Use of fungi to improve bioconversion of activated sludge

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Water Research 39 (2005) 2935–2943 www.elsevier.com/locate/watres

Use of fungi to improve bioconversion of activated sludge Sarkar Mannana,, A. Fakhru’l-Razia, Md Zahangir Alamb a

Department of Chemical and Environmental Engineering, Faculty of Engineering, University Putra Malaysia (UPM), 43400 UPM Serdang, Selangor, D.E., Malaysia b Department of Biotechnology Engineering, Faculty of Engineering, International Islamic University Malaysia (IIUM), Jalan Gombak, 53100 Kuala Lumpur, Malaysia Received 6 December 2004; received in revised form 18 April 2005; accepted 21 April 2005 Available online 5 July 2005

Abstract The present study was designed to evaluate the potential of microbial adaptation and its affinity to biodegradation as well as bioconversion of soluble/insoluble (organic) substances of domestic wastewater treatment plant (DWTP) sludge (activated domestic sludge) under natural/non-sterilized conditions. The two filamentous fungi, Penicillium corylophilum (WWZP1003) and Aspergillus niger (SCahmA103) were used to achieve the objectives. It was observed that P. corylophilum (WWZP1003) was the better strain compared to A. niger (SCahmA103) for the bioconversion of domestic activated sludge through adaptation. The visual observation in plate culture showed that about 95–98% of cultured microbes (P. corylophilum and A. niger) dominated in treated sludge after 2 days of treatment. In this study, it was also found that the P. corylophilum was capable of removing 94.40% of COD and 98.95% of turbidity of filtrate with minimum dose of inoculum of 10% v/v in DWTP sludge (1% w/w). The pH level was lower (acidic condition) in the fungal treatment and maximum reduction of COD and turbidity was observed (at lower pH). The results for specific resistance to filtration (SRF) showed that the fungi played a great role in enhancing the dewaterability and filterability. In particular, the strain Penicillium had a more significant capability (than A. niger) of reducing 93.20% of SRF compared to the uninoculated sample. Effective results were observed by using fungal inoculum after 2 days of treatment. The developed LSB process is a new biotechnological approach for sludge management strategy. r 2005 Elsevier Ltd. All rights reserved. Keywords: Activated sludge; Adaptation; Bioconversion; Dewaterability; Domestic wastewater sludge; Filamentous fungi

1. Introduction Domestic wastewater sludge is composed of the solids/organic matter (approximately 96–99% water) generated from private or community wastewater Corresponding author. C/o Dr. Jubaida Rumana (Dina),

241/2 South Pirerbag (3rd Floor) Mirpur, Dhaka-1216, Bangladesh. Tel.: +88 02 8012032; fax: +88 02 8125813. E-mail addresses: [email protected], mannan.bd@ gmail.com (S. Mannan), [email protected] (A. Fakhru’l-Razi), [email protected] (M.Z. Alam).

treatment processes (Gray, 1989; US EPA, 1999). The sludge treatment and disposal in the proper way are probably the most costly and difficult task, not only in Malaysia but also all over the world. Kadir and Velayutham (1999), have reported that Indah Water Konsortium (IWK) produces approximately 3.8 millions m3 of sewage sludge annually in Malaysia. It is estimated that the cost of management is more than USD 0.26 billion annually. Sludge production in Malaysia is expected to increase in the future and to double by the year 2020. Also in a report of the US Environmental Protection Agency (US EPA, 1999), the

