XUE Xu-dong, PENG Dang-cong, GOU Miao

(Key Laboratory of Ministry of Education of Northwest Water Resource and Environmental Ecology , School of Environmental and Municipal Engineering，Xi′an University of Architecture and Technology , Xi′an 710055 , China)

0 Introduction

Continuous system is widely used in wastewater treatment, influent, effluent and reaction work together in the reactor. Substances remain the same in the system and microorganisms stay in unchanged surrounding with a steady state. In batch system, the operation is different from continuous system. Influent, effluent and reaction are separated in a cycle[1]. And the anaerobic sequencing batch reactor (ASBR) is extensively used because of low price and easy handling[2-4]. In ASBR, the substance concentration is maximum after influent, and decrease with the reaction[5]. Microorganisms are exposed to variable substrate concentrations over a cycle, resulting in high rates of substrate conversion, efficient biomass flocculation and settling[6]. These dynamics in the reactor give an environment in which the production of reserve polymers might provide a competitive advantage to those microorganisms capable of producing storage products[7]. In published studies, the microorganisms have been shown to accumulate carbohydrates as glycogen in the cell. For example, glycogen storage and degradation have been observed[8,9]. Microbial accumulations of glycogen and glycogen-like compounds have been contact with the excess carbon source and nutrient deficiency in the reactor[10]. But in the continuous system, microorganisms do not absorb carbohydrate into cells because of the invariable substances concentration.

The object of this study was finding the different degradation ways of the carbohydrate between continuous and batch system in anaerobic condition, the percent of each metabolic product and the different construction of biogas.

1 Materials and Methods

1.1 Laboratory-scale reactor

Two jacketed reactors with the same working volume (4.5 L) were used for both batch and continuous systems. Reactors were operated at 35±1 ℃ with recirculation water, using intermittent agitation (30 s every 5 m at 300 rpm), and hydraulic retention time (HRT) was 1 d. Gas bags were used for biogas collection. The continuous system ran constantly, and the ASBR was operated with 8 h cycles consisting of feeding (30 min), reaction (400 min), settling (30 min), effluent (10 min) and idle (10 min). Floc sludge in the reactor which was inoculated from wastewater treatment plant has been cultured for more than 1 year.

Hydrogen utilization rate was determined by modified serum bottle method. The quantitative sludge was injected into a serum bottle with the free space full of hydrogen. A peristaltic pump was used for internal circulation aeration, and a gas bag with full of nitrogen which was controlled by a valve was placed to balance the pressure in the bottle because the pressure was decreased while hydrogen was used. Then gas constitution in the bottle at different time was analyzed with GC.

1.2 Substrate and nutrient solution

Substrate was 4 kg chemical oxygen demand (COD)/m3·d with glucose feeding, C∶N∶P was controlled at 300∶5∶1. Nutrient solution was: NaCl 120，MgCl2·6H2O 49，Na2SO443，FeSO4·7H2O 5.53，MnCl2·4H2O 2.02，CaCl2·2H2O 0.59，ZnCl20.67，NiCl2·6H2O 0.65，CuCl2·2H2O 0.16，CoCl2·6H2O 0.48，H3BO30.063，Na2MoO4·2H2O 0.004 5 (in milligram per liter)[11].

1.3 Analytical methods

COD, ammonia nitrogen, total suspended solid (TSS) and volatile suspended solid (VSS) were determined according to standard methods[12]. pH was determined by glass-electrodes method. Partial pressure of hydrogen and volatile fatty acid (acetic and propionic) were analyzed with GC (Agilent 6 890 N). The specific methanogenic activity (SMA) of formate, acetate, propionate and butyrate were analyzed in serum bottles. Glycogen in cells was determined according to anthrone colorimetry method. And hydrogen utilization rate was determined by modified serum bottle method.

1.4 Calculations

In the system, COD balance can be evaluated with formula 1 and 2.

CODinfluent=CODsoluble+CODmethane+CODbiomass+CODglycogen

(1)

CODremove=CODinfluent-CODsoluble=CODmethane+CODbiomass+CODglycogen

(2)

CODinfluent——total COD in the influent water;

CODsoluble——soluble COD in the reactor, mostly were VFA;

CODmethane——the cumulative methane production determined by Gas flow counter and GC, the former determine total gas production, the latter detect the percent of methane, then dividing by 0.395 L methane/g COD (35 ℃, 1atm).

CODbiomass——COD used for biomass production, calculated by ammonia nitrogen decrement.

