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Physiological activities associated with biofilm growth in attached and suspended growth bioreactors under aerobic and anaerobic conditions a

a

a

b

Iffat Naz , Shama Seher , Irum Perveen , Devendra P. Saroj & Safia Ahmed a

a

Department of Microbiology, Quaid-i-Azam University, Islamabad 45320, Pakistan

b

Faculty of Engineering and Physical Sciences, Centre for Environmental and Health Engineering (CEHE), University of Surrey, Surrey GU2 7XH, UK Accepted author version posted online: 22 Jan 2015.Published online: 30 Jan 2015.

Click for updates To cite this article: Iffat Naz, Shama Seher, Irum Perveen, Devendra P. Saroj & Safia Ahmed (2015) Physiological activities associated with biofilm growth in attached and suspended growth bioreactors under aerobic and anaerobic conditions, Environmental Technology, 36:13, 1657-1671, DOI: 10.1080/09593330.2014.1003614 To link to this article: http://dx.doi.org/10.1080/09593330.2014.1003614

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Environmental Technology, 2015 Vol. 36, No. 13, 1657–1671, http://dx.doi.org/10.1080/09593330.2014.1003614

Physiological activities associated with biofilm growth in attached and suspended growth bioreactors under aerobic and anaerobic conditions Iffat Naza , Shama Sehera , Irum Perveena , Devendra P. Sarojb and Safia Ahmeda∗ a Department

of Microbiology, Quaid-i-Azam University, Islamabad 45320, Pakistan; b Faculty of Engineering and Physical Sciences, Centre for Environmental and Health Engineering (CEHE), University of Surrey, Surrey GU2 7XH, UK

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(Received 21 June 2014; accepted 27 December 2014 ) This research work evaluated the biofilm succession on stone media and compared the biochemical changes of sludge in attached and suspended biological reactors operated under aerobic and anaerobic conditions. Stones incubated (30 ± 2°C) with activated sludge showed a constant increase in biofilm weight up to the fifth and seventh week time under anaerobic and aerobic conditions, respectively, where after reduction ( > 80%) the most probable number index of pathogen indicators on ninth week was recorded. Reduction in parameters such as biological oxygen demand (BOD) (47.7%), chemical oxygen demand (COD, 41%), nitrites (60.2%), nitrates (105.5%) and phosphates (58.9%) and increase in dissolved oxygen (176.5%) of sludge were higher in aerobic attached growth reactors as compared with other settings. While, considerable reductions in these values were also observed (BOD, 53.8%; COD, 2.8%; nitrites, 28.6%; nitrates, 31.7%; phosphates, 41.4%) in the suspended growth system under anaerobic conditions. However, higher sulphate removal was observed in suspended (40.9% and 54.9%) as compared with biofilm reactors (28.2% and 29.3%). Six weeks biofilm on the stone media showed maximum physiological activities; thus, the operational conditions should be controlled to keep the biofilm structure similar to six-week-old biofilm, and can be used in fixed biofilm reactors for wastewater treatment. Keywords: biofilm growth; stone media; attached growth process; suspended growth process; wastewater treatment

1. Introduction Deteriorating fresh water quality and growing demands of water for human consumption necessitate the development and implementation of cost-effective, efficient wastewater treatment technologies. Compared with different physical and chemical wastewater treatment systems, biological systems have been gaining much attention currently due to low operational cost, easy handling and comparatively less harmful effects exerted on the corresponding environment.[1] Biological treatment processes are generally of two types: suspended and attached growth.[2] In the suspended growth process, activated sludge technology is the most commonly used in industrialized countries, in which biological solids are removed by sedimentation. Poor settling of these solid pollutants can lead to increased solids treatment costs, increased effluent solids concentrations, decreased disinfection efficiencies, and increased risks to downstream ecosystems and public health.[3,4] However, the attached growth process is a well-established technology, in which solid media are added to suspended growth reactors to provide attachment surfaces for biofilms, thereby increasing microbial concentrations and rates of contaminant degradation.[4–7] Biofilm systems have several advantages such as operational flexibility, reduced hydraulic retention time, resilience to changes

*Corresponding author. Email: safi[email protected] © 2015 Taylor & Francis

in the environment, high active biomass concentration, enhanced ability to degrade recalcitrant compounds as well as a slower microbial growth rate resulting in lower sludge production.[2,8–10] Biofilms take advantage of a number of removal mechanisms such as biodegradation, biosorption, bioaccumulation and biomineralization.[11,12] These biofilm reactor configurations both fixed and moving bed based on the state of the support material are applied in wastewater treatment. In fixed bed systems, the biofilm is formed on static media such as rocks, plastic profiles, sponges, granular carriers or membranes.[13] The liquid flow through the static media supplies the microorganisms with nutrients and oxygen. Moving bed systems comprise all biofilm processes with continuously moving media, maintained by high air or water velocity or mechanical stirring.[14] Biofilms can be developed on all types of surfaces (biotic and abiotic) in most moist environments.[15] It consists of diverse microorganisms where usually bacteria predominate and linked detrital substance that adheres to the substrates in water.[16] During biofilm progression, bacteria move with mass transport towards the substratum by chemotaxis or Brownian motion, resulting in a temporary bacteria–surface association.[17] The initially adhered cells secrete extracellular polymeric substances (EPS),