0043-1354/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.04.074

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quantity of municipal biosolids produced annually in the United States has increased dramatically, from roughly 4.6 millions dry tons in 1972 (Bastian, 1997) to 6.9 millions dry tons in 1998. Future biosolids production is expected to increase to 8.2 million dry tons in 2010. At present, the management cost of sludge is USD 35–38 per dry ton (US EPA, 1999). Most biosolids (wastewater sludge) undergo additional treatment on site before they are used or disposed of to meet regulatory requirements that protect public health and the environment, to facilitate handling and reduce costs. For these reasons, the governments, other authorities, agencies and researchers are searching for the best suitable and sustainable alternative processes/ methods for future waste-management strategies (Cameron et al., 2000; Alam et al., 2003a). Liquid state bioconversion (LSB) of domestic wastewater treatment plant (DWTP) sludge is a new biotechnological approach for the biodegradation, bioseparation and biosolids accumulation (biosolids production) of soluble and insoluble pollutants (Alam et al., 2003a). Moreover, the treated sludge supernatant (by LSB) can be disposed without any further treatment as it meets the standard values of discharge (ILBS, 1999). Biodegradation and bioconversion is a natural decay or degradation process in which microbial communities play very important roles to break down or alter the structure of wastewater sludge ingredients/constituents such as metals, organic and inorganic substances (Akthar and Mohan, 1995; Field et al., 1993; Feijoo and Lema, 1995; Palma et al., 1999; Coulibaly, 2002). In fact, the biodegradation of wastewater constituents enhances the bioseparation (settling characteristic of solids), biosolids accumulation (biosolids production by fungal treatment), filterability as well as dewaterability considerably (Alam and Fakhru’l-Razi, 2003; Alam et al., 2003a). The dewaterability by fungal treatment can be defined as biodewaterability. In the LSB process, isolated filamentous fungi entrap/ become immobilized on the biodegradable solid particles. It compresses the treated sludge with its filamentous (hyphae) mycelia that modify the porosity structure of biosolids and enhance the bioseparation, dewaterability and filterability (Hamdi and Ellouz, 1992; Friedrich et al., 1996; Alam and Fakhru’l-Razi, 2003). However, the LSB process has been developed with controlled conditions on a laboratory scale (Alam et al., 2001, 2003a). In the LSB, the sample of sludge was sterilized prior to starting the process/experiment. In addition, wheat flour was used as a co-substrate (carbon source) in the treatment. These are the main limitations of the LSB process. There is a current interest in the present research into the use of Penicillium corylophilum and Aspergillus niger to enhance the LSB of DWTP sludge under non-sterilized/natural conditions through the adaptation, with the improved dewaterability/filterabil-

ity and removal of COD and turbidity of treated sludge. This research has mainly focused on the use of potential fungi (Alam et al., 2004) for the bioconversion of organic substances in sludge/activated sludge.

2. Materials and methods 2.1. Sample collection and preparation The DWTP sludge of 0.7–1.00% (w/w) of total suspended solids (TSS), initial pH of 6.65–6.9 was collected from the domestic wastewater treatment plant, IWK, Kuala Lumpur, Malaysia. The required concentration of sludge such as 1.0% w/w of TSS was prepared by adjusting the moisture of the original sludge collected. The wastewater sludge of 1% w/w concentration (TSS) with the optimum initial pH of 5.5 was used throughout the experiment to evaluate the adaptability of microbes and their potential in bioconversion of DWTP sludge (Alam et al., 2003c). 2.2. Microorganism The microorganisms, A. niger SCahmA103 (IMI 385267) and P. corylophilum WWZP1003 (IMI385277) were used in the study. These strains were obtained from the laboratory stock of Biochemical Engineering lab, University Putra Malaysia (UPM), Malaysia. The species were isolated from the relevant sources (wastewater and sludge cake) and screened and optimized for its potential as the mixed culture based on the biodegradation and bioseparation for the treatment of domestic wastewater sludge in LSB process under controlled (sterilized) conditions (Alam et al., 2001, 2003b; Fakhru’l-Razi et al., 2002a). The cultures were maintained on 3.9% w/v potato dextrose agar (PDA media, Merck, Germany) slants. The strains are stored at 4 1C and sub-cultured once in a month for further use. 2.3. Inoculum (cultured inoculum) Spore suspension was prepared according to the method of Fakhru’l-Razi et al. (2002b). Its concentration was measured (A. niger was 6.5
106 spores/ml and P. corylophilum was 9.7
106 spores/ml) with hemocytometer. A 2% w/v of wheat flour containing sterilized distilled water medium was inoculated with 2% v/v spore suspension and then processed for 48–72 h in a rotary shaker with 150 rpm at optimum temperature of 33–35 1C for pellet formation (Alam et al., 2003a, c). The pellets cultured were used as an inoculum at 5, 10, 15, and 20% (v/v) to evaluate the LSB process of DWTP sludge under natural conditions. The concentrations of cultured inoculum of A. niger and P. corylophilum were 11.97 and 12.907 g/L, respectively.