CODglycogen——COD of glycogen was calculated as 1.066 7 g COD/g glycogen.

CODremove——COD removed from the mixed liquor.

Some parameters were shown in Tab.1.γ1,γ2andγ3were different parts of glycogen in the microorganisms.ε1,ε2,ε3andε4stand for the percentage of CODglycogen, CODbiomass, CODmethaneand CODsolublein CODinfluent.

In the Tab.1：Xstois the stored glycogen in microorganisms,Xgis the total glycogen in microorganisms.Xstand for MLVSS.

Hydrogen utilization rate (HUR) can be evaluated with formula 3

(3)

In the formula, 2 is molecular weight of hydrogen, 22.4 is molar volume of gas

C0—— percentage of hydrogen in the bottle at beginning of reaction

C1—— percentage of hydrogen in the bottle after while of reaction

V——volume of the free space

T——reaction time when get the sample

X——quantity of the sludge

Tab.1 Parameters in the research

2 Results and Discussion

In batch system, MLVSS was 8.81 g/L, SRT was controlled at 15 d with 0.15 g/L SS in effluent and 225 mL/d mud removing, and SVI was 51.08 mL/g. In continuous system, there were plenty of filamentous microorganisms in the rector because of the low substrate, MLVSS was 5.01 g/L, SRT was controlled as same as the batch system with 0.25 g SS /L in the effluent and 75 mL/d mud removing, and SVI was higher than batch system (95.81 mL/g). In continuous system, reaction goes continuously without cycle, but in order to compare with batch system easier, the data of 8 h (a cycle in batch system) was taken out as a cycle.

2.1 Glycogen

Glycogen in different systems was shown in Fig.1. In continuous system, reactor was stayed in an unchanged surrounding, glycogen in microorganisms remained the same, there was no glycogen stored in microorganisms,γ1approximated to 0, andγ2was as same asγ3(meanvalue 137.19 mg/gVSS). In batch system, a mass of substrate got into the reactor during influent, a part of substrate was turned to VFA while the other was stored in the microorganisms as glycogen,γ1(63.39 mg/gVSS) andγ2(244.77 mg/gVSS) reached the maximum at the end of the influent. With the reaction carried on, glycogen was exhausted gradually by the microorganisms, at the end of the reaction,γ2(181.38 mg/gVSS) decreased to the minimum, and was as same asγ3. As reported, the glycogen stored by granular sludge was 63～129 mg/gVSS[13]and 2.6～18 mg/gVSS in extracellular polymers in UASB[14]; also it was similar to the glycogen (111.2 mg/gVSS) in ASBR with granular sludge[15]. But in this research, it was floc sludge in reactor, glycogen in the microorganisms reached up to 134～245 mg/gVSS which was much higher than reported. Different sludge shape and structure mean different microbial quotient and component in the reactor, and bring about the big different of glycogen in the microorganisms. Moreover,γ3in batch system was obvious higher than it in continuous system, and there was not any stored glycogen in continuous system. It indicated that there was a part of stored glycogen stayed in cells for keeping the microorganisms′ normal existence when substrate was not enough. This part of glycogen occupied 24% ofγ3in batch system. In continuous system, this part of glycogen was inexistence without the variational substrate.

Fig.1 Glycogen change a cycle in different systems

2.2 COD, pH and VFA

In two different systems, each of the substrate concentration showed special slope-change trend because of different influent. In batch system, influent last half an hour, COD (418.84 mg/L, Fig.2 (a)) and VFA reached the highest, and pH (6.66, Fig.2 (a)) decreased to the least at the end of the influent. VFA was constructed mostly of acetic and propionic, with the maximums were 96.66 mg/L and 186.14 mg/L respectively in Fig.2 (c). After the influent, COD and VFA reduced gradually with the reaction going on. At the end of the reaction, COD, acetic and propionic decreased to the least, which were 115.23 mg/L, 5.08 mg/L and 48.91 mg/L respectively, pH raised up to 6.95 (Fig.2 (a) and (c)), as same as the COD, VFA and pH before influent, then the reactor processed to the next cycle. In continuous system, substrate went into the reactor continuously at low and unchanged level, COD, VFA and pH remained stable, VFA was consisted of propionic and a few of acetic, and each meanvalue of COD, acetic, propionic and pH were 67.40 mg/L, 41.78 mg/L, 4.51 mg/L and 6.85 respectively (Fig.2 (b) and (d) ). In different systems, the percentage of propionic in VFA was different. Propionic was the main component of VFA in batch system, but that was opposite in continuous system, there was only a few of propionic. Because the SMA of propionic in two systems were very different from each other, which in batch system was higher than in continuous system. It caused much of substrate transformed to acetic, and the concentration of propionic was very low in continuous system.

