshold. The threshold of DP for oxygen consumption was in the range of 0.0031 to 0.0040 mgP/L. The pseudo first-order biodegradation rate constant of DOM was estimated to be 0.0022 d -1 in 1980s and 0.0014 d -1 in 2000s based on DP in the northern basin of Lake Biwa. Consequently, DOM in 2000s was expected to be 1.6 times higher than that in 1980s from mass balance analysis. Since the ratio of COD Mn observed in 2000s to that in 1980s in the northern basin of Lake Biwa was 1.3, it was likely that the deterioration of bioactivity for DOM degradation contributed to the accumulation of COD Mn in Lake Biwa. Keywords: bioactivity, biodegradation, dissolved organic matter, phosphorus INTRODUCTION Lake Biwa is located in Shiga Prefecture, which is in the center of Honshu Island (Fig. 1), and is the largest lake in Japan. It has a surface area of 674 km 2 , a volume of 27.5 km 3 , a maximum depth of 104 m, a mean depth of 41 m, a catchment area of 3,174 km 2 and a residence time of 5.5 years (International Lake Environment Committee, 2001). Lake Biwa is divided into two basins, south and north. The southern basin is shallow and small with a mean depth of 4 m and a volume of only 0.2 km 3 , but the northern basin is deep and large with a mean depth of 43 m and a volume of 27.3 km 3 . Thus, most of the water is stored in the northern basin. Lake Biwa is very important as a drinking water source for more than 14 million residents. Therefore, the national government and the local government have made efforts for the preservation of the water environment of Lake Biwa. As a result, the annual mean biochemical oxygen demand (BOD) and total phosphorus (TP) concentration have been improved from 0.8 mgO 2 /L and 0.011 mgP/L in 1979 to 0.5 mgO 2 /L and 0.008 mgP/L in 2009, respectively (Fig. 2; Shiga Prefecture, 2010). Although the chemical oxygen demand measured by acidic permanganate method (COD Mn ; Japanese Industrial Standard, 1998) has also decreased from 2.4 mgO 2 /L in 1979 to 1.9 mgO 2 /L in 1984, COD Mn increased again after 1984 and plateaued around 2.7 mgO 2 /L after 1998 (Shiga Prefecture, 2010). Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 216 - Fig. 1 - Location of Lake Biwa Fig. 2 - Trend in water quality of the northern basin of Lake Biwa. Data originated from Shiga Prefecture (2010) Many researchers have discussed the cause of the diremption phenomenon of BOD and COD Mn in Lake Biwa but COD Mn loadings from the watershed were unable to explain this phenomenon (Hayakawa, 2005; Kishimoto, 2008). As Matsui (1975) reported that particulate organic matter did not affect COD Mn , the possibility of accumulation of dissolved organic matter (DOM) is pointed out (Imai, 2002). On the other hand, Takahashi (1999) reported that dissolved organic carbon (DOC) in 1997 did not increase Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 217 - in comparison with that in 1985. However, as the particle retention diameter of filter used for separating dissolved matter and particulate matter in 1985 was larger than in 1999 in Takahashi's report, it is difficult to conclude that DOC in Lake Biwa did not increase between 1985 and 1997. In addition, even if DOC in two water samples are the same, COD Mn of both samples are generally different, because the ratio of COD Mn to DOC is generally changed by the composition of organic matter in the sample. Therefore, it can be pointed out that DOM measured by COD Mn might increase in Lake Biwa without DOC increase. There are several reports on the characterization of DOM in Lake Biwa. Hori et al. (1998) tried to fraction DOC based on the adsorption characteristics onto hydrous iron oxide at pH ranging from 4 to 6. As a result, the adsorption active DOC seasonally changed in the range of 0.7 to 1.2 mgC/L and was correlated with COD Mn , whereas the adsorption inert DOC showed a steady concentration of around 0.4 mgC/L and less correlation with COD Mn . Imai et al. (1998) discussed the origin of DOC in Lake Biwa by the content of six DOC fractions and the ratio of UV/DOC. As a result, DOC in Lake Biwa was inferred to be mainly affected by primary production. Aoki et al. (2004) investigated the behavior of humic substances and other DOM and implied that the increase in COD Mn was attributed to the contributions not only of humic substances but also of hydrophilic acids. These reports indicate that DOM in Lake Biwa consists of stable and fluctuating fractions and is influenced not only by humic substances but also by hydrophilic acids, which are thought to be provided by primary production. From the viewpoint of mass balance, the four possible pathways to increase DOM in the lake are as follows: (1) increase in the DOM loading from the watershed, (2) increase in internal production of DOM, (3) decrease in DOM discharge