J. Cent. South Univ. (2013) 20: 2472−2477 DOI: 10.1007/s11771-013-1759-5

Feasibility of using lysozyme to reduce excess sludge in activated sludge process

SONG Yong(宋勇)1, 2, SHI Zhou(施周)1, CHEN Shi-yang(陈世洋)1, LUO Lu(罗璐)1

1. Key Laboratory of Building Safety and Energy Efficiency of Ministry of Education (College of Civil Engineering,

Hunan University), Changsha 410082, China 2. Department of Biological and Environmental Engineering, Changsha College, Changsha 410003, China

© Central South University Press and Springer-Verlag Berlin Heidelberg 2013

Abstract: Lysozyme reaction was developed as a novel technique for minimizing the amount of excess sludge in the sequential batch reactor (SBR). In the present work, excess sludge taken from a SBR system was treated by lysozyme reaction and then returned to the reactor. The quality of the effluent water and characteristics of the activated sludge in the SBR were analyzed to determine the effectiveness of the reduction process. The results show that excess sludge production could be reduced to almost 100% in the first 30 d of operation and could be reduced to further by 40% in the succeeding 20 d or so. In these time periods, the average removal efficiencies of the chemical oxygen demand and total nitrogen are 87.38% and 52.78%, respectively, whereas the average total phosphorous in the effluent is nearly 17.18% greater than that of the effluent of the reference system. After 50 d of operation, the sludge floc size is in the range of 20 to 80 μm, which was smaller than the size prior to the start of the hydrolysis and the ratio of mixed liquor volatile suspended solids/mixed liquor suspended solids increases from 86% to 90%. Key words: cell lysis; sludge minimization; sequential batch reactor ; lysozyme; ATP

1 Introduction

Biodegradation is the most important treatment process for industrial and domestic wastewater purifications in which large amounts of excess sludge are produced daily. In addition, the excess sludge contains toxic, harmful and refractory material. Currently, landfills and/or incineration are used in its ultimate disposal. However, secondary pollution problems are produced in these processes. At the same time, treatment and disposal of excess sludge can account for up to 60% of the total operational costs of a wastewater treatment plant [1]. Thus, increased attention has been given to the minimization of excess sludge in the wastewater treatment process. Various technologies have been proposed to reduce sludge production, including physical, chemical, and biological processes. In 1994, YASUI and SHIBATA [2] used ozone gas to dissolve the excess sludge in traditionally activated sludge reactors. In their approach, a fraction of the recycled sludge was treated by ozonation, and the ozonated sludge was then fed back to the aeration tank for biological treatment together with the wastewater. In their experiment, sludge reduction

could be reduced to almost 100%. Since then, the effects of introducing ozonated excess sludge into a variety of activated sludge reactors were evaluated, including sequential batch reactor (SBR) and membrane bioreactors [3−8]. These studies also proved that sludge ozonation treatment was a potential solution to the excess sludge problem. Other studies, such as mechanical disruption using ultrasounds [9−12], chemical hydrolysis using chlorination [13], photo-fenton, and acid or alkali [14−15], had also been widely researched and sludge reduction was achieved in their experiment.

The above mentioned studies are based on the understanding that the bacterial cell wall of the activated sludge is destroyed. The bacterial cell wall can also be effectively hydrolyzed by lysozyme, hence, potentially offering a new way for the reduction of excess sludge using lysozyme in the activated sludge process. The present work investigated the feasibility of this approach by laboratory experiments. A small amount of sludge gathered from the SBR was disintegrated with lysozyme and then returned to the SBR. The effectiveness of the excess sludge reduction and the effluent quality were subsequently investigated.

Foundation item: Project(51078130) supported by the National Natural Science Foundation of China; Project(10C0419) supported by the the Education

Department of Hunan Province, China Received date: 2012−06−06; Accepted date: 2012−08−18 Corresponding author: SONG Yong, PhD; Tel: +86−13187220390; E-mail: 461862116@qq.com

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2 Material and methods 2.1 Experimental devices

Two identical SBRs were operated. The working volume of each SBR was 12 L. The operation cycle was 6 h, in which 4 h was for aeration, 1.5 h for sludge settling, and 0.5 h for decanting and the rest of the procedure. Dissolved oxygen was controlled at the level of 4 mg/L during aeration. 2.2 Sludge cultivation

For the start-up of the two reactors, the returned sludge of a municipal wastewater treatment plant (Xingsha, Changsha, China) was inoculated and cultivated with synthetic wastewater to domesticate the two reactors. The wastewater used in the experiments was synthetic wastewater. It had a chemical oxygen demand (COD) of 449 mg/L, total nitrogen (TN) of 24.3 mg/L, and total phosphorous (TP) of 4.6 mg/L. The SBRs reached steady operation after 25 d of culturing. Meanwhile, the effluent COD was 35−50 mg/L, TN was below 13 mg/L, and TP was about 1.1−2 mg/L. The color of the sludge was brown, and metazoans were found by microscopic examination in the activated sludge. These showed that the activated sludge matured. When the mixed liquor suspended solids (MLSS) was maintained at 3 000 mg/L in each SBR, the generation rate of the excess sludge was around 300−400 mg/(L·d). Thus, sludge retention time was about 10 d. After culturing, column A was kept as the reference system without lysozyme, and column B was used as the testing system for the lysozyme test. 2.3 Lysozyme dosing method

