Introduction
The upflow anaerobic sludge blanket (UASB) reactor has been employed in industrial and municipal wastewater treatment for decades since it was developed by Lettinga G and his co-workers in the 1970s (
Lettinga et al., 1980). The major differences between UASB system and other anaerobic technologies are as follows: 1) the wastewater flows up in the reactor; 2) the sludge does not have to be stirred; and 3) there is a three-phase separator at the top of the reactor. Compared with other anaerobic technologies, UASB has some remarkable advantages such as high organic loadings, great efficiency, low energy demand, short hydraulic retention time (HRT) and easy reactor construction. One of the main obstacles for the application of UASB is long start-up time, usually 2-8 months, needed for the formation of granules. Many efforts have been made to accelerate the start-up of UASB reactors (
Ghangrekar et al., 1996;
Kazuaky et al., 1997;
Tsuyoshi et al., 1997;
Yu et al., 2001;
Kuan-Yeow et al., 2004).
The UASB system is highly dependent on its granulation process with a particular organic wastewater. Anaerobic granular sludge is the core component of an UASB reactor. Compared with floccular sludge, granular sludge could be retained in the reactor at a higher gas production rate and higher velocity of upflow because of the much better settling. Some studies of granular sludge formation have already been made (
Kuan-Yeow et al., 2004;
Kazuaki et al., 1998;
Delia, 2001;
2003). However, to date no work has been done with the diosgenin wastewater.
Diosgenin is extracted from
Dioscorea Zingiberensis and has been used to synthesize steroid for medicines such as pregnenolone and progesterone. The wastewater from diosgenin industry is characterized by high concentration of chloride ion (8000-10000 mg/L) and refractory organics (COD up to 20000-30000 mg/L). All previous studies on treatment of this wastewater have been conducted only in China (
Shan et al., 2003;
Wang and Hu, 2004;
Li et al., 2004;
Liu et al., 2004;
Zhang et al., 2003;
Chen et al., 1999).
Shan et al. (2003),
Li et al. (2004) and
Zhang et al. (2003) used UASB reactors for diosgenin wastewater treatment, but none of them has made detailed work on UASB start-up and granular sludge formation. The objective of this investigation was to find a new way of enhanced granulation in a UASB reactor for diosgenin wastewater treatment. A model for the granular structure was also proposed based on the observation.
Materials and methods
Materials and the UASB reactor
The UASB reactor was designed specifically for this investigation. Figure1 illustrates the plexiglass UASB reactor with the internal diameter of 200 mm and a height of 1000 mm. Like other UASB reactors, the reactor designed for this work comprises water inlet and distribution system, tank and three-phase separator. The most special part of the reactor is the filler between the sludge blanket and the settling area, making a mass of anaerobic microorganisms to be immobilized on it. The significance of the immobilization is that the retention time of the anaerobic sludge in the reactor was extended, and the treatment efficiency was enhanced.
The anaerobic sludge for inoculation was obtained from a brewhouse. The chloride-resistant bacteria were selected from the sludge that was obtained from several approaches. The wastewater obtained was pretreated by internal-electrolysis and diluted with water to reach the required COD concentration (5000-13000 mg/L). Certain mass of chloride was added to simulate the high chloride characteristics of diosgenin wastewater (4000-7000 mg/L) and the pH value was adjusted to 7.5-8.5 by adding limewater. Then the wastewater was settled for 24 h before it was introduced into the UASB. Its pH was double checked to be kept at 7.5-8.5. If not, limewater would be added again.
Procedure to start up the UASB
The anaerobic sludge obtained from a brewhouse was settled for 7 days. Then the supernatant was removed, and the residual sludge was inoculated into the UASB reactor. The volume of the sludge for inoculation is 9L
The sludge was settled in the UASB for 48 h at a room temperature of 9°C and a water bath temperature of 13°C. The temperature of the water bath was gradually raised to 37°C, which is the optimal temperature for mesophilic anaerobic reaction, and this temperature was kept throughout the experimental period.
The UASB reactor was tested with the prepared diosgenin wastewater. The wastewater was introduced into the reactor by a constant flow rate pump of HL-2. The volume and COD concentration of the wastewater were adjusted according to the running situation. During the start-up process, the suspension of the selected chloride-resistant bacteria was gradually introduced.
Analytic methods
The influent COD, chloride concentration and the effluent COD concentration were measured with the standard methods (
The Monitoring and Analytic Methods for Water and Wastewater Compiling Committee, 2002). The pH value was measured by pH meter HI8314 (Hanna, Italy). Scanning electron microscope KYKY-1000B and microscope BX40F4 (Olympus, Japan) were used to observe the granular sludge.
Results and discussion
Start-up of the UASB
The start-up of UASB reactor lasted for 40 days. During these days, the influent COD and chloride concentration were gradually raised from 5000 mg/L to 13000 mg/L and from 4000 mg/L to 7000 mg/L respectively. From day 35 to day 40, the sludge color in the UASB reactor turned black. The production of biogas was also obviously enhanced, implying that the function of the reactor was substantially accelerated. On day 35, granular sludge was found in the reactor, indicating successful start-up of the UASB reactor.
The mechanism for granule formation
A lot of work has been done on granulation process in the UASB reactor. However, there is no consensus about the mechanism triggering granulation. Different models have been proposed (
Liu et al., 2003). The initial stages of the formation of anaerobic granules follow the same principles as biofilm formation of bacteria on solid surfaces. There exists strong evidence that inert carriers play an important and positive role on granulation. Most researchers concluded that
Methanosaeta concilii is a key organism to granulation (
Hulshoff et al., 2004).