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2.4. Experimental design and procedure In the study, the inoculum size of 10%, 15% and 20% represented by S210, S215 and S220 were used. For example, S220P meant that the sludge (sludge-2 containing 1% w/w of TSS) was treated by P. corylophilum with an inoculum size of 20% (v/v), and in the same way S220A meant that it was treated by A. niger with an inoculum size of 20% (v/v). A total of five experiments with 1% (w/w) of TSS (Sludge-2) of wastewater sludge were conducted to evaluate the fungal potentiality in the LSB process under natural conditions through their adaptation. The experiments were conducted in a 250 ml Erlenmeyer flask containing 100 ml of sludge samples. The inoculated and uninoculated samples were incubated in a rotary shaker (Innova 4000, New Brunswick Scientific Co. Inc. Edison, USA) using 150 rpm at 3372 1C for 5 days of treatment with sampling each day. The initial pH (6.65–6.90) of the sludge samples was adjusted by sulfuric acid to 5.5.

Eq. (1) can be written as   mrc t=V ¼ V ¼ b  V. 2A2 P

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(2)

Taking the slope of the line as b, SRF can be calculated from the formula:  2  2A P r¼ b. (3) mc The filtrate (from the SRF test) of the treated and untreated (control) samples was collected for COD analysis, which was done according to the Standard Methods (APHA, 1999). The HACH Turbidimeter 2100N (HACH, USA) was used to measure turbidity of the filtrate. All analyses were performed on data using the Microsoft Excel, Microsoft Office 2003 software package (Microsoft Corporation, USA). The data obtained were the average of three replicates.

2.5. Analytical methods

3. Results and discussion

The supernatant of treated and untreated (control) sludge were cultured on the Petri dish containing PDA media to evaluate adaptability of the P. corylophilum and A. niger after each days treatment with visual observation. The plate culture was incubated at room temperature for 7 days for clear observation of adaptation. The pH was measured by a pH meter (Fisher, USA) with the help of manual instructions. Specific resistance to filtration (SRF) test, known as the Buchner funnel test, is one of the most commonly employed tests for the evaluation of dewaterability or filterability of wastewater and/or wastewater sludge. The filtration was performed using a 90 mm diameter Whatman ]1 filter paper at an applied vacuum pressure of 300 mmHg (Alam and Fakhru’l-Razi, 2003). The volume of the filtrate (50 ml) was recorded as a function of time for every 10 ml of filtrate collected. Specific resistance to filtration (SRF ¼ r) was determined using a plot of filtration time/filtrate volume (t/V) vs. filtrate volume (V). SRF was calculated from the following formula (Carman, 1938) by using the slope (t/V vs. V) of the line:

3.1. Visual observation on microbial treatment of DWTP sludge

t=V ¼

ðmrc Þ mRm Vþ , 2A2 P AP

(1)

where r is the specific resistance to filtration (m/kg); A is the area of filter (m2); P is the pressure of filtration (N/ m2); m is the viscosity of the filtrate (N s/m2); V is the volume of filtrate (m3); t is the filtration time (s); C* is the weight of dry solids per volume of filtrate (kg/m3) and Rm is the resistance on the medium (1/m). For compressible sludge, ignoring Rm, which is very small (compared to the resistance on the sludge cake),

The supernatants of the treatment and uninoculated sample were cultured on Petri dishes containing PDA media every day to evaluate their adaptability compared to the control. Fig. 1 shows the adaptability of the fungi after 2 days of treatment in DWTP sludge by LSB under natural conditions. In Fig. 1(b), 95–98% of the existing microorganisms were dominated by P. corylophilum with 10% , 15% and 20% of inoculum dose of treatment, respectively, compared to the control (Fig. 1(a)). Moreover, almost 98% of existing microorganisms were dominated by A. niger (Fig. 1(c)) in treatment after 2 days for different inoculum dose (10–20%). However, it was observed that there was no significant dominating percentage as compared to the control in PDA plate when the sludge was treated with 5% of inoculum dose (data were not shown) for both treatment with Penicillium and Aspergillus. The 5% of inoculum dose in treatment might be insufficient to adapt in sludge under natural conditions. Adapted liquid culture seen in shake flask experiment after 2 days of treatment are presented in Fig. 2(a), (b) and (c) for control, P. corylophilum and A. niger, respectively. In the treatment, the soluble and insoluble solids of the sludge were entrapped with the filamentous body [of the fungal biomass (pellets)]. They might have increased their biomass by changing their secondary metabolism by using soluble/insoluble solids of sludge. As result, the free water was released, which enhanced the filterability as well as dewaterability of the treated sludge compared to the control.