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forms a cohesive, three-dimensional polymer network that interconnects and transiently immobilizes biofilm cells.[18] The shift from reversible to irreversible attachment is relatively rapid, i.e. few minutes or less.[19] Surface attached bacterial cells use the nutrients in the conditioning film and the aqueous bulk to grow and produce more EPS resulting in the formation of microcolonies.[20] Eventually, the microcolonies expand to form a layer covering the surface.[21] Detachment is the last step, occurred naturally or artificially and dispersal of biofilm colony cells is an essential stage of the biofilm life cycle. It enables biofilms to spread and colonize new surfaces.[22] Biofilm formation, detachment, composition, etc. on support media during its succession can be studied by using different techniques such as gravimetric analysis,[23] microscopic analysis (light microscopy,[24] scanning electron microscopy (SEM),[13,25] confocal electron microscopy [26]), spectroscopic analysis (Fourier transform infrared, nuclear magnetic resonance) [27] and various molecular methods (GEL Electrophoresis,[28] 16S ribosomal RNA, Denaturation Gradient Gel Electrophoresis, Fluorescence in situ hybridization).[29,30] Biofilm carrier media are selected based on size, porosity, density, resistance to erosion and chemicals.[31] The filter media should have a larger surface area and large pores and is generally made of crushed rock, gravel, polystyrene packing, tyre derived rubber and plastic modules.[32] Tilley et al.[33] recommended that the rock particles should be uniform such that 95% of the particles have a diameter between 7 and 10 cm. The size is not too critical; however, it is important that the media are uniform to allow sufficient ventilation through the void space, locally available and cost effective. The use of stone media seems to be a viable option because of the economic advantage, ease of availability and the high potential for biofilm attachment. Its use as a filter media in attached growth reactors for treatment of wastewater is not a new idea.[7] However, a very little literature is available about conventional methods used to study biofilms in such bioreactors and about comparative studies regarding wastewater treatment efficiencies of fixed film reactors and suspended growth reactors.[34] Therefore, the present research study was designed to use stones as a biofilm supporting media in attached growth reactors for detailed investigations of three fundamental phases under aerobic and anaerobic conditions. First, monitoring of the growth of biofilm during succession on stone media exposed to activated sludge; second, the evaluation of the most competent phase of biofilm, i.e. physiologically/metabolically active and free of pathogenic indicators during its development on stone media and finally, the assessment of stability of biofilm growth supporting matrix media by SEM under aerobic and anaerobic conditions, to be applied for biological treatment of wastewater in attached growth reactors. Moreover, the comparative study of attached growth and suspended growth reactors for the removal of organic matter and nutrients from activated

sludge under aerobic and anaerobic conditions was also considered in detail, to be used in future for respective pollutants removal. 2. Materials and methods 2.1. Media selection and evaluation Stones (pebbles) having an average surface area (4πr2 ) of 21.23 cm2 and total occupied volume (4/3π r3 ) of 9.2 cm3 were collected from a fresh water stream. For quantification of elements in stone medium, X-ray photoelectron spectroscopy (XPS) analyses (ThermoFisher Scientific Theta Probe spectrometer, East Grinstead, UK) were performed. As a mono-chromated Al Kα X-ray source (hν = 1486.6 eV), an X-ray spot of ∼ 400 μm radius was used to obtained XPS spectra. Survey spectra were acquired by using pass energy of 300 eV. High-resolution core level spectra for C 1s, O 1s, Ca 2p and N 1s were acquired with a pass energy of 50 eV, while all other highresolution core level spectra were acquired by using a pass energy of 80 eV. All spectra were charge referenced against the C 1s peak at 285 eV to correct for charging effects during acquisition. Quantitative surface chemical analyses were calculated from the high-resolution core level spectra following the removal of a non-linear (Shirley) background. The manufacturers ‘Avantage’ software was used, which incorporates the appropriate sensitivity factors and corrects for the electron energy analyzer transmission function. 2.1.1. Development of biofilm on stone media Biofilm was developed on sterilized stone media by using activated sludge as a seed, sampled from the aerobic wastewater treatment plant (Islamabad, Pakistan). After initial physico-chemical and microbiological characterization of sludge, stone media were incubated in the activated sludge in small reactors, i.e. glass jars (height = 19.05 cm, radius = 6.95 cm) with a volumetric capacity of 2891.93 cm3 and the biofilm was allowed to grow on stone media under aerobic and anaerobic conditions at 30°C (Figure 1). Exactly the same experimental set-up having sludge without stones; considered as suspended growth reactors were operated under similar conditions (in parallel). The experimental jars were covered with perforated gauze and were slowly and periodically agitated to maintain aerobic conditions. While anaerobic conditions were created by packing stone media and sludge in paraffin-sealed jars initially spurged with nitrogen gas (5 min). All the experiments were conducted in batch mode, without the addition of fresh sludge till nine weeks of experiments in order to explore the changes in the attached growth as well as in the suspended growth bioreactors. 2.2. Characterization of the sludge The sludge samples were collected at certain time intervals (0, 3, 6 and 9 weeks) from the reactors and were subjected to microbiological and chemical analyses.

