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Int. J. Global Warming, Vol. 6, Nos. 2/3, 2014

Monitoring and examination of adaptation period of microorganisms in membrane-bioreactor system treating fruit juice industry wastewaters Güler Turkoğlu Demirkol Environmental Engineering Department, Faculty of Engineering, Istanbul University, 34320, Avcılar, Istanbul, Turkey E-mail: [email protected]

Sevgi Gunes Durak Environmental Engineering Department, Faculty of Civil Engineering, Yıldız Technical University, Davutpasa Campus, 34220, Esenler, Istanbul, Turkey E-mail: [email protected]

Süleyman Ovez* Faculty of Civil Engineering, Environmental Engineering Department, Istanbul Technical University, Ayazaga Campus, 34469, Maslak, Istanbul, Turkey E-mail: [email protected] *Corresponding author

Nese Tufekci Faculty of Engineering, Environmental Engineering Department, Istanbul University, 34320, Avcılar, Istanbul, Turkey E-mail: [email protected] Abstract: In this study, fruit juice industry wastewaters by using MBR process have been monitored and examined considering effective microorganisms and changes in biodiversity during the treatment period. Microbiological examinations have been carried out using conventional microbiological methods and microscopic observations. Eukaryotic and prokaryotic microorganisms and also floc structure of the activated sludge were identified and characterised. On the other hand, environmental parameters effective on

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Monitoring and examination of adaptation period of microorganisms

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microbial diversity of MBR process treating fruit juice wastewaters have been identified and discussed. Fruit juice wastewaters supported almost all kinds of microorganisms, primarily filamentous fungi Aspergillus spp., and ciliated protozoa Coccidia spp. and eliminated nematodes from the MBR system. Keywords: Aspergillus spp.; Coccidia spp.; nematodes; filamentous microorganisms; fruit juice wastewater; MBR; microbiological monitoring. Reference to this paper should be made as follows: Demirkol, G.T., Durak, S.G., Oves, S. and Tufekci, N. (2014) ‘Monitoring and examination of adaptation period of microorganisms in membrane-bioreactor system treating fruit juice industry wastewaters’, Int. J. Global Warming, Vol. 6, Nos. 2/3, pp.160–174. Biographical notes: Güler Turkoğlu Demirkol is a Research Assistant in Istanbul University, Faculty of Engineering Department of Environmental. She is also a doctoral student in the same department. Sevgi Gunes Durak is a Research Assistant in Yildiz Technical University, Faculty of Civil Engineering Department of Environmental. She is a doctoral student in Istanbul University, Environmental Engineering Department. Süleyman Ovez is an Associate Professor Doctor in Istanbul Technical University, Faculty of Civil Engineering Department of Environmental. His study fields are microbiology, environmental microbiology, biological and chemical treatment, sea biology and ecology, and environmental ecology. Nese Tufekci is a Professor Doctor in Istanbul University, Faculty of Engineering Department of Environmental. Her study fields are waste water treatment methods. This paper is a revised and expanded version of a paper entitled ‘Monitoring and examination of adaptation period of microorganisms in membranebioreactor system treating fruit juice industry wastewaters’ presented at the International Conference on Recycling and Reuse 2012 (R&R, 2012), Istanbul, Turkey, 4–6 June 2012.

1

Introduction

In wastewater systems, diversity of microorganisms is so broad and numerous including eukaryotic and prokaryotic cells. Activated sludge system is one of the most applied technologies that exhibit a matrix structure, which includes biomass components coupled with the metabolically active eubacteria and eukaryotic microorganisms. Biological wastewater treatment systems are often operated under non-steady-state conditions due to frequent changes in inflow rate and composition (Laspidou and Rittmann, 2002; Ni et al., 2009; Ni and Yu, 2012). Wastewater effluents from the fruit juice industry contain primarily high concentrations of organic materials, which are occasionally discharged into the municipal wastewater collection system and processed in wastewater treatment plants along with domestic wastewater. Major problems in the treatment of raw effluents from the fruit juice industry are low pH values, imbalance of nutrients, and the very considerable fluctuations in the amount of effluent and waste matter produced (Austerman-Haun et al., 1997; Dold et al., 1980; Elmaslar-Ozbas et al., 2006). The

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characterisation of the wastewaters can be summarised as follows 4,000–10,000 mg/L COD, 2,000–5,000 mg/L of BOD, and nearly 6−7 mg/L TKN, 2−4 mg/L NH3-N, 0.5−1 mg/l TP and 200−1,000 mg/L of MLSS for fruit juice industry. The combination of membrane separation with the process of biological reactor is called a membrane-bioreactor (MBR). Because membrane filtration and separation is used the gravitational settling tank in the conventional activated sludge process is not needed. The advantages offered by MBR over conventional treatment technologies are well known and the main of them are given below: 1

complete removal of solid

2

effluent disinfections

3

separation of hydraulic retention time (HRT) and sludge retention time (SRT)

4

higher loading rate capability and longer SRT

5

lower/zero sludge production

6

rapid start-up

7

more compact size

8

lower energy consumption.