In the testing system, 1.2 L of the mixture was collected before the end of each operation cycle. The supernatant was removed after 1.5 h of settling, and 0.8 g lysozyme (20 000 U/mg，Amresco0663) was added into the settling sludge. A magnetic stirrer was used to facilitate the reaction between the sludge and lysozyme. The reaction was carried out for 30 min under 30 °C. The lysozyme-treated sludge was then poured into column B where the effluent had been decanted. 2.4 Analytical methods

COD, TN, TP, mixed liquor volatile suspended solids (MLVSS) and MLSS were determined in accordance with standard methods [16]. The pH was measured with a Hach Accurate pH meter. Particle size distributions of the sludge were measured via a laser light scattering method. Biomes in the activated sludge were determined through a biological microscope. The intracellular ATP concentration was measured with an

ATP detection kit (Beyotime, China). The dehydrogenase activity (TTC-ETS) was determined using a method that has been reported elsewhere [17]. All chemicals used were of analytical grade. 3 Results and discussion 3.1 Sludge increments in SBRs

To investigate the feasibility of lysozyme for reducing excess sludge production, 70 d of laboratory- scale experiments were performed. During the 70-day operation, the MLSS in the two systems was maintained at 3 000 mg/L, and excess sludge was collected from the two systems in every cycle. Figure 1 shows the daily excess sludge generation rate of the two systems.

Fig. 1 Excess sludge generation during operation time.

In the reference system, the daily sludge production

rate during the operation period remained at around 350 mg/(L·d), whereas in the testing system, the sludge lysis step had a different effect on the excess sludge production rate at a different time. The decrease in sludge production in the testing system appeared very quickly after the start of the lysozyme treatment. During the first four days, the generation rate of the excess sludge was reduced from 339 mg/(L·d) to zero. The following 30 d indicated no discharge of excess sludge, and MLSS was maintained between 2 500 to 3 000 mg/L. Such reduction in excess sludge was significant; 100% reduction could potentially be achieved. In the next phase, at around 20 d, the activated sludge in the testing system started to grow slowly and reflected nearly 45% reduction compared with that of the reference system. In the third stage, the reduction of the excess sludge in the testing system was no longer obvious. This poor reduction of the excess sludge signified a potential problem in the process. Therefore, maintaining the effectiveness of excess sludge reduction in the third stage should be the focus of future studies. 3.2 Effluent qualities

The water treatment performances of these two

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systems are shown in Fig. 2(COD), Fig. 3(TN) and Fig. 4(TP).

As shown in Fig. 2, in the initial two days, the effluent COD of the testing system was higher than 100 mg/L as a result of an increase in COD loading and the inability of the bacterial population to adapt to the new type of COD produced by the lysozyme. The new type of COD emerged when the effluent COD in the

Fig. 2 Variation of effluent CODs in two systems during

operation time

Fig. 3 Variation of effluent TNs in two systems during

operation time

Fig. 4 Variation of effluent TPs in two systems during operation

time

testing system decreased significantly in the subsequent period and was stabilized at around 50 mg/L. Even in the period of no excess sludge, the removal efficiency of the COD maintained at high level in the testing system. In conventional biological wastewater treatment, 40% of the carbon is oxidized to CO2 and 60% is converted into biosolids. In the testing system, the returned sludge was hydrolyzed and the cell contents were released as feed organics. Hence, the organic loading rate to the testing system was much higher than that of the reference system. Therefore, the effluent COD of the testing system was slightly higher than that of the reference system, but it was lower than the discharge standards. This indicated that the COD treatment capability was completely effective by feeding with lysozyme-treated sludge.

The TN in the effluent of the two reactors is plotted in Fig. 3. The average TN in the effluent of the testing system was almost the same as in the reference system. When compared with the influent, approximately 52.78% of the TN was removed from the testing reactors. In 2007, DYTCZAK et al [18] studied the effect of denitrification in SBR, where the returned activated sludge was ozonated. They found that denitrification could be improved by ozonation with its potential to provide an ideal carbon source for denitrification. The lysozyme could also conduct lysis and provide the carbon source. However, the degradation rate of nitrogen would not be improved significantly.

Meanwhile, the average TP in the effluent of the testing system was higher than that of the effluent of the reference system (Fig. 4). The same phenomenon had been reported by other researchers using different reduction methods [9−19]. Theoretically, the P-removal bacteria absorb P in the aeration period and release P in the anaerobic period. P is removed from the SBR via effluent and excess sludge discharge. In the testing system, the inability of P to be withdrawn with the excess sludge resulted in higher TP in the effluent. Other methods, such as chemical precipitation and crystallization, should be combined for phosphorus removal.