from the lake, and (4) decrease in the biodegradation rate of DOM in the lake. However, the possibilities of the pathways (1) and (3) to happen are thought to be weak because the decrease in COD Mn loadings and the increase in COD Mn discharge have been reported (Hayakawa, 2005; Kishimoto, 2008). For pathway (2), the decrease in chlorophyll concentration during 1980s (Ichise et al., 1999) and the shift of phytoplankton species from larger ones to smaller ones have been reported in Lake Biwa (Kishimoto et al., 2009). Although the former is a factor lowering the internal production, the latter is a factor enhancing the internal production, because small phytoplankton species generally show higher productivity (Kagami and Urabe, 2001). Accordingly, it is difficult to judge that pathway (2) happens or not. Gurung and Urabe (1999) reported that the addition of 2.5µM of phosphorus enhanced the growth of heterotrophic bacteria in the surface water of Lake Biwa, although the enhancement effect of phosphorus addition was inhibited by low temperature in hypolimnion. Yoshida et al. (2004) reported that the addition of 2µM of phosphate augmented the biodegradation of glucose by benthic bacterial community in Lake Biwa, but the effect was not observed for coastal surface water due to a high phosphate concentration of 4.78µM originating from the native water. These reports indicate that phosphorus is one of the limiting nutrients for the biodegradation activity of the bacterial community in limnetic zone of Lake Biwa. As shown in Fig. 2, TP in the northern basin of Lake Biwa is lower than 2µM and has been decreasing. Accordingly, pathway (4) with the decrease Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 218 - in phosphorus concentration is unable to be denied. In this research, we evaluated the influence of phosphorus concentration on the biodegradation of DOM and discussed the possibility of pathway (4) to happen in the northern basin of Lake Biwa. MATERIALS AND METHODS Materials Lake Biwa water was sampled from a depth of 60 m at Imazuokichuo point (Fig. 1) on Oct 5, 2009, because hypolimnetic DOM in Lake Biwa was stable and refractory (Hayakawa and Takahashi, 2002). Prior to biodegradation test, the lake water was filtrated by a glass fiber filter with a particle retention of 1.0 µm (Whatman GF/B; Whatman, Japan). Secondary effluent from a sewage treatment plant (Konan-chubu Sewage Treatment Plant, Otsu, Japan) was used as a source of trace nutrients. Filtration with 0.2 µm pore size membrane filters was applied to the secondary effluent before its use as the source of trace nutrients. The water quality of the filtrated lake water and secondary effluent are summarized in Table 1. Phosphate buffer solution, which contained 21.75 mg/L of dipotassium hydrogenphosphate, 8.5 mg/L of potassium dihydrogenphosphate, 44.6 mg/L of disodium hydrogenphosphate dodecahydrate and 1.7 mg/L of ammonium chloride, was prepared for the adjustment of phosphorus concentration. All chemicals were guaranteed analytical grade obtained from Nacalai Tesque (Japan) and used without further purification. Water quality data of Lake Biwa from 1979 to 2009 were obtained from the public database prepared by Lake Biwa Environmental Research Institute (2011). Biodegradation Test Procedure Experimental procedure for biodegradation was as follows: Adequate amount of phosphate buffer, filtrated lake water and, if necessary, 5 mL of filtrated secondary effluent were poured into a 102 mL BOD bottle. Then, the BOD bottle was incubated in the dark for 5 days at a temperature of 20ºC. In addition, biodegradation of secondary effluent filtrated was also performed as follows: 5 mL of the filtrated lake water and 97 Table 1 - Water qualities of filtrated secondary effluent and Lake Biwa water Parameter Secondary effluent Lake Biwa water pH Electrical conductivity (EC) 5-day Biochemical oxygen demand (BOD) COD by permanganate method ( COD Mn ) COD by dichromate method (COD Cr )* Dissolved nitrogen (DN) Dissolved phosphorus (DP) 7.0 430 µS/cm 0.43 mgO/L 4.4 mgO/L 5.7 mgO/L 7.55 mgN/L 0.026 mgP/L 7.1 126 µS/cm N.D. 2.0 mgO/L 3.3 mgO/L 0.42 mgN/L 0.005 mgP/L N.D.; not detected. * Measured after concentration with a rotary evaporator Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 219 - mL of the filtrated secondary effluent were poured into a 102 mL BOD bottle and incubated under the same condition aforementioned. Initial and final dissolved oxygen (DO), pH, and initial dissolved phosphorus (DP) were measured. Analytical methods used in this study were as follows: DO was measured with a DO meter (LDO HQ10, Hack, USA), pH with a pH meter (B-212, Horiba, Japan), DN by persulfate digestion - automated cadmium reduction method (American Public Health Association et al., 1998), DP by