On the other hand, negative aspects of MBR systems, however, include membrane fouling and concentration polarisation (which are to some extent exacerbated by membrane fouling) (Choi et al., 2002; Chang et al., 2001; Defrance et al., 2000; Hong et al., 2002; Clech et al., 2003). Microbial structures of activated sludge and MBR systems are a mix culture of almost all kinds of microorganisms including bacteria, protozoa, fungi, algae, viruses and other multicellular microorganisms. The most important and dominated component of these mix culture microbial system are bacteria and they make up about 95% of the activated sludge and also MBR system biomass. These single celled micro-organisms grow in the wastewater by consuming bio-degradable materials such as proteins, carbohydrates, fats and many other compounds. In the active sludge, the other organisms particularly the presence of particular types of protozoans is related to effluent quality and plant performance. Most flagellates absorb dissolved nutrients. Flagellates and bacteria both feed on organic nutrients in the sewage so as the nutrient level declines they have difficulty out compete the bacteria for soluble food so, their numbers begin to decrease. While bacteria and flagellates compete for dissolved nutrients, ciliates compete with higher microorganisms including nematodes and rotifers for bacteria. The presence of ciliates and other attached protozoa indicates a good sludge, because they dominate after the floc has been formed and after most of the organic nutrients have been removed. Rotifers, metazoan are rarely found in large numbers in wastewater treatment processes. Rotifers and also nematodes contribute to the removal of effluent turbidity by removing non-flocculated bacteria (http://dnr.wi.gov/regulations/opcert/documents/wwsgactsludgeadv.pdf; Metcalf and Eddy, 2004).


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In biological treatment processes, the effluent contains some portion of organic material originating from soluble microbial products (SMP) that is closely related with extracellular polymeric substances (EPS), although the biological activity is maximised. These SMP should be considered important target materials if additional processes are necessary to satisfy the required water quality for the intended reuse. In this study, the characteristic of SMP and EPS were investigated using a submerged MBR, which is one of the most promising water treatment process (Janga et al., 2006). Biological treatment systems are affected by many environmental parameters primarily temperature and oxygen concentration. Increased temperatures negatively affect oxygen solubility in water causing unfavourable conditions to microbial systems and high costs to operation of the biological treatment systems. These systems should be operated properly because of their high cost investments and also positive environmental effects. MBR systems are one of the most favourable and desired systems protecting our environment in last one to two decades. Sustainable management and proper operation of the MBR systems need continuous monitoring and optimum environmental conditions for each microbial living thing. The aim of this research is to monitor and identify the microorganisms who can be indicators to MBR system treating fruit juice wastewater. These microorganisms are usually good indicators of system during the operation enabling reveal the system performance and control. For these aims monitoring the system for a long time period helps and be foreseen future remedial actions and preventive measures for the system.

2

Material and method

In this research, because of the deficiency of nitrogen and phosphorus in the fruit juice wastewaters as feeding source, these nutrients have been supplied into the system. Microbiological examinations have been carried out two to three times in a week depending on general physical appearance of the activated sludge quality and changes in biodiversity of the eukaryotic and prokaryotic filamentous microorganisms in the microscopic examination and monitoring. Floc of the activated sludge have been dispersed by over aeration of the system and fungi cells and filaments have been multiplied rapidly and over developed under the lack of the dissolved oxygen condition, under 2 mg/L O2 values. In addition to conventional microbiological parameters i.e., ciliated and flagellated protozoa, rotifers, nematodes, fungi, and other higher organised living things have been examined microscopically. First examinations and observations have revealed that remediating and recovering the bulking problem by the fungi filaments problem has not taken long time. Positive and negative environmental parameters effective on microbial development of MBR process treating fruit juice wastewaters have been identified.