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Fig. 1. Cultures of adapted fungi on PDA plate after 2 days of treatment: (a) control; (b) Penicillium corylophilum, b-1 for 10%, b-2 for 15%, b-3 for 20% of inoculum; and (c) Aspergillus niger, c-1 for 10%, c-2 for 15%, c-3 for 20% of inoculum.

3.2. pH of treated and untreated sludge The pH values for the microbial treatment of wastewater sludge are shown in Fig. 3. In treatment with P. corylophilum, the pH decreased to 3.4, 3.5 and 3.5 from the initial value (pH 5.5) for 10%, 15% and 20% of inoculum dose, respectively, after 2 days of treatment (Fig. 3(a)). It was maintained in a decreasing trend for

up to 5 days of fermentation for all percentages of inoculum doses of Penicillium applied. No remarkable difference was observed for the different doses (10%, 15% and 20%) of inoculum over the total period of treatment, whereas the pH was decreased by up to 2 days of treatment with A. niger (Fig. 3(b)) and it was increased until the final days of treatment. The lowest pH values were recorded as 3.7, 3.4 and 2.4 after 2 days

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Fig. 2. Adapted liquid culture seen in shake flask experiment after 2 days of treatment: (a) Penicillium corylophilum (WWZP1003); (b) control; and (c) Aspergillus niger (SCahmA103).

10

Penicillium corylophilum 8 S210P S220P

6

S215P Control

4 2 (a)

0 pH

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1

3

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5

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3.3. Turbidity of treated and untreated sludge

Aspergillus niger 8 S210A S220A

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3 2 Fermentation Period,day

The pH value was affected by the fungal treatment and, during the population growth of microbes in treatment, the pH level was changed because the fungal growth may have led to excretion of acidic metabolites (Fakhru’l-Razi et al., 2002b). This might suppress or reduce the intensity of the growth in alkaline pH of sludge. In addition, final pH for control was 7.54 after 5 days of treatment because the control sample had several existing types of microorganisms and in controlled conditions (temperature, agitation and pH) the existing microorganisms might produce several types of unknown metabolics (acidic or alkaline).

4

5

Fig. 3. pH values in treated sludge during the fungal treatment by (a) Penicillum corylophilum and (b) Aspergillus niger with different dose of inoculum.

of treatment and the highest values were recorded as 8.10, 7.8 and 6.6 after final days of treatment by Aspergillus with 10%, 15% and 20% of inoculum dose, respectively. The acidic metabolites were produced with fungal growth after 2 days of treatment with A. niger.

The effects of fungal treatment on removal of turbidity are shown in Fig. 4. The closer value of turbidity in the filtrate of the treated sample against distilled water was estimated as lower turbidity of sludge (Alam et al., 2003b). The minimum value of turbidity means the minimum presence of TSS. The lowest turbidity of the filtrate of the treated sample was recorded after 2 days of treatment. The lowest observed values of turbidity in NTU were 1.07, 1.10 and 1.06 for 10%, 15% and 20% of inoculum dose of P. corylophilum, respectively (Fig. 4(a)), which were very close to that of distilled water (0.82 NTU). In the treated sludge, turbidity decreases were observed to be 98.95%, 98.91% and 98.95% for Penicillium for 2 days of the treatment period and, from then on until the final days of treatment, there was no significant difference in the change in removal percentage for the number of days of treatment period or as a percentage of inoculum dose of Penicillium. For A. niger, the lowest turbidity (2.1, 2.1 and 2.087 NTU) of the filtrate were observed after 2 days of treatment and the decreases in percentage of

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(a)

(a) Penicillium

Penicillium

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Turbidity, NTU

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CODx102, mg/L

S210P S215P S220P Control % Removed by S210P % Removed by S215P % Removed by S220P

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Turbidity Removal (%)

100 75

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COD Removal (%)

2940

60 40

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20

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Fig. 4. Turbidity removal (%) of filtrate by the treatment of (a) Penicillium corylophilum and (b) Aspergillus niger with different dose of inoculum in treated sludge.