This study basically supported the mechanism of inert nuclei model. In the presence of inert microparticles in an UASB reactor, anaerobic bacteria could be attached to the particle surfaces to form the initial biofilm, namely embryonic granules (Fig. 2). The mature granules can be further developed through the growth of these attached bacteria under given operation conditions. The inert nuclei model suggests that the presence of nuclei or micro-size biocarrier for bacterial attachment is the first step towards anaerobic granulation (
Liu et al., 2003).
In this study, limewater was added to adjust pH, which made it possible for calcium to function as nuclei. The granulation was enhanced with the increase of nuclei.
The new model for granulation
Anaerobic granules with diameters of 0.5-3.0 mm occurred in the reactor after 35 days of operation. The granules had various irregular shapes, i.e., spherical, elliptical, bacilliform, nail-like, platelet-like and so on, and most of them were irregularly spherical and elliptical. Most of the granules were grey, with taupe, offwhite, and primrose and white mixtures.
The development of granular sludge from dispersive anaerobic microorganisms to a macroscopic granule composed of anaerobic microorganisms is an extremely complicated physicochemical and microbiological process. The mechanism has not been fully understood, although great efforts have been made worldwide.
In this study, the granules in different stages of granulation were observed carefully as shown in Fig. 3. According to the observation, a new model for granular sludge formation using diosgenin wastewater as substrate was proposed, in which the process of granulation consists of the following six continuous stages.
1) Semi-embryonic granule formation
A mass of microorganisms adhere to either a microorganism or an infinitesimal particle, which could be called as an inert nucleus, with the help of extracellular polymer (ECP) secreted by these microorganisms. Then the semi-embryonic granule was formed.
2 ) Embryonic granule formation
With hydraulic agitation, single semi-embryonic granules collide with other microorganisms and they adhere to each other. The inert nucleus is enwrapped by plenty of microorganisms, and this attachment results in the formation of embryonic granules.
3) Single-nucleus granule formation
Single embryonic granules collide with other microorganisms with hydraulic agitation, and these microorganisms adhere to the surface of the embryonic granule and enwrap it to form a single-nucleus granule.
4) Multi-nuclei granule formation
Embryonic granules collide with each other, and adhere together to form a granule. Such a granule has no inert nuclei, and it is called a multi-nuclei granule. This type of attachment is the primary way of granule formation.
5) Granule growth
Microorganisms in incipient granules reproduce constantly, making the granule grow gradually. At the same time, other microorganisms keep on adhering to the granules, which also help the granules grow. But the main factor for the growth of granules at this stage is the reproduction of microorganisms.
6) Granule maturation
Biogas is produced continuously by methanogenic bacteria. There will be some micropores at the place where methanogenic bacteria are concentrated, and these micropores will be the channels for gas discharging. This appearance implies that the granule is matured.
Mature granules rely on the inner microorganisms’ propagation to maintain their stabilization and the metabolism of the systems. Extracellular polymer (ECP) which is secreted by microorganisms forms a porous ‘protective membrane’ on the surface of the granule (Fig. 4). As a result, the exterior of the granules looks like translucent jelly.
Among the above 6 stages of granulation, the formations of semi-embryonic granule and embryonic granule are the two most important processes, and they can be achieved by enwrapping and growing of methanothrix or by aggregating various bacteria. The aggregation of granules needs suitable conditions such as hydraulic loading and biogas agitation.
The new model for the granule structure
The effects of ECP and microbial composition on the strength of granules were the focus of previous studies on granule structures (
Quarmby and Forster, 1995). In this investigation, abundant micropores of different sizes were observed on the granules using scanning electron microscope (SEM) (Fig. 5). A model for these granule structures is proposed here on the basis of the SEM observation. It is suggested that microorganisms rely on, and support, each other to form microspores. Countless microspores of different sizes were found on the surface and led further into the inside of the granules. The bigger microspores could be called pores, the main function of which is to act as channels for wastewater substrate to contact with the internal parts of granules. On the other hand, they act as a passage for biogas discharge. Thus the microorganisms in the interior of the granules could conduct their metabolism successfully through these microspores, and the granules’ metabolism is subsequently accomplished.
Conclusions
The UASB reactor filled with diosgenin wastewater was started up with high concentration of chloride (4000-7000 mg/L) and COD (5000-13000 mg/L). Anaerobic granular sludges were formed on day 35, indicating an accelerated start-up of the reactor.
According to the observation of granular sludge at different periods of granulation, a new model of granule formation was proposed. This model divides the granulation process into 6 continuous periods: semi-embryonic granule formation, embryonic granule formation, single-nucleus granule formation, multi-nuclei granule formation, granule growth and granule maturation. Among these, semi-embryonic granule formation and embryonic granule formation are the two most important steps.
A new model for the granule structure was proposed in this study, featured by countless microspores of different sizes which occurred on the surface, and led to the inside of the granules. These microspores act as the channels for wastewater substrate to reach the inner granules, and the passage for biogas. Thus the microorganisms in the interior of granules could conduct their metabolism successfully through these microspores, and granules¡¯ metabolism is subsequently accomplished.
Higher Education Press and Springer-Verlag Berlin Heidelberg
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