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2.1 MBR system In this study, treatment of peach juice wastewater by laboratory-scale MBR system and start-up problems are investigated. MBR, made of stainless steel (active volume 50 L), has nearly internal diameter of 30 cm. Figure 1 shows the experimental setup for a flat-type submerged MBR system. Six vertical flat plates were installed in the bioreactor. The pressure gauge was installed in order to monitor the variation of the trans-membrane pressure between the membrane and a suction pump. To maintain a constant level in the reactor, a peristaltic pump providing influent wastewater and a level sensor were used. Air was supplied through the diffuser below the membrane module. Figure 1

Table 1

Lab-scale MBR system (see online version for colours)

Using equipment and models

Using equipment Water level alarm sensor

Model Foks Ind. Elec. Co. Ltd. (24 V)

Scales

Kern 573 (2000 g)

Blower

Side channel blower (0.20 W)

Pumps

Antech (25 W)


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Microorganisms and changes in biodiversity during the start-up and treatment period of peach fruit juice industry wastewaters by using MBR process have been monitored and examined. The seed activated sludge was taken from Bahcesehir Domestic Wastewater Treatment Plant and fed into the Lab scale (35 L) MBR reactor. The fruit juice wastewater that was taken from reactor has been prepared synthetically and daily 2,000 mg/L COD, 100 mg/L NH4Cl and 20 mg/L KH2PO4 fed to the MBR reactor. COD, TSS, VSS, SVI, pH, and O2 as daily and TKN, NO2-N, NO3-N, NH3-N, SMP, EPS, total phosphorus, and orthophosphate as weekly have been analysed and examined for the effluent. Determination of the COD, TSS, VSS, SVI, TKN-N, NO2-N, NO3-N, TP and orthophosphate parameters proceeded according to Standard Methods for the Examination of Water and Wastewater (APHA, 1995). COD analysis performed by COD cell test method photometric spectroquant colorimeter picco COD/CSB by Merck. The extraction of bound EPS from the cell surface was performed with a cation exchange resin (DOWEX) according to the method of Frolund et al. (1996). Determination of pH and dissolved oxygen parameters proceeded according to Standard Method 4500 (−H+) and were measured by HACH HQ 40d multi. Microscopic analyses were performed using an Olympus BX50 Model research microscope with attachments of the bright field and the phase contrast microscopy, Ikegami camera and spot analysis computer programme. Microbiological analyses including eukaryotic and prokaryotic species were identified by using schemas, pictures, examination of wet mount and stained slides of original samples. Other microbiological and morphological characteristics of microorganisms were examined and compared to those were defined by Jenkins et al. (2004). Physical, chemical and microbiological parameters were carried out in two separate periods of detection because of worsen effluent quality and distorted activated sludge structure. When the quality of effluent decreased and the structure of activated sludge distorted, first activated sludge was replaced and inoculated with a new sludge. First period covered 32 days, and the second period covered 88 days.

3

Results and discussion

Physical analyses: During the operation period measured temperature-pH, SVI and MLSS-MLVSS are given in Figure 2. Temperature-pH values have been working at stabile conditions. MLSS and MLVSS values were tried to be in the range of a certain value by sludge removing. SVI values decreased over time. Chemical analyses: Total phosphorous and ortho-phosphorous, and nitrogen values of fruit juice wastewater are given in Figure 3. Also, COD values of first and second studies are given individually. Cause of becoming wastewater unqualified, COD values has increased. Nitrogen and phosphorous values of fruit juice are given Table 2. Daily organic load is 2,000 mg/L COD. According to 100/5/1 rate, 2,000/100/20 mg/L C/N/P nutrients were added to the reactor.

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G.T. Demirkol et al. (a) Temperature and pH values (b) sludge volume index values and (c) mixed liquor suspended solids-mixed liquor volatile suspended solids

(a)

(b)

(c) Figure 3

(a) Total phosphorus and ortho-phosphorus (b) nitrite, nitrate, ammonia and TKN (c) COD concentrations values (d)

(a)

(c)

(b)

(d)

Monitoring and examination of adaptation period of microorganisms Table 2

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COD, pH, nitrogen, chloride and orto-phosphorous values of peach fruit juice

Fruit juice charac.

pH (%1 COD (1/400 COD (1/500 peach dilut.) dilut.) f. juice)

NH3-N

TKN-N

Cl-

Orto-P (1/400 dilut.)

mg/L

188000

175

368

800

75.2

190000

7.00

In addition, after 24 hours according to COD, treatment yield was determined rate of 90%. Table 3

SMP-EPS values

Carbohydrate

EPS(mg/L)

SMP (mg/L)

287,142

110,632

85,66

34,55

Protein Zeta potential (mv)