turbidity were 97.91%, 97.91% and 97.93% for 10%, 15% and 20% of inoculum dose, respectively (Fig. 4(b)). Moreover, after the final days of treatment, no significant changes in reduction of turbidity were observed. Alam et al. (2003c) and Fakhru’l-Razi et al. (2002b) have made similar observations for the bioconversion of domestic wastewater sludge in shake flask experiments with sterilized conditions. 3.4. COD removal of filtrate of treated sludge The removal percentage of COD of the filtrate during the microbial treatment of wastewater biosolids is presented in Fig. 5. The chemical oxygen demand (COD) is a measure of oxygen equivalent of the organic matter as well as microorganisms in the wastewater (Pipes and Zumda, 1997). Thus, the COD is a very important factor in evaluating the organic content of DWTP sludge. The results showed that the COD removal was highly influenced by the microbial treatment of sludge. P. corylophilum had more potential to reduce the COD comparing with A. niger. The COD removal (maximum) of filtrate was recorded as 94.40%, 94.00% and 94.30% after 2 days of treatment by P. corylophilum for 10%, 15% and 20% of inoculum dose compared to uninoculated sludge, respectively. The maximum COD removal of filtrate by Penicillium in wastewater treatment under control condition was

0

0 0

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2

3

4

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Fermentation Period,Day

Fig. 5. Removal (%) of chemical oxygen demand (COD) of filtrate in fungal treatment of wastewater sludge by using (a) Penicillium corylophilum and (b) Aspergillus niger with different dose of inoculum.

reported as 93% (Alam et al., 2004). In the present study, the COD removal efficiency was slightly decreased after 3–5 days of treatment by Penicillium but there was no significant difference. In addition, it was observed that the COD removal was not affected significantly after 2 days of treatment regardless of the percentage of inoculum dose (for 10% and 20%) of Penicillium (Fig. 5(a)). The maximum COD removal of the filtrate was observed as 80.40%, 74.80% and 73.40% after 2 days of treatment by SCahmA103 for 10%, 15% and 20% of inoculum dose, respectively (Fig. 5(b)). In addition, the COD removal (%) was below 60% by A. niger for 10% 15% and 20% of inoculum dose at the end of treatment. According to Alam et al. (2004), maximum COD removal recorded was 86% by SCahmA103 in LSB process under controlled conditions. However, it was observed that the best result was recorded for the low dose of inoculum (10% of dose was more effective than 20% of dose) more probably because of less biomass content than a higher dose of inoculum. After 2 days of fermentation period, the treatment might be affected by some factors such as malnutrition in treatment, agitation rate, etc., which increased the breaking down of pellets as well as the COD value in treatment.

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3.5. Dewaterability/filterability of treated sludge Dewatering is the most common type of biosolids treatment process (US EPA, 1995). It increases the treatment efficiency of sludge treatment plant. SRF was determined to assess the dewaterability and filterability of the sludge. The SRF reductions of treated sludge by Penicillium and Aspergillus with different percentages of inoculum applied compared to the untreated sludge are shown in Fig. 6. The filterability of treated sludge was highly influenced by the fungal treatment (Fakhru’l-Razi et al., 2002b; Alam et al., 2003b). The maximum decreased percentages of SRF recorded were 91.20%, 91.60% and 93.20% after 2 days of fungal treatment and the values were slightly decreased after final days of treatment by P. corylophilum (Fig. 6(a)). In addition to the sludge treatment with 10%, 15% and 20% of inoculum dose of A. niger, the maximum decreased percentage of SRF values were observed to be 87.30% 88.20% and 90.10% after 2 days of treatment (Fig. 6(b)). In addition, the magnitude of the SRF decreased percentage was slightly reduced after final days of treatment, but not significantly for both species (Penicillium and Aspergillus). In Fig. 6(a), there was no significant difference in SRF removal between 10% and 20% of inoculum dose of treatment by Penicillium. The lowest SRF value, 0.097
1012 m/kg after 2 days of treatment with 20% of inoculum dose of Penicillium, was detected for sludge (1% w/w). This was much lower than untreated (control) sludge 1.424
1012 m/kg. Alam et al. (2003a) examined SRF in 1% w/w of DWTP sludge when treated with fungi in LSB process under controlled conditions and noted that the lowest value

100

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S210P S215P S220P Control % Decreased by S210P % Decreased by S215P % Decreased by S220P