Fig.2 COD, pH and VFA change in a cycle in different systems

2.3 Sludge production

The main way to remove ammonium nitrogen is biomass production in anaerobic system. In this research, we determine COD which used for biomass production by decreased ammonium nitrogen and the molecular formula of microorganism (C5H7NO2)[16]. ammonium nitrogen in the influent was 63.23 mg/L in both systems and the change in a cycle were shown in Fig.3. In batch system, a mass of ammonium nitrogen was utilized in the influent; the concentration was 10.91 mg/L (160.75 mg/L CODbiomass). Then ammonium nitrogen decreased gradually with the reaction going on; in the final phase of the reaction, it went up a little because biomass decomposed itself in lacking substrate surrounding. The concentration of ammonium nitrogen was 3.27 mg/L (251.24 mg/L CODbiomass). In continuous system, ammonium nitrogen was held in a low level because of the slow influent, mean concentration of it was 4.22 mg/L, (249.45 mg/L CODbiomass).

Fig.3 Ammonium nitrogen change in a cycle in different systems

2.4 Biogas

Biogas production is the main method to remove the COD in anaerobic system. In batch system, all the substrate went into reactor in the influent; enough substrate ensured the microorganisms produced biogas with full speed, and the variation curve of cumulative biogas production was a straight-line at beginning of the reaction (Fig.4). Plenty of carbon dioxide were produced because of the fermentation, the percentage of methane reduced to the least (50.5%, Fig.5) at the end of influent. As the reaction going on, the speed of biogas production was obviously slow after 3 hs because the concentration of acetic decreased to a very low level (less than 10 mg/L, Fig.2 (c)); the percentage of methane increased with the hydrogen and carbon dioxide were used by microorganisms. At the end of the cycle, cumulative biogas production was 3.1 L (Fig.4), the percentage of methane rose up to 56.1% (Fig.5). In continuous system, the speed of biogas production remained unchanged, the variation curve looked like a straight-line all over the reaction, the cumulative biogas production was 3.11 L (Fig.4) in 8 hs, almost the same as the batch system; meanvalue of the percentage of methane was 54.4% respectively (Fig.4).

Fig.4 Cumulative gas production in a Fig.5 Percentage of methane in biogas cycle in different systems change in a cycle in different systems

2.5 Hydrogen partial pressure

Hydrogen partial pressure is a very important parameter in anaerobic system. It impacts the speed of VFA transformation in the system. In batch system, hydrogen partial pressure reached the maximum (14.3 Pa, Fig.6) after influent, and then it reduced with the reaction and decreased to 3.5 Pa at the end of the cycle. In continuous system, Hydrogen partial pressure was about 20 times to batch system and unchanged (about 291.3 Pa). It was reported the highest hydrogen partial pressure was about 200 Pa in anaerobic system[17], and hydrogen yield in continuous system was higher than batch system[18]. But in our research, the hydrogen partial pressure was much higher than reported. This phenomenon could be explained by two points.

Fig.6 Hydrogen pressure change in Fig.7 SMA and HUR in different systems a cycle in different systems

(1) Different hydrogen utilization rate (HUR, Fig.7), each of them was 8.954 mg/h·gVSS (1.719 g COD-H2/gVSS·d) in batch system and 3.659 mg/h·gVSS (0.703 g COD-H2/gVSS·d) in continuous system. Low utilization rate cause more hydrogen accumulated in the reactor.

(2) It was reported that 41%～46% of the substrate in the influent was stored in the microorganism as glycogen, only 34%～38% was hydrolyzed to VFA[19], less substrate hydrolyzed meant less H2production during the hydrolyzing. In our research, the storage substrate was about 50% (Tab.2).

2.6 Specific methanogenic activity (SMA)

SMA is an important physical sign to describe the methanogenesis ability of methanogen. It indicates the maximum methane production of unit mass of sludge in unit time and the utilizationof different VFA in the reactor. SMA in different system was shown in Fig.7. In batchsystem, SMA of acetic, propionic and butyrate were 0.521 5 g COD-CH4/gVSS·d, 0.382 8 g COD-CH4/gVSS·d, 0.453 5 g COD-CH4/gVSS·d respectively. In continuous system, they were 0.544 6 g COD-CH4/gVSS·d, 0.076 8 g COD-CH4/gVSS·d, 0.289 0 g COD-CH4/gVSS·d Total SMA of batch and continuous system were 1.357 8 g COD-CH4/gVSS·d and 0.910 4 g COD-CH4/gVSS·d, MLVSS in batch system (8.81 g/L) was higher than continuous system (5.01 g/L). That means the treatment capacity in batch system was higher than continuous system, and with the different MLVSS in two systems, sludge in continuous system always worked on high burden, this is not good for a stable system.