−17.3

Total EPS concentrations were generally one order of magnitude higher than those of SMP. As a general trend, concentrations of both protein and carbohydrate fractions of EPS and SMP increased with increasing MLSS levels. Such increase was more pronounced for EPS fractions when considered on a mass basis. Microbiological analyses: Microscopic examinations of activated sludge can help to assess the condition of the biomass in an aeration basin and the settleability of the sludge. It can also aid in the identification of filamentous bacteria that may cause problems in wastewater treatment plants. When floc particles first develop in the activated sludge process, that is, at a relatively young sludge age, the particles are small and spherical. Because filamentous organism do not develop or elongate at relatively young sludge ages, the floc-forming bacteria can only ‘stick’ or flocculate to each other in order to withstand shearing action. Bacterial flocculation and the absence of filamentous organisms result in pin-point small spherical floc particles and associated with turbidity and worsen effluent quality. As the sludge age increases and the short filamentous organism within the floc particles began to elongate, the floc forming bacteria form macro structure and provide firm and resistance to activated sludge floc. These organisms provide increased resistance to shearing action and permit a significant increase in the number of floc-forming bacteria in the floc particles. The presence of long filamentous organisms results in a change in the size and shape of floc particles. The floc particles increase in size to medium and large and change from spherical to irregular (http://dnr.wi.gov/regulations/opcert/documents/wwsgactsludgeadv.pdf).

4

Prokaryotic microorganisms

•

Eubacteria: There have been a lots of gram negative and gram positive bacteria in the bulk solution of the MBR system especially in higher dissolved oxygen concentrations. Numbers of eubacterial cells in bulk solution have been gradually increased and caused higher turbidity and worsen effluent quality after addition of extra nutrients (N and P) and higher amount of dissolved oxygen (above 5 mg/l O2).

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G.T. Demirkol et al. These free eubacteria cells, G-bacteria group usually observed and detected in the form of diplococci, tetracocci (tetrad) and sarcina (packets) as seen in Figure 6.

The important species of filamentous bacteria including Type 021N (gram negative, like a pink coloured necklace), Type 0041 (gram positive within the floc or gram positive extending into the bulk solution) and Type 0675 (gram positive usually found in the floc) have been identified in the system (Figures 8 and 9). The numbers of Type 0041 and Type 0675 filamentous bacteria species have been counted and observed as ‘excessive’ numbers or as numerical value ‘6’ and Type 0021 has been counted as much as ‘common’ or ‘3’ according to Jenkins et al. subjective scoring of filamentous abundance system. According to this abundance of the filamentous bacteria, ‘excessive’ amounts usually cause bulking and foaming problems in the activated sludge systems and filament numbers found in bulk solution more than on flocs (Jenkins et al., 2004). The filamentous bacteria species have been greatly influenced by imbalanced nutrient concentrations (C, N, P) and the sludge age of the system (over 15 days). Another filamentous eubacteria species gram positive chain form of Bacillus spp. has been occasionally seen in the system as ‘some’ or ‘2’ according to Jenkins et al. subjective scoring of filament abundance system [Figure 9 (f)]. They have been found as laid near or on the flocs especially under micro aerophilic conditions, oxygen value under 1−2 mg/L.

5

Eukaryotic microorganisms

•

Ciliated protozoa Coccidia spp. has been dominated in the first period of the MBR system treating fruit juice wastewaters. They have reached enormous number (about 105 cells/ml) and producing pale pink coloured appearance in the bulk solution. This situation changed morphological structure of the activated sludge and the effluent quality deteriorated rapidly and other eukaryotic microorganisms appeared in the system. Biological diversity of the eukaryotic cells increased and Euplates spp., Paramecium spp. Lionotus spp. and other small ciliated, attached and flagellated protozoa species have been usually observed under wet mouth and stained slides during the examination and microscopic monitoring of the sludge. Attached protozoa including Vorticella spp., Epistylis spp., and Podophrya spp. forming single or three to eight colonial cells, and Vaginicola spp. is an attached ciliate and has been found as attached to flocs and extended towards to bulk solution and consuming free bacteria in the bulk solution [seen some of them in Figure 7(b)]. These attached and stalked protozoa are actually good indicators of the well operated activated sludge systems. However, they have been seen and increased their numbers during the first period of the system after deterioration of effluent quality starting 20th day of operation.

•

Algae: Single spherical shaped algae cells Cyclotella spp. found and increased their numbers reaching about 1,000/ml in bulk solution after addition of nitrogen and phosphorus together with increasing oxygen concentration [seen in Figure 4(b)].


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•

Fungi: Filamentous fungus, Aspergillus spp. has dominated the system reaching unbelievable numbers much more than filamentous bacteria reached to ‘excessive’ in the MBR. Fungi species actually likes very much little bit acidic conditions in pH level between 6−6.5 and also degrades fruit sugars and absorbing organic matters. Fruit juice industry wastewater is just ideal for those conditions and supports growth rate of fungi species. As they can be seen in Figures 4, 5, 7, 10 and 11 covered all flocs and even completely the MBR bulk solution. These filaments have formed very dense and thick biomass (hyphae) stimulating and causing incredible biodiversity in the system.