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Microbiological and chemical analyses of sludge before experimentation and evaluation of stone media by XPS

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Aerobic conditions at 30ºC (Batchreactors covered with perforated material and periodically shaken)

Attached growth reactors (Stone media incubated with sludge)

Suspended growth reactor (Sludge incubated without stone media)

Estimation of physiological activities of microbes by characterization of sludge after 3rd, 6th and 9th weeks of incubation (Microbiological and chemical analyses)

Estimation of the growth of biofilm by weight, optical density and scanning electron microscopy after rd 3 , 6th and 9th weeks of incubation

Anaerobic conditions at 30ºC (Batch reactors tightly sealed with paraffin after sparged with nitrogen gas)

Attached growth reactors (Stone media incubated with sludge)

Suspended growth reactor (Sludge incubated without stone media)

Estimation of physiological activities of microbes by c haracterization of sludge after 3 rd, 6 th and 9 th weeks of incubation (Microbiological and chemical analyses)

Estimation of the growth of biofilm by weight, optical density and scanning electron microscopy after rd 3 , 6th and 9th weeks of incubation

Figure 1. Flow sheet of the experimental set-up and operation.

2.2.1. Quantification of pathogenic indicators Coliforms and Escherichia coli were enumerated from the sludge samples using the most probable number (MPN) test. For fecal coliforms MacConkey’s broth was used in the multiple tube technique. Positive tubes showing colour change and gas production were sub-cultured on nutrient agar and mannitol salt agar plates and incubated at 44.2°C for 24–48 h. Positive isolates (showing growth) were confirmed by microscopy (Gram’s staining and shapes analysis). For the investigation and enumeration of E. coli, lactose broth was used for MPN index determination. Positive tubes were sub-cultured on Eosin methylene blue and nutrient agar plates. Positive isolates were confirmed by microscopic observations.

2.2.2. Quantification of chemical parameters The chemical oxygen demand (COD), determined by the kit method; high range 14,541 and low range 14,560 COD kits (Merck Co., Germany), absorbance of

digested sample (sludge) was taken by spectroquant (Pharo ® 100 Spectroquant Merck, Germany). Biological oxygen demand (BOD) was estimated by five-day BOD test (5210 B), while 4500-P, 0375 barium chrometery, 4500 NO2 –N and 4500 NO3 –N methods were used for the determination of orthophosphate, sulphates, nitrites and nitrates according to standard methods.[35] The pH was determined by a digital pH meter (D-25 Horiba) and dissolved oxygen (DO) by a digital DO meter (MM-60R, TOA-DKK). All the analyses were performed in triplicates and the mean values were compared by the t-test and p < .05 was considered as the minimum value for statistical significance using Microsoft Excel 2007. 2.3. Monitoring of biofilm growth on stone media The adhesion and detachment of microbes on media in attached growth reactors were analysed by gravimetric (weight, optical density (OD)), microscopic and biochemical techniques under both aerobic and anaerobic conditions.

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2.3.1. Estimation of biofilm weight Dry and wet weights of biofilm from stone were measured in triplicates during its progression till nine weeks duration. The wet weight of bacterial growth on media was obtained by using a digital weighing balance (Scout™ pro, OHAUS) after soft rinsing with distilled water and gentle blotting. On the other hand, biofilm dry weight measurements were conducted as previously described [36] with some modifications. 2.3.2. Estimation of biofilm OD The OD of the biofilm grown on stone media was measured after each week until nine weeks time. The stones with biogrowth were gently taken (avoid force) from experimental set-up under aseptic conditions and delicately washed with distilled water for the removal of sludge particles. The biofilm was removed in 0.9% saline and absorbance of dissolved biofilm was taken at 550 nm. 2.3.3.