persulfate digestion method (Japanese Industrial Standard, 1998), COD Cr by closed reflux - colorimetric method (American Public Health Association et al., 1998) and COD Mn by effective colorimetric method (Ishii and Urano, 1999). All the equipment used were prewashed with diluted hydrochloric acid (1+11) to avoid phosphorus contamination. Evaluation of oxygen consumption Oxygen consumption for 5-day incubation (OC obs ) was calculated as the difference between DO before and after incubation. Then, oxygen consumption by the lake water (OC) was estimated from equation (1). OC[mgO 2 /L] OC obs r SE OC SE r LW (1) where, r SE is the ratio of the filtrated secondary effluent to the volume of the BOD bottle, r LW is the ratio of the filtrated lake water to the volume of the BOD bottle, and OC SE is the oxygen consumption by the filtrated secondary effluent. RESULTS AND DISCUSSION Biodegradation of DOM in Lake Biwa Experimental results of the biodegradation test using Lake Biwa water are demonstrated in Fig. 3. As shown in Fig. 3, single addition of phosphorus enhanced the OC. However, simultaneous addition of phosphorus and the treated secondary effluent much more enhanced the OC compared to single addition of phosphorus. Accordingly, Fig. 3 - Relationship between OC and DP for the biodegradation of DOM in Lake Biwa. Natural microorganism community in Lake Biwa water was responsible for biodegradation Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 220 - biodegradation activity in Lake Biwa water was thought to be limited by both the phosphorus concentration and other micronutrients. In general, the rate of biochemical reaction such as biodegradation depends on substrate concentration, and it is expressed by the Michaelis-Menten relationship (Sawyer et al., 2003). However, the data in Fig. 3 seems to have a threshold on DP. Therefore, the data were fitted into equation (2) using the Levenberg-Marquardt algorithm programmed into DeltaGraph software (RedRock Software, USA). OC OC max C DP T DP K+ C DP T DP (2) where, OC max is the maximum value of OC, C DP is DP concentration, T DP is a threshold of C DP , and K is a half saturation constant for C DP –T DP . The fitting curve obtained is also shown in Fig. 3 and the constants of the obtained regression lines and the coefficient of determination are summarized in Table 2. The simultaneous addition of phosphorus and the treated secondary effluent showed 8.5 times higher OC max than in the case of single addition of phosphorus. However, the other constants, K and T DP , were not very much different. Thus, the curve-fitting results demonstrated that the threshold of DP for biodegradation was in the range of 0.003 to 0.004 mgP/L and biodegradation activity dropped to half of the maximum value when DP was in the range of 0.006 to 0.007 mgP/L. Effect of biodegradation activity change on DOM concentration in Lake Biwa Effect of biodegradation activity change on DOM concentration in Lake Biwa is discussed here. When a part of the water body in Lake Biwa is considered as a control volume, the following mass balance equation is established. V dC dt L in L out kCV (3) where, C is DOM concentration [mgO 2 /L], L in is organic load to the control volume [mgO 2 /d], which contains an influent load and an internal production, L out is missing load except biodegradation [mgO 2 /d], k is a pseudo-first order biodegradation rate constant [d –1 ], V is the control volume [L], and t is time [d]. When a steady state is established, equation (3) is transformed into equation (4). C= L in L out kV (4) Thus, DOM concentration is inversely proportional to k, when (L in –L out )/V is constant. From the biodegradation test, k can be determined by the following equation. k= lnC 0 lnC 5 5 (5) Table 2- Constants of regression lines shown in Fig. 3 Filtrated secondary effluent OC max [mgO 2 /L] K [mgP/L] T DP [mgP/L] K+T DP [mgP/L] r 2 Added Not added 1.02 0.12 0.0043 0.0023 0.0031 0.0040 0.0074 0.0063 0.94 0.69 Average – 0.0033 0.0035 0.0068 – equation type: OC OC max C DP T DP K+ C DP T DP Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 221 - where, C 0 is the initial DOM concentration [mgO 2 /L] and C 5 is DOM concentration after the 5-day biodegradation test [mgO 2 /L]. The C 5 under a facultative DP concentration can be estimated from equations (2) and (6) C 5 = C 0 – OC (6) Consequently, k under a facultative DP concentration can be obtained from equations (5) and (6). From Table 2, OC max of 0.12 mgO 2 /L, K of 0.0033 mgP/L and T DP of 0.0035 mgP/L were used for the estimation of k in Lake Biwa. Although DP concentration is monitored these days, there were few DP data before 1999. Therefore, the ratio of DP to TP was estimated from DP and TP data in 1986, 1990 and 1999-2009 at Imazuokichuo-point and Minamihiraokichuo-point