1. Introduction

Figure 1. Schematic process diagram and layout of the CSTP.

Figure 1. Schematic process diagram and layout of the CSTP.

2. Materials and Methods

2.1. CSTP Pilot Scale

The pilot scale of CSTP was designed and constructed with a capacity of 1.4 m³/d which consists of grit chamber, primary clarifier, anaerobic biofilter, anoxic chamber, MBBR 1 and 2, and secondary clarifier unit Figure 1. SAP was done in a batch mode Figure 2. Seeding and analysis of all parameters of SAP were done in the Final Project Laboratory, Environmental Engineering Department (EED), Institut Teknologi Nasional (Itenas), Bandung, Indonesia; acclimatization was executed in the MBBR units of pilot plant CSTP located in the EED.

Figure 2. Seeding and acclimatization process.

Figure 2. Seeding and acclimatization process.

2.3. Acclimatization Procedure

Substrate was added in three stages to avoid shock loading of susceptible and unstable microorganisms [17]. Air was continuously injected during the acclimatization process, and temperature was in accordance with the MBBR condition which was operated under aerobic condition therefore aerobic microorganisms were expected to grow Figure 2 [18].

2.3.1. Adding Substrate

Details of substrate additions are shown in Figure 2.

2.3.2. Adding Kaldnes Media

Kaldnes media were also added into MBBR during the acclimatization process (Stage 2 & 3) to enhance the specific surface area where new microorganisms could attach and grow onto resulted from the addition of substrate during the acclimatization process. Kaldnes media were only added in Stage 2 & 3 due to the additions of substrate in Stage 2 & 3 were quite high, hence the microorganisms were expected to grow significantly; thus, more media were required.

The available kaldnes media was 7.4 kg with the specific gravity of 150 kg/m³ and were divided proportionally with the volume of each MBBR unit (270 L and 240 L of MBBR 1&2 subsequently) (Table 1), based on Equation 1, thus each unit had the same filling ratio i.e., the ratio of the volume of the added media towards the water volume in the unit. Hence the organics removal efficiency by microorganisms in each unit was expected to be proportional.

$$\mathrm{Kaldnes}\mathrm{volume}\mathrm{in}\mathrm{MBBR}1\mathrm{or}2=\frac{\mathrm{MBBR}1\mathrm{or}2\mathrm{volume}}{\mathrm{MBBR}\mathrm{total}\mathrm{volume}}\mathrm{x}\mathrm{Kaldnes}\mathrm{total}\mathrm{volume}$$

(1)

Similar values of filling ratio (≈10%) in each MBBR unit were obtained using Equation 2.

$$\mathrm{MBBR}\mathrm{filling}\mathrm{ratio}=\frac{\mathrm{Kaldnes}\mathrm{total}\mathrm{volume}}{\mathrm{MBBR}\mathrm{volume}}\mathrm{x}100\%$$

(2)

Table 1. Addition of substrate & kaldnes media in seeding & acclimatization processes.

Table 1. Addition of substrate & kaldnes media in seeding & acclimatization processes.

Process	Substrate Added (L)		Kaldnes Media Added (L)
	MBBR 1	MBBR 2	MBBR 1	MBBR 2
Seeding	53 L+7 L microorganisms = 60 L @ 30 L		13 L @ 6.5L
Acclimatization Stage 1	15	15	-	-
Acclimatization Stage 2	30	30	8	8
Acclimatization Stage 3	45	45	11.5	8.5
Total	120	120	26	23

2.3.3. Analyzed Parameters

Parameters analyzed every day during the acclimatization process were Chemical Oxygen Demand (COD) and Mixed Liquor Volatile Suspended Solid (MLVSS) concentrations [9,10,11,19]; and pH and temperature in influent and effluent samples (Table 2) [9,10]. Microorganisms were always fed with 20 gr glucose for every 200 mg/L drop of COD [20]. All chemicals used were purchased from PT Merck Tbk, Indonesia, and were of reagent grade. Sampling was done only in MBBR 2 as each MBBR unit was treated equally.

Table 2. Analyzed parameters.

Table 2. Analyzed parameters.

No.	Parameter	Analytical Method
1.	COD	Closed reflux titrimetric (Indonesian National Standard (INS) No. 06-6989.2-2004) [21]
2.	MLVSS	Gravimetric (INS No. 06-6989.3-2004) [22]
3.	pH	Potentiometric electrode (INS No.06-6989.11.2004) [23]
4.	Temperature	Potentiometric electrode (INS No.06-6989.11.2004) [23]

3. Results and Discussion

3.1. Seeding Process

Seeding was halted on day-6 when the colour of kaldnes media changed from white (before) into brown (after seeding) (Figure 3). The change in colour suggested the attachment of microorganisms on the kaldnes carriers to create the biofilm [24]. It is to note that Lemire et al., (2015) who used similar K1 seed carriers but from a different manufacture (Veolia Water Technologies) obtained shorter period of seeding (5 days). This may due to the used of the growth medium (soy broth, salts, and yeast extract) as compared to none used in this study [14].