Estimation of biofilm heterotrophic plate count per mL Heterotrophic plate counts (HPC) of selected microbes in biofilms were determined by the conventional serial dilution method. The biofilms suspension was serially diluted up to 10−5 and plated on selective media (nutrient agar, Eosin methylene blue agar, MacConkey agar, Salmonella Shigella agar, Pseudomonas Citrimide agar, mannitol salt agar and blood agar) for microbial counts. Pure cultures thus obtained were further identified and characterized according to the Bergey’s manual.[37] 2.3.4. Scanning electron microscopy The copper stubs (10 × 10 mm) were washed with deter® gent, dried by drier KADA 85 U/SMD, containing heat gun at 200°C for 2 min before sample loading. The dried samples (finely cut surfaces of stone media) were fixed on the stub with carbon tape, sticky on both sides to cover the edges of the sample. Silver paste conduction (SPI-CHEM) was also applied to ensure the conduction of the electron beam. Then, high voltage and vacuum of 10−2 ATM were created in sputtering for gold coating by using the SPIMODULE (sputter coater). A high voltage (25 mA current for 50 s) was used to create plasma for the deposition of gold on the target sample. The sample was then loaded on the holder and it was placed in the chamber under the column (15 min vacuum was created in the chamber). Finally, the surface morphology of the biofilm and then of the carrier was observed on the screen under 3000 and 5000 × magnification. 3. Results and discussions 3.1. Stone media evaluation by XPS It is very important to discover the elemental composition of support media for the determination of the compatibility

of microbes with it. Presently, XPS analysis for the evaluation of stone media was undertaken by Thermo Scientific XPS Theta Probe spectrometer with monochromated Al Kα radiation with a 400 μm spot size. The intensity of photoelectrons as a function of binding energy is shown in Figure 2(a). Two peaks appeared on C 1s spectrum at 284.8 and 289.7 eV representing adventitious carbon and carbonate, respectively (Figure 2(b)). Peaks observed for O 1s, Ca 2p3/2 , Ca 2p1/2 and Si 2p at 531.5, 347.1, 350.6 and 103 eV, respectively (Figure 2(c–e)). The stone contains C 1s (38%), O 1s (49%), Ca 2p (12%) and Si 2p (1%). This suggests that the stone medium’s main mineral is calcium carbonate (CaCO2− 3 ) and it has no hazardous effects on developing biofilms on its surface and is quite durable.[38]

3.2.

Characterization of sludge during experimental period Succession of the biofilm on stone media and free living bacteria exerted various microbiological and physicochemical changes in the sludge. Qualitative and quantitative variations in microbes were due to its fixation in the form of the biofilm or its removal. While changes in physico-chemical parameters of sludge were due to the metabolic activities of both suspended and attached microorganisms.

3.2.1.

MPN index of indicator pathogens

Microbiological characterization was performed before and after nine weeks in suspended and attached growth reactors, by determination of the MPN index of two types of pathogen indicators, i.e. E. coli and fecal coliforms. Fecal coliforms were considered because they are the most commonly used bacteria indicators to monitor water quality.[39] Initially, MPN/100 mL of fecal coliforms in sludge (95% confidence limit for various combinations of positive and negative results) was > 1100. The presumptive and confirmatory tests were also positive for MPN positive tubes and after nine weeks of incubation, its range was 21 and 15 (98% and 98.6% reduction) under aerobic and anaerobic conditions, respectively, in the biofilm reactor (Table 1). However in the suspended growth reactor, the MPN index was 240 and 460 (78.18% and 58.18% reduction, respectively) under aerobic and anaerobic conditions, respectively, after nine weeks of incubation. Similarly, MPN/100 mL of E. coli of the activated sludge sample showed an MPN index of > 1100 and after nine weeks of interval its value dropped to 28 and 240 (97.4% and 80% reduction) in biofilm reactors, 220 and 240 (80% and 78.2% reduction, respectively) in suspended growth reactors under aerobic and anaerobic conditions, respectively. Comparatively, less reduction in fecal coliforms and E. coli was observed in anaerobic reactors.

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Figure 2. (a) X-ray photoelectron spectrum (XPS) of stone media indicating cluster of elements, (b) peak of C 1s, (c) O 1s, (d) Ca 2p and (e) Si 2p.

Various researchers have also noticed that coliforms removal efficiency remained low in anaerobic systems. [40,41] However, considerable reduction of MPN/100 mL of pathogenic microbes in both experimental sets, under aerobic and anaerobic environments, revealed depletion of organic matter contents in the sludge over a period of nine weeks. A positive correlation of indicator bacteria with organic matter (BOD) was also reported.[42] However, the

indicators showed highly significant reduction (p < .005) in the biofilm reactors in the presence and absence of air due to the removal of inorganic and organic pollutants in biofilms reactors or due to retention in the biofilm by adsorption.[43] Later it was associated with detachment and deactivation or death of the microbes.[44] The removal efficiency of pathogenic indicators has also been indirectly linked with parameters such as DO, pH and temperature.[45,46]

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Table 1.