in the northern basin of Lake Biwa (Lake Biwa Environmental Research Institute, 2011). As a result, the ratio was 0.53 ± 0.06 (average ± standard deviation). Accordingly, DP concentration in the northern basin of Lake Biwa was inferred to be TP concentration shown in Fig. 2 multiplied by 0.53. Fig. 4 shows the estimated DP and k from 1979 to 2009. Since Lake Biwa has 5.5 years of hydraulic retention time, 5 year moving averages were also plotted in Fig. 4. As shown in Fig. 4, k dropped between 1990 and 1995. The averages of k in 1980s, in which a retroversion of the trend of COD Mn was observed, and in 2000s were 0.0022 d –1 and 0.0014 d –1 , respectively. Since the DOM concentration was inversely related to k, it is expected that DOM concentration in 2000s will be about 1.6 times higher than in 1980s. As the averages of COD Mn observed in 1980s and in 2000s were 2.1 and 2.7 mgO 2 /L, respectively, the ratio of COD Mn in 1980s against that in 2000s was 1.3, which is in the same order as the ratio of the reciprocal of k aforementioned. The value of k evaluated in this study is a trial estimation under the assumption of a complete mixing flow and a constant (L in –L out )/V. Therefore, it is expected that the k evaluated will be different to some extent from the actual value, but the fact that the ratio of the reciprocal of k is in the same order as the ratio of COD Mn implies a contribution of biodegradation activity drop with a decrease in DP to the increase in COD Mn . However, further study on the internal production will be required for the exact evaluation of the role of biodegradation activity in the accumulation of COD Mn in Lake Biwa. Fig. 4 - Yearly changes in the estimated DP and k in the northern basin of Lake Biwa where lines show 5-year moving averages Journal of Water and Environment Technology, Vol. 9, No.2, 2011 - 222 - CONCLUSIONS One of the possible hypotheses for the accumulation of COD Mn in Lake Biwa is the deterioration of bioactivity for DOM degradation. Accordingly, the influence of phosphorus concentration on the biodegradation of DOM was discussed in this research, because the phosphorus concentration in Lake Biwa has been decreasing for the past 30 years. The obtained results are summarized as follows: (1) Enhancement of oxygen consumption in the biodegradation test by DP and filtered secondary effluent was observed in Lake Biwa water with the natural bacterial community. Although the enhancement effect of the filtered secondary effluent was much higher than that of DP, the addition of the filtered secondary effluent did not affect the dependency of oxygen consumption on DP very much. (2) The response-curve of the oxygen consumption on DP was expressed by a modified Michaelis-Menten type equation with a threshold. The threshold of DP for oxygen consumption was estimated to be in the range of 0.0031 to 0.0040 mgP/L. (3) The pseudo first-order biodegradation rate constant of DOM was estimated to be 0.0022 d -1 in 1980s and 0.0014 d -1 in 2000s based on DP in the northern basin of Lake Biwa. Consequently, DOM in 2000s was expected to be 1.6 times higher than that in 1980s. Since the ratio of COD Mn observed in 2000s to that in 1980s in the northern basin of Lake Biwa was 1.3, it was likely that the deterioration of bioactivity for DOM degradation contributed to the accumulation of COD Mn in Lake Biwa. ACKNOWLEDGEMENT Parts of this work were performed under the Grant for Water Quality Preservation Research "Effect of coexistent substances on biodegradation of recalcitrant organic matter in a lake. (FY2009-2010, Project leader: N. Kishimoto)" supported by Lake Biwa-Yodo River Water Quality Preservation Organization. REFERENCES American Public Health Association, American Water Works Association and Water Environment Federation (1998). Standard Methods for the Examination of Water and Wastewater, 20th edn, American Publich Health Association, Washington DC, USA. Aoki S., Fuse Y. and Yamada E. (2004). Determinations of humic substances and other dissolved organic matter and their effects on the increase of COD in Lake Biwa, Anal. Sci., 20, 159-164. Gurung T. B. and Urabe J. (1999). Temporal and vertical difference in factors limiting growth rate of heterotrophic bacteria in Lake Biwa, Microbial Ecol., 38, 136-145. Hayakawa K. (2005). Behavior of organic matter in Lake Biwa, Annual Rep. 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Annual Conf. Jpn. Soc. Limnol., 69, 249. (in Japansese) . there were few DP data before 199 9. Therefore, the ratio of DP to TP was estimated from DP and TP data in 198 6, 199 0 and 199 9-20 09 at Imazuokichuo-point and. 199 8) has also decreased from 2.4 mgO 2 /L in 197 9 to 1 .9 mgO 2 /L in 198 4, COD Mn increased again after 198 4 and plateaued around 2.7 mgO 2 /L after 199 8