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SRFx1012, m/kg

In this study, it was observed that the cultured inocula (pellets) in treatment were beginning to start breaking down after 3 days of treatment which might increase the soluble and insoluble biomass in the solution, which in turn might increase the COD of the filtrate, observed to be significant, when the sludge was treated by A. niger (rather than P. corylophilum). The COD removal by fungal treatment with considerable values (60–90%) has been studied by many authors in domestic wastewater (Fakhru’l-Razi et al., 2002b; Alam et al., 2003c, 2004), apple distillery waste (Friedrich et al., 1987), olive mill waste (Hamdi et al., 1991; Robles et al., 2000) and starch processing wastewater (Jin et al., 1999). The soluble and insoluble organic substances in sludge were reduced significantly by fungal growth and its secondary metabolites (Friedrich et al., 1983). The COD value of 2 days of treatment by Penicillium (with 10% inoculum dose) was 92.20 as mg O2/L and, after completion of treatment, it was 88 as mg O2/L, which meets the water quality Class-B according to Interim National Water Quality Standards (INWQS) for Malaysia (ILBS, 1999).

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40 20 0

0 0

1

2 4 3 Fermentation Period,day

5

Fig. 6. Reduction (%) of specific resistance to filtration (SRF) of fungal treated sludge by (a) Penicillium corylophilum and (b) Aspergillus niger with different dose of inoculum to evaluate the dewaterability/filterability of treatment.

was 1.4
1012 m/kg after 6 days of treatment. The filamentous fungi immobilized/entrapped the solid particles of DWTP sludge by their filaments, compressed the porosity structure of sludge cake, and accelerated the filtration process (Hamdi and Ellouz, 1992; Fakhru’lRazi et al., 2002b). To evaluate dewaterability, SRF has been studied by many authors and achieved a significantly lower value after treatment from its initial value such as in domestic wastewater sludge with fungal treatment (Sorensen and Hansen, 1993; Novak et al., 1999; Fakhru’l-Razi et al., 2002b; Alam et al., 2003a); aerobic/anaerobic sludge with treatment of polymer dose (Arhan et al., 1996); raw sludge by using cationic polyelectrolyte dose (Lotito et al., 1993; Lee and Liu, 2000.) and sludge with oxidative treatment (Mustranta and Viikari,1993).

4. Conclusions Based on the present study the following conclusions can be drawn: 1. For the adaptability of microbes in treatment, almost 95–98% of existing microorganisms were dominated by P. corylophilum and A. niger.

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2. The P. corylophilum was more suitable for biodegradation and bioremediation in LSB process under natural conditions than A. niger. The Penicillium inoculum dose of 10% enhanced the treatment efficiency significantly by increasing filterability/ dewaterability and by decreasing COD and turbidity in treatment of DWTP. The COD removal of filtrate of treated sludge with 10% of inoculum dose by Penicillium recorded was 94.40% while it was 80.40% for A. niger after 2 days of treatment. In addition, the COD value of treated sludge was much lower (88–92.8 as mg O2/L) than the untreated sludge (1640 as mg O2/L) by Penicillium. 3. Turbidity removal of filtrate by Penicillium was observed as 98.95% after 2 days of the treatment period. The lowest turbidity value of treated sludge was 1.07 NTU, which is very close to that of distilled water (0.82 NTU) by Penicillium. 4. The lowest value of SRF was recorded as 0.11
1012 m/kg, which was about 13 times lower than the untreated sludge (1.36
1012 m/kg), for 10% of inoculum dose of P. corylophilum after 2 days of treatment 5. It can be concluded that the developed LSB process is a new biotechnological approach for sludge management strategy. The potential fungi in the sludge and developing optimum process conditions for LSB may be able to solve the global sludge problem; however, it still needs special consideration on research in lab scale.

Acknowledgements Support for this research was provided by IWK-UPM project-9 under the Industrial Funded Research Project, University Putra Malaysia (UPM), Malaysia. The authors are grateful to the Department of Chemical and Environmental Engineering, UPM, Malaysia and IWK, for their co-operation, lab facilities, stock microorganisms, research fund and supply of sludge sample during the study. The authors thank to Mr. Derek E. Emerson, UK, Mr. Ying Kei Thomas Ha, Hong Kong and Mr. Munir H. Khan, Malaysia, for their cooperation and help in the spell and grammar check in the preparation of manuscript.

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