MPN index of E. coli and fecal coliforms in the activated sludge before and after a period of nine weeks. Fecal coliform

E. coli MPN index/100 mL Comparative analysis of sludge Attached growth reactor

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Suspended growth reactor

3.2.2.

Conditions Before colonization (sludge) Aerobic

After nine weeks

> 1100

95% confidence limit Lower 150

MPN index/100 mL

95% confidence limit

Upper 4800

> 1100

Lower 150

Upper 4800

210

35

470

21

4

47

Anaerobic Aerobic

28 220

10 35

150 470

15 240

3 36

44 1300

Anaerobic

240

36

1300

460

71

2400

Changes in the concentrations of chemical parameters

The biodegradative capabilities of the biofilm in attached growth reactors and suspended growth reactors were evaluated at regular intervals in terms of changes in the initial concentrations of chemical parameters and nutrients in the activated sludge under aerobic and anaerobic conditions. Initially, the average concentration of COD in the sludge was 292 mg/L in all the experimental sets under both aerobic and anaerobic conditions. Under aerobic condition, 74.5% and 33.5% (74.5 and 194 mg/L, respectively) reduction was noticed in COD after nine weeks in attached and suspended growth reactors, respectively. It revealed that 40.9% reduction in COD was due to biofilm bacteria and 33.5% owing to free living microbes of sludge (Table 2). Under anaerobic condition after nine weeks, the COD values showed a significant decline (70.1%; p < .001) in attached growth reactors (Figure 3(b)). While only a 42.8% decrease was observed in the suspended growth set-up. Whereas more COD reduction (42.8%) was noticed by free living bacteria as compared with biofilm (27.2%) under anaerobic conditions (Table 2). The mean BOD5 of the activated sludge at the start of experiment was 198.6 mg/L which was significantly reduced (p < .05) over a period of nine weeks under both aerobic and anaerobic conditions (Figure 3(c) and Table 2). The net BOD reduction was 70.9% after nine weeks of biofilm development on media, out of which 47.7% (more) is contributed by bacterial growth on stone media and 23.2% (suspended growth reactor) owing to free living microbes. The reduction in BOD under aerobic conditions was attributed to high biodegradation of organic matter by oxidation. Under anaerobic conditions, a considerable drop (p = .037) in BOD5 was reported by biofilm (14.2%) after nine weeks (Figure 3(c)), while a 53.8% decline in the concentration of BOD was due to free microbes of the sludge out of net 68.1% reduction. Here, the reduction of BOD5 was owing to anaerobic digestion of organic matter.

There was a linear relationship between the elimination rate of pollution indicators (COD, BOD5 and pathogens) with time interval from sludge under aerobic and anaerobic conditions. The average concentration of DO in the activated sludge was 1.02 mg/L in both experimental sets. After nine weeks of incubation, DO levels considerably raised to 3.4 and 1.6 mg/L (233.3% and 933.3%, respectively) in attached growth and suspended growth reactors, respectively, under aerobic and anaerobic conditions. Figure 3(e) revealed a considerable increase (p = .054) of 1.8 mg/L of DO, contributed by biofilm alone (176.5%). The concentration of DO is inversely related to the concentrations of COD and BOD under aerobic environments. In anaerobic conditions, DO levels dropped (p > .05) from its initial values to 1.0 and 0.6 mg/L in suspended and attached growth reactors, respectively (Figure 3(f)). Since no additional oxygen was provided, DO concentration decreased and further conformed by the falling of pH from 7.02 to 6.8. There was not much change in pH values during the experiments. Under aerobic conditions in the attached growth reactor, pH remained in the neutral range (7.04– 7.01) While a change in pH from 7.04 to 6.8 was noticed in anaerobic environments (Figure 4(a)). Similarly in suspended growth reactors more change in pH towards the acidic window was noticed in an anaerobic conditions, i.e. changed from 7.04 to 6.2 (11.9%) over a period of nine weeks incubation as compared with aerobic conditions (Figure 4(b)). Most probably this change is due to the conversion of organic pollutants by biofilm and free living microbial activates to acid products in anaerobic circumstances. Moreover, change in pH under both aerobic and anaerobic conditions over a period of nine weeks in batch reactors may be due to simultaneous reduction of BOD, COD and nitrification, where some of the heterotrophs respiration-derived CO2 is available for the nitrifiers and will therefore reduce the alkalinity of the medium. It was also previously reported as BOD and COD concentrations

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Figure 3. (a) Changes in the concentration of BOD, (c) COD and (e) DO of sludge under aerobic conditions while (b) indicating changes in the levels of BOD, (d) COD and (f) DO in sludge under anaerobic conditions.