Figure 3. Observed kaldnes media (a) Before seeding (b) After seeding.

Figure 3. Observed kaldnes media (a) Before seeding (b) After seeding.

In the MBBR system, biomass plays a key role to control the performance of the system. The attachment of biomass to the carriers’ surface is particularly very important hence was studied by many other researchers [9,10,12].

The kaldnes media were weighed before and after seeding; and the difference was found to be 0.074 gr/carrier showing the growth of biomass on the surface of the carrier [9]. Other researchers weighed the biomass after the acclimatization [9].

The fundamental target of MBBR is to immobilize the biomass on the carrier. Plastic (polyethylene, polypropylene) carriers are designed accordingly so as to protect biofilm loss and promote the growth of biofilm on the surface of the media [25]. The result of this study showed that cylindrical polyethylene carriers were proved to provide area for the growth and stable attachment of microorganisms. Other researchers also claimed the same results [9,10,14]. Freitas et al. (2022) who used a cylindrical shaped polypropylene carrier obtained the similar proof as well [26].

The attachment of the biomass on the carriers in this study was only observed visually and confirmed by biomass weight. It is suggested that the active attachment of the biomass further observed microscopically (by using scanning electron microscope (SEM) etc.) as shown by other research [14].

3.2. Acclimatization Process

Table 3 shows the results of Stage 1-3 acclimatization process i.e., the changes of measured parameters of pH, temperature, COD, and MLVSS.

Table 3. Results of analyzed parameters during acclimatization process.

Table 3. Results of analyzed parameters during acclimatization process.

Acclimatization	Day	COD (mg/L)	COD Removal Rate (%)	MLVSS (mg/L)	pH	Temperature (oC)
Stage 1	1	1,144.6	-	644.9	7.65	26.0
	2	886.2	22.6	316.0	7.50	26.5
	3	646.2	43.5	594.2	7.77	25.5
	4	738.5	35.5	562.0	7.65	26.0
	5	400.0	65.1	794.0	7.15	25.9
Stage 2	1	965.4	-	824.0	6.54	26.9
	2	733.9	24.0	478.0	7.31	25.8
	3	576.6	40.3	414.0	7.34	24.3
	4	368.7	61.8	562.0	7.53	25.1
Stage 3	1	1,009.0	-	909.0	7.10	24.7
	2	654.5	35.1	638.0	6.88	25.3
	3	344.8	65.8	316.0	6.50	26.7
	4	475.7	52.9	488.0	6.54	25.8
	5	275.9	72.7	770.0	7.15	25.9
	6	206.9	79.5	908.0	7.31	26.9
	7	275.8	72.7	972.0	7.77	26.0

3.2.1. pH

pH measured was in the range of 6.50-7.77; which was in-line with the optimum pH for carbon oxidation, i.e., 6.50-8.50 [27]; the growth of bacteria in general, i.e., 6.50-7.50 [1]; nitrification bacteria, i.e., 7.20-8.00 [28]; and polyphosphate-accumulating organisms (PAOs), i.e., 5.00-6.50, or 6.50-7.00 (5.5>8.5) [29]. Therefore, it is postulated that the organotroph microorganisms were considered to be well alive (and grow) in the system under this condition.

3.2.2. Temperature

Temperature measured was in the range of 24.30-26.90°C showing the conditions where mesophilic bacteria are active enough to degrade organics, i.e., 25-40°C [30]; nitrification bacteria can live, i.e., 23.00-30.00°C; and are optimum, i.e., 25.00-30.00°C [31]. However, this range did not comply with the range of temperature where PAOs bacteria can survive, i.e., 5-20°C [32]. Nevertheless, the temperature values supported the environment where organic degrading microorganisms could grow well.

3.2.3. Stage 1 Acclimatization Process

COD, MLVSS, pH and temperature were analyzed every day to monitor the process of microorganisms’ growth, so as the formed microorganisms would be able to adapt with and survive in their new environments displayed by the stability of the removal rate of COD, and the constant growth rate of microorganisms.

On day-1, COD was 1,145 mg/L and the acclimatization stage was terminated on day-5 when COD was 400 mg/L, and MLVSS was 794 mg/L (Figure 4); resulting in the removal rate of 65.1%. Tchobanoglous et al. (2014) stated that the optimum removal rate in the biological process generally was about 60-70%; thus, the result was somehow expected [1]. In addition, acclimatization (in day-5) was halted to avoid the further decrease of COD resulting in the lack of the remaining substrate for microorganisms.

Figure 4. COD and MLVSS in stage 1 acclimatization.

Figure 4. COD and MLVSS in stage 1 acclimatization.