After the lysozyme-treated sludge was returned to the testing system, the influent of the testing system contained more debris, soluble organics, TN and TP than the reference system; however, the effluent quality of the testing system (except TP) did not deteriorate. The results indicated that the testing system effectively removed the COD and TN. In order to find the reason, the biological activity pertaining to the degradation of these components was examined. In this work, the dehydrogenase activity and intracellular ATP concentrations were chosen as indicators of the sludge microbial activity after the lysozyme-treated sludge was

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fed back into the bioreactor (Fig. 3). The dehydrogenase activity of the testing system

was obviously higher than that of the reference system after 15 d (Fig. 5). During the 70-day operation period, the average dehydrogenase activities of the testing system and the reference system were 29.79 and 23.82 mg/(g·h), respectively. The increase in dehydrogenase activity suggests that the microbes in testing system increased their ability to decompose the organics and the bacteria could use the organics more easily.

Fig. 5 Variation of TTC-ETSs in two systems during operation

time.

Furthermore, the average intracellular ATP concentration in the testing system throughout the 70-day operation period was 13.53 nmol/mg MLSS, which was higher than that of the reference system (10.18 nmol/mg MLSS) (Fig. 6). The influent of the testing system contained a greater amount of cell debris and soluble organics; however, the system also contained higher levels of ATP which increased by 32.91% compared with the reference system, indicating that the testing system showed better metabolic reactivity. Overall, these results showed that feeding the soluble organics and insoluble organics contained in the lysozyme-treated sludge may enhance the microbial

Fig. 6 Variation of ATPs in two systems during operation time

activity in activated sludge, which maintained the performance of the wastewater treatment. 3.3 Sludge characteristics

The color of the activated sludge in the testing system changed from brown to dark brown during the experiment. Figure 7 shows the color of the sludge in each SBR when the mixed liquor suspended solids was maintained at 3 000 mg/L.

Fig. 7 Change in color of sludge suspension in two systems

after 30 d (left: testing system, right: reference system)

Some amount of foam appeared on the surface of

the water in the testing system. The foam was considered an indication of the disintegrated cells and the absorbed protoplasmic polymers. Microscopic observation of the activated sludge flocs was conducted at different stages. The results showed that bacterial species were nearly the same in the two systems at different stages.

The ratio of MLVSS/MLSS demonstrated the organic content in the activated sludge. The ratio was measured to investigate the accumulation of inorganic materials in the activated sludge. When the ratio decreased, inorganic materials, such as aluminum and iron, accumulated in the sludge. The activated sludge system then slowly deteriorated. Figure 8 shows the ratios of MLVSS/MLSS in the two systems. The ratio of MLVSS/MLSS in the testing system was higher than that the reference system during the experiment. The increment was from 86% to 90% in the first 20 d and

Fig. 8 Change of MLVSS/MLSS ratios during operation time

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then stabilized at a mean value of 90%. Some part of the inorganic materials may have been solubilized, while some were removed with the effluent in the testing system. These findings were inconsistent with the results previously reported in other literature [20−21].

Particle size was also observed in other studies where different activated sludge reactors and different types of wastewater were used. SABY et al [13] used chlorination to reduce the excess sludge. In their experiments, the sludge floc size was in a range of 10 to 20 μm before chlorination. After 20 d, the size reduced to below 5 μm. YOON et al [12], on the other hand, used MBR-US for zero sludge production. In the MBR-US system, the average sizes changed from 132 to 95 μm. Figure 9 presents the changes of the particle size of the sludge floc in the testing system. Prior to the start of the hydrolysis, the sludge floc size was in a range of 40 to 45 μm. After 50 d, the size was in a range of 35 to 40 μm, indicating that the particle size of the sludge decreased slightly.

Fig. 9 Comparison of particle size distribution in activated

sludge prior to lyolysis study and after recirculation of lysised

excess sludge for 50 d

However, the filterability of the sludge of the testing system became much worse than that of the reference system. More than 18 min was needed to filter the 100 mL mixture of the testing system, while about 9 min was needed for the mixture of the reference system. The ability of the sludge to settle was almost the same in the two systems. The sludge volume index (SVI), defined as the sludge volume per gram of dry solid after 30 min of sedimentation, was slightly higher in the testing system than that in the reference system. SVI fluctuated at around 75 mL/g in the testing system. 4 Conclusions

1) The results of the current study shows that excess sludge production in the SBR can be prevented with lysozyme. In the first stage, excess sludge reduction is

significant and can potentially reach 100% reduction. In the second stage, the excess sludge production reduces more than 40%. However, the effluent COD does not worsen in the two stages, which shows that the COD treatment capability is almost unaffected by feeding with lysozyme-treated sludge.

2) The mechanism analysis shows that the dehydrogenase activity and intracellular ATP concentration in the testing system are increased, indicating that the testing system has a better ability to digest organics. No change is found in the biological species communities as seen through the biological microscope. Thus, this method is feasible for excess sludge reduction. The principal disadvantage of the study is that the effectiveness of excess sludge reduction slowly deteriorated as the experiment progressed. Finding new dosing methods or inducers is necessary to solve this problem.

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(Edited by HE Yun-bin)