decrease, the DO concentration increases which trigger the process of nitrification.[47,48] 3.2.3. Changes in the concentrations of nutrients The average level of nitrites (NO2 ) in the activated sludge was initially 0.126 mg/L. After biofilm development on stone under aerobic conditions its levels showed a net reduction of 23.1% in nine weeks duration. While in the suspended growth apparatus, the final concentration was 0.024 mg/L in the sludge indicating 7.3% reduction in nitrites concentration due to free living microbes. Thus, more (17.8%) reduction in nitrites concentration was exerted by the aerobic biofilm (Figure 5(a)) and illustrated by the existence of slow growing nitrifying bacteria, i.e. nitrobacter. Nitrification plays an important role in aerobic wastewater treatment,[47] while an anaerobic condition causes the half-life of nitrifies.[48] Whereas under anaerobic conditions the total reduction in nitrites was 26.9% after nine weeks of biofilm formation on stone media. More reduction (15.4%) was

noticed due free living microbes (suspended growth reactors) as compared with biofilm (11.5%) (Figure 5(b) and Table 2). On the other hand, nitrates levels increased from 0.372 to 0.881 mg/L and 0.489 mg/L in the suspended growth reactor and in attached growth reactor, respectively, under aerobic conditions (Figure 5(c) and Table 2). Szatkowska et al.[49] reported multifunctional biological reactions in the biofilm under aerobic environment such as simultaneous nitrification and denitrification. Under anaerobic conditions biofilm fixation enhanced the NO3 increase (54.1%) as compared with suspended growth reactors (31.7%). While the per cent increase was less than the aerobic biofilm (105.5%) on stone surfaces (Figure 5(d)). Overall, a 22.4% increase in nitrates in the sludge was attributed to the biofilm (Table 2). Thus, indicating the second step of the process of nitrification by Nitrobacteria.[50] The attached growth process has more capacity of biomass and nitrogen removal than the suspended growth process. There could be an anoxic zone inside the media and that can lead to simultaneous nitrification–denitrification.[6,51]

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I. Naz et al. Table 2. Physiological activities of microbes (suspended and biofilm) under aerobic and anaerobic conditions over a period of nine weeks. % Change in sludge Aerobic conditions Time (weeks) Parameters (mg/L)

Experimental set-up

BOD5

Suspended growth reactor Attached growth reactor Biofilm Suspended growth reactor Attached growth reactor Biofilm Suspended growth reactor Attached growth reactor Biofilm Suspended growth reactor Attached growth reactor Biofilm Suspended growth reactor Attached growth reactor Biofilm Suspended growth reactor Attached growth reactor Biofilm Suspended growth reactor Attached growth reactor Biofilm

COD

NO2 NO3 PO4 SO4

3rd

6th

9th

3rd

6th

9th

↓14.5 ↓43.4 ↓28.8 ↓8.5 ↓32.0 ↓23.4 ↑20.6 ↑81.4 ↑60.8 ↓6.3 ↓28.8 ↓22.2 ↓2.1 ↑6.9 ↑4.8 ↓8.6 ↓26.4 ↓17.8 ↓9.0 ↓38.7 ↓29.7

↓23.8 ↓57.3 ↓33.3 ↓22.3 ↓61.3 ↓39.0 ↑33.3 ↑164.7 ↑131.4 ↓18.2 ↓46.8 ↓28.6 ↑13.8 ↑82.1 ↑68.3 ↓1.9 ↓51.5 ↓49.6 ↓20.3 ↓57.9 ↓37.6

↓23.2 ↓70.9 ↓47.7 ↓33.5 ↓74.5 ↓41.0 ↑56.9 ↑233.3 ↑176.5 ↓20.6 ↓80.9 ↓60.2 ↑31.4 ↑136.9 ↑105.5 ↓1.8 ↓60.7 ↓58.9 ↓40.9 ↓69.2 ↓28.2

↓28.3 ↓46 ↓39.8 ↓19.9 ↓40.6 ↓20.7 ↓0.0 ↓1.9 ↓1.9 ↓11.9 ↓19.7 ↓7.8 ↑2.4 ↑3.8 ↑1.4 ↓20.2 ↓39.4 ↓19.2 ↓16.5 ↓43.1 ↓26.6

↓42.7 ↓55.6 ↓12.9 ↓30.9 ↓59.1 ↓28.2 ↓0.9 ↓31.4 ↓30.4 ↓16.7 ↓30.6 ↓13.9 ↑19.1 ↑30.9 ↑11.8 ↓37.4 ↓62.4 ↓25.0 ↓50.9 ↓55.7 ↓4.8

↓53.8 ↓68.08 ↓14.2 ↓42.8 ↓70.1 ↓27.2 ↓1.9 ↓60.8 ↓59.8 ↓28.6 ↓48.8 ↓20.2 ↑31.7 ↑54.1 ↑22.4 ↓41.4 ↓64.2 ↓22.8 ↓54.9 ↓84.2 ↓29.3