•

Metazoa: Only rotifer species, Philodina spp. has been determined during the wet month slide examinations. Rotifers usually have been found in steady-state conditions attaching to activated sludge flocs and consuming free and attached bacteria and some organic particulates in the activated sludge systems. Philodina spp. has been observed very motile in flocs and also moved from floc to neighbour floc [Figure 7 (b)].

Nematodes have identified and found very motile in the activated sludge flocs especially during the long mean cell residence time operation of the system. They are multiple celled ring worms increased their numbers by producing spherical or ellipsoidal eggs. However, their maximum growth rates are very low and survival in the activated sludge depends on free bacteria and organic particulate turbidity that’s why they can be washed out in rapid systems. One nematode spp. has been observed during the first and second inoculation and adaptation period of the activated sludge. They disappeared from the system after a week starting up period and adaptation period of the activated sludge both renewed culture and inoculations.

5.1 First samples Figure 4

General view of active sludge (a) (original wet mount, 100X) (b) dominant protozoan, intestinal Coccicidia (fast contrast microscopy, original wet mount slide, 100X) (see online version for colours)

(a)

(b)

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G.T. Demirkol et al. (a) Type 0041 filamentous bacteria and fungus Aspergillus spp. (gram staining, 400X), (b) dominant filamentous fungus Aspergillus spp., Type 0041 filamentous bacteria (gram staining, 400X) (see online version for colours)

(a) Figure 6

(b)

(a) Type 0021 (fast contrast microscopy, gram staining, 1000X) (b) Type 0021, diplococci, tetracocci and sarcina group filamentous bacteria (gram staining, 1000X) (see online version for colours)

(a)

(b)

5.2 Second samples Figure 7

(a) and (b) General view of MBR activated sludge (wet mount, 100X) (see online version for colours)

(a)

(b)

Monitoring and examination of adaptation period of microorganisms Figure 8

General view of (a) MBR activated sludge (b) filamentous microorganisms (gram staining, 400X) (see online version for colours)

(a) Figure 9

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(b)

(a) Filamentous microorganisms on attached growth (gram staining, 1000X), (b) gram positive filamentous Bacillus spp., and other g-bacteria (gram staining, 1000X) (see online version for colours)

(a)

(b)

Figure 10 General view of MBR (a) dominancy of fungus Aspergillus spp. (wet mount, 400X), (b) (gram staining, 100X) (see online version for colours)

(a)

(b)

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Figure 11 (a) Aspergillus spp. (gram staining, 400X) (b) filamentous microorganisms (gram staining, 400X) (see online version for colours)

(a)

(b)

Figure 12 (a) Aspergillus spp., Type 0021, gram negative sarcina and g-bacteria (gram staining, 1000X), (b) Type 0041 (gram staining, 1000X) (see online version for colours)

(a)

6

(b)

Conclusions

Fruit juice wastewaters support almost all kinds of microorganisms including prokaryotes and eukaryotes because of their high organic contents and carbohydrates. Biological treatability of the fruit juice wastewaters in an MBR system has been greatly influenced by provided dissolved oxygen concentration and external nitrogen and phosphorus. Because of lack of enough N and P concentrations in the fruit juice wastewaters have affected shift of the microbial communities in the activated sludge and effluent quality has been deteriorated during the adaptation periods. When dissolved oxygen concentrations have been decreased to below 2 mg/l and increased to above 5 mg/l caused some effluent quality problems, including high COD concentration, imbalanced N and P content and solid separation problem. Increasing sludge age MCRT over 15 days has caused almost uncontrolled growth of filamentous bacteria (Type 021N, Type 0041 and Type 0675) and filamentous fungus (Aspergillus spp.) reaching ‘excessive’ amounts in the system causing bulking and foaming problems. Microbial diversity shifts affected the quality of the effluent positively or negatively depending on dominant species.


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On the other hand, imbalanced C, N, P ratio in fruit juice industry wastewaters, caused domination and increase in concentration of ciliated protozoan, especially for Coccidia spp. Depending on the influent characteristics of wastewater, the external supply, deficiency or lack of nutrients has caused serious microbial diversity shift in the MBR system. These microbial alterations have also resulted disappearing Nematodes and increasing in amount of SMP and EPS production by microorganisms in the MBR system treating fruit juice wastewater comparing to amounts of SMP and EPS production in conventional biological treatment system microorganisms.

Acknowledgements This research is supported by Istanbul University Scientific Research Projects.

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