3.2.4. Stage 2 Acclimatization Process

In Stage 2.30 L, new substrate was added to each MBBR unit to avoid the lack of nutrition for the microorganisms, as only 400 mg/L substrate (as COD) remained in Stage 1. On day-1, COD was 965 mg/L, and MLVSS was 824 mg/L. On day-4, acclimatization was terminated once COD reached 368.68 mg/L, and MLVSS was measured of 562 mg/L (Figure 5). The removal rate was 61.8%, as expected, and was in conformity with Tchobanoglous (2014) [1].

Figure 5. COD and MLVSS in stage 2 acclimatization.

Figure 5. COD and MLVSS in stage 2 acclimatization.

3.2.5. Stage 3 Acclimatization Process

With the same previous reason, new substrate was added to each MBBR in Stage 3. COD on day-1 was measured of 1,009 mg/L while MLVSS was 909 mg/L. Acclimatization was halted on day-7 once COD reached 276 mg/L, while MLVSS measured was of 972 mg/L (Figure 6). The removal rates of COD on day-5-7 were 72.66%, 79.50%, and 72.66% respectively with the differences of less than 10%; in-line with Tchobanoglous (2014) who stated that acclimatization was done once the constant removal rate of COD was achieved or the difference of removal rate was nearly 10% [1]. This was also in agreement with Maulidiany (2015) and Rifaldi (2015) who stated that stabilized or less than 10% fluctuation of COD reflected high activity rate of bacteria to degrade the organics [19,20]. In addition, the 73-79.50% removal rates were the optimum removal rates as defined by Tchobanoglous (2014) [1].

Figure 6. COD and MLVSS in stage 3 acclimatization.

Figure 6. COD and MLVSS in stage 3 acclimatization.

All this showed that the acclimatization process was considered to be completed, and the microorganisms well adapted to the MBBR system, hence could be further applied to treat septage using CSTP. The whole acclimatization process was 16 days in total; and with seeding, the total process of growing and adapting microorganism in the MBBR system was 22 days. This acclimatization time was shorter than Adabju (2013) that reported 30 days of acclimatization period. This might be due to the K1 kaldnes carriers used in this study was smaller (diameter = 9.1 mm) than Adjabu (2013) (diameter =4.5 cm); resulted in higher surface area (=500 m²/m³), hence promoting faster adaption of microorganisms to the carriers [33].

3.2.6. The Growth of Biomass

MLVSS was used as a proxy to find out the bacterial growth during the acclimatization process. However, in this study, the measured MLVSS could not represent the whole biomass grown in the MBBR, as MBBR is an attached growth system. For the attached growth system, 90% microorganisms would attach to the carriers, so the measured MLVSS only represented the number of suspended microorganisms, i.e., 10%, hence the attached microorganisms could not be measured in this study [34]. However, the growth of biomass analysis was still be done using the assumption that by having the constant removal rate of COD, microorganisms were considered able to grow, proliferate, and stabilized, hence could be employed to degrade organics in the MBBR system.

By using this assumption, the growth phases of microorganisms were clearly shown in Stage 3; the acclimatization process was thus halted as the stabilized growth was observed. In Stage 3, a lag phase occurred on day 1-3, shown by the decrease of MLVSS. On day-1, substrate was just added, so as available food for microorganisms was excessive, thus the rate of microorganisms’ growth and proliferation was limited by their ability to adapt to degrade the food [35]. The decrease in MLVSS concentration indicated the adaptation process of microorganisms towards their environments (to cultivate the substrate); both the decrease of organics in septage and also the increase growth of new microorganisms (that were adaptable with the new environments) on the kaldnes media. The growth of microorganisms was marked by the reduction of COD on day 1-3 describing the removal of organics (by microorganisms) used for their metabolisms and protoplasm synthesis processes [36,37].

After the microorganisms could well adapt with their new environments, constant increases of the exponential growth of microorganisms occurred (constant growth rate), on day 3-5, thus the removal of organics occurred in the same rate as shown by the constant exponential increase in the MLVSS, and the decrease in COD concentration [36,37]. On day 5-7, the growth rate of microorganisms started to constantly decline (declining growth rate), hence, the removal of organics reduced in the same rate as indicated by the decreasing of MLVSS enhancement and of COD constantly [36,37]. Stationary and endogenous phases were not detected as the acclimatization process was terminated on day-7.

For Stage 1 & 2, the growth phases of microorganisms were not clearly seen, due to the acclimatization processes were already halted on day-5 and 4 for Stage 1 & 2 subsequently. However, similar to Stage 3, in Stage 1 & 2, a lag phase also existed where the reduction of MLVSS occurred (on day 1-2 for Stage 1, and day 1-3 for Stage 2), accompanied by the decrease of COD.

For Stage 1, the growth rates of microorganisms were fluctuated; increased on day 2-3 and 4-5; yet decreased on day 3-4; indicating the growth of microorganisms was not yet stabile. For Stage 2, the growth rates of microorganisms went up on day 3-4; however, the growth of microorganisms could not be seen further as the acclimatization process was stopped on day-4.

4. Conclusions

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