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A decrease in organic and inorganic carbon positively influences the growth of nitrifiers and thus ultimately triggered the process of nitrification. However, the low concentration of nitrates in anaerobic apparatuses (both attached and suspended process) can be attributed to denitrification. Knowles [52] reported when available oxygen is exhausted (low DO concentrations) and nitrates are accessible, the reduction of NO3 to NO2 , N2 O or N2 chiefly occurs. Thus, NO3 reduction in the present research might be due to Bacillus, Pseudomonas and many members of Enterobacteriaceae, identified in the sludge as well as in the biofilm developed from sludge on stones. Along the same line, it was previously reported

that nitrates ammonification is carried out by facultative anaerobic bacteria belonging to the genera Bacillus, Citrobacter, Pseudomonas and Aeromonas, or members of the Enterobacteriaceae.[53] The concentration of PO4 was initially very low (0.016 mg/L) in the sludge and it consistently reduced over a period of nine weeks both under aerobic and anaerobic conditions (p = .051). The average reduction in its level in the sludge of suspended and attached growth reactors was 1.8% and 51.5%, respectively, in aerobic experimental sets. Thus, illustrating the highest removal efficiency (58.9%; p = .037) of biofilm-forming phosphate accumulating organisms in aerobic environments (Table

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2). Bajekal and Dharmadhikari [54] confirmed by microscopic observation that the removal of PO4 was due to the intracellular accumulation by the organisms. In sludge without support media, the PO4 decline was very slow (0.02 mg/L after ninth week) as compared with stone media (0.005 mg/L after ninth week of biofilm formation) under aerobic surroundings (Figure 6(a)). Kerrn-Jespersen and Henze [55] also reported rapid phosphorus uptake in

aerobic conditions, Helness and Ødegaard [56] investigated simultaneous nitrification and phosphorus removal under aerobic conditions than under anoxic conditions. Anaerobically more reduction (41.1%) of PO4 occurred in the sludge of the suspended growth reactor as compared with aerobic conditions (Figure 6). In the current study a considerable uptake of phosphate can be due to the process of denitrification.[57,58]

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Figure 7. (a) Changes in weight (wet and dry) of the biofilm during succession on stone surfaces from sludge under aerobic and (b) anaerobic conditions over a period of nine weeks.

Initially, the mean level of SO4 in the activated sludge was 0.22 mg/L. Its concentration considerably decreased to 0.13 and 0.06 mg/L (40.9% and 69.2%, respectively) after nine weeks in suspended and attached growth reactors sludge (p = .05) under aerobic conditions (Figure 6(c)). A proficient concentration (0.06 mg/L; 28.2%) of SO4 was removed (p = .037) solely by the biofilm on stone media (Table 2). From these results it was logical that an increase in DO concentration of the sludge also has a positive effect on the removal of SO4 . On the other hand, its concentration showed diminution due to free living microbes (54.9%) in the suspended growth process, free living plus fixed bacteria on stone media (84.2%) and solely biofilm (29.3%) over a period of nine weeks under anaerobic conditions (Figure 6(d) and Table 2). Free living microbes (p = .03) were responsible for more SO4 removal (0.099 mg/L after ninth week) than biofilm-forming microbes (0.049 mg/L after ninth week). The decrease in sulphate concentration was an indication of the presence of sulphate-utilizing bacteria both free and fixed film conditions.[59] In anaerobic circumstances a pungent smell was observed in both suspended and attached growth apparatuses. It was also previously reported that sulphate reduction causes unpleasant odours in the sewerage system and also affects methane production in the anaerobic process.[60] About 8% sulphates were removed under anoxic conditions (15 min) from complex wastewater in a sequencing batch reactor with aerobic suspended growth of microbes.[61] However, Table 2 indicates that biofilm formation on stone media enhances (84.2%) the removal of SO4 from sludge (0.035 mg/L after nine weeks). Under aerobic conditions, the removal of SO4 was slow; it might be due to the combined activity of sulphur oxidizing bacteria (SOB) and sulphate reducing bacteria (SRB) present in sludge and in biofilm. According to Odom et al.[62] SOBs are widely detected in the environment. These bacteria play an important role in carbon, sulphur and nitrogen cycles. Normally, the SOBs oxidize the sulphur into sulphate and then SRB reduce this sulphate into sulphide (H2 S). The sulphate removal may be correlated with nitrites and phosphates removal by microorganisms. Walker and Nicholas [63] purified a nitrite reductase ( > 600-fold) from extracts

geared from briskly denitrifying Pseudomonas aeruginosa. This enzyme require either PO4 or SO4 for the highest activity.

3.3.

Monitoring of biofilm growth in the attached growth reactor

The successive phases of biomass on the stone were characterized with three main complementary approaches, quantification of the attached biomass by measuring its weight, OD and microscopic observation. 3.3.1.