Hydrogen Utilization Rate (HUR) shows the ability of sludge to use hydrogen in the reactor. HUR was 1.719 g COD-H2/gVSS·d in batch system and 0.703 g COD-H2/gVSS·d in continuous system. Low HUR cause more hydrogen accumulated in the reactor. As we know, hydrogen pressure which ensure propionic metabolism is less than 10 Pa. High partial pressure of hydrogen limited the propionic metabolism ability in continuous system and SMA of propionic in continuous system was much less than batch system.

2.7 Utilization of the substrate in different system

The results (Tab.2) showed that 36.61% of total COD was transformed to VFA (acetic and propionic,ε3+ε4) in the influent and 50.17% was stored (ε1), a part of the VFA was changed to biogas (11.56% of total COD,ε3), others stayed in the mixed liquor (25.05%of total COD,ε4). This phenomenon is different from the absorption and utilization of VFA in biological phosphorus removal. In biological phosphorus removal, VFA are absorbed and stored as PHB by phosphate accumulating organisms (PAO) in anaerobic surrounding, and the PHB is oxidized in oxic zone. But in ASBR, glucose storage and fermentation work together, and the storage is major (50.17% of total COD). At the end of the reaction in batch system, most of glycogen and VFA were transformed to biogas (79.44% of total COD,ε3) and microorganism cells (20.94% of total COD,ε2),ε1andε4decreased approximately to 0. In continuous system, it was as same as the end of reaction in batch system; glucose was immediately transformed to biogas (79.34 of total COD,ε3) and microorganism cells (20.66% of total COD,ε2), glycogen and VFA were not detected in the reactor. The way of substrate utilization is different in two systems because of different influent. Glycogen storage is an important way to remove COD from the mixed liquor at beginning of the reaction in batch system. And the final way to remove COD is similar, all of the glucose is transformed to biogas and microorganism cells.

Tab.2 Parameters in different time and system

2.8 Metabolic method of different systems

In traditional continuous system, the degradation complex organic (carbohydrate) is 3 steps: (1) substrate is changed to VFA by acid-producing bacteria; (2) VFA is transformed to acetic and hydrogen by hydrogen-producing acetogens; (3) methanogen use the acetic and hydrogen to produce methane. In batch system, the degradation of substrate has two ways to transform, one is VFA, and the other is glycogen stored in the microorganisms, then the glycogen is transformed to VFA gradually. The storage of glycogen can decrease the accumulation of VFA, which gives good condition to hydrogen-producing acetogens and methanogen, keep the balance of anaerobic system, and ensure the methanogenesis.

We name the microorganism which store the glycogen rapidly as glycogen-accumulating organism. In batch system, more percent of glycogen-accumulating organisms exist in system, more glycogen will be stored, less VFA will be accumulated, and more organic load will be disposed. So the glycogen-accumulating organism contributes much to steady the ASBR system.

3 Conclusions

(1) The way of substrate utilization is different in two systems. The degradation of substrate has two ways to transform in batch system, one is VFA (36.61%), another is glycogen (50.17%), and there isn′t storage in continuous system. At last, the removal ways of COD in two systems are almost the same, biogas (79.34%～79.44%) and microorganism cells (20.66%～20.94%). Glycogen storage decreased the VFA production with the substrate was fed in.

(2) Hydrogen partial pressure was obviously different in two systems (291.3 Pa in continuous system and 14.3 Pa in batch system) because of different HUR (1.719 g COD-H2/gVSS·d in batch system and 0.703 g COD-H2/gVSS·d in continuous system) and glycogen storage (0 in continuous system and 50.17% in batch system). Low HUR caused hydrogen accumulated and low SMA of propionic in continuous system.

(3) Total SMA in two systems was 1.357 8 g COD-CH4/gVSS·d in batch system and 0.910 4 g COD-CH4/gVSS·d in continuous system, and the MLVSS in continuous system (5.01 g/L) was less than batch system (8.81 g/L). That means sludge in continuous system worked on a higher burden than batch system. This was disadvantageous in a stable and long-time running system.

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