Assessment of biofilm weight

Under aerobic condition, the wet weight of the biofilm showed a significant increase (p = .002) till the seventh week (0.45 ± 0.01 g) and then followed by a sharp decrease and reached 0.29 ± 0.01 g after nine weeks. While almost the same percentage increase was observed in the dry weight, it was increased till the seventh week (mean: 0.15 g) and then reduced and attained a value of 0.05 g (Figure 7(a)). These results also showed that the total water content of bacterial cells was approximately 91.6%. The dry matter content of bacterial cells was 8.4%. Biofilms are considered as hydrogels because the main component of biofilms is water, which can make up to over 90% of the wet weight.[64] However, generally 20% dry matter content appears to be an accepted standard value for bacterial cells.[65] While under the anaerobic condition, the wet weight of the biofilm showed a slow increase until the third week (0.39 ± 0.01 g) followed by a gradual decrease to 0.23 ± 0.01 g after nine weeks. The dry weight showed a regular increase from the first week (mean: 0.02 g) till the fifth week (mean: 0.09 g), followed by a decrease to 0.04 g after the ninth week (p = .0005) as shown in Figure 7(b). Approximately 90.4% of water and 9.6% of dry matter content in the bacterial cells were revealed by these results. According to Schmitt and Flemming,[66] the biofilms largely consist of water (70– 95% wet weight), held by the highly hydrated extracellular polymers (EPS, 70–95% dry weight) in which the microorganisms are entrenched. During biofilm formation,

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the weights (wet and dry) of the biofilm increased due to increasing growth of the microorganisms that was also shown by increased OD of biofilm.

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3.3.2. Assessment of biofilm OD OD measurement light strength lessening, reported as spectrophotometric absorbance, is proportional to the concentration of bacteria within definite ranges of cell size and shape.[67] Under aerobic conditions, the OD(550) of biofilm increased from the first (0.1452 ± 0.001) to seventh (0.80446 ± 0.0051) week followed by a sudden decrease (ninth week biofilm has an OD of 0.463 ± 0.006) While in the anaerobic biofilm, OD(550) increased from 0.0381 ± 0.0023 to 0.752 ± 0.009 in seven weeks and then drastically reduced to 0.344 ± 0.007 after nine weeks (Figure 8). The most reliable and oldest method to quantify total biofilm was OD and there was a linear relation between OD and dry weight.[68] In the present study the increase in biofilm weight and OD(550) was confirmed by SEM, which showed constant amplification in microbial population on the matrix media with the passage of time. 3.3.3. Assessment of HPC of biofilm Heterotrophs stay alive by utilizing readily available organic matter from their surroundings and transfer it into CO2 in the presence of oxygen. Determination of HPC/mL of these microbes in the activated sludge before and during the experimentation is a very important factor for their quantification. Before subjected to experimentation, immediately after sampling of sludge, 14 different bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Enterobacter aerogenes, Staphylococcus aureus, Salmonella typhimurium, Proteus vulgaris, Alcaligenes faecalis, Micrococcus luteus, Streptococcus lactis, Shigella dysenteriae, Klebsiella pneumoniae, Corynebacterium xerosis and Bacillus subtilis) were characterized on the basis of their morphology (on specific media), light microscopy and physiological tests. Several microbial invaders were recognized and characterized during

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Figure 9. (a) Changes in HPC/mL of the biofilm during succession on stone surfaces from sludge under aerobic and (b) anaerobic conditions over a period of nine weeks.

biofilm configuration, a number of primary colonizers were gram negative, flagellated and appeared to be more aggregated rather than being a monolayer cells. However, four species, i.e. E. coli, P. aeruginosa, S. typhimurium and S. dysenteriae, were selected to be monitored by their HPC/mL on stone media under aerobic and anaerobic conditions over a period of nine weeks in order to investigate microbial shift from the pathogenic biofilm to beneficial organisms (Figure 9(a) and 9(b)). In the aerobic biofilm, E. coli colony counts were increased until the third week (3.4 × 103 HPC/mL), while the anaerobic biofilm showed a gradual increase until the fifth week and its count reached to 2.2 × 103 /mL. The S. typhimurium showed its appearance until the fifth week (4.0 × 102 /mL) and then disappeared under aerobic conditions, while in anaerobic conditions it appeared only in second and third weeks (2.0 × 102 and 4.0 × 102 , respectively). S. dysenteriae colonies were noticed from the third week until the sixth week in the aerobic biofilm in the range of 2.0 × 102 to 6 × 102 /mL (Figure 9(a)). Whereas the anaerobic biofilm has maintained S. dysenteriae colonies in only second and third with HPC/mL of 2.0 × 102 and 3.0 × 102 , respectively. P. aerouginosa started to appear after the fourth week (1 × 102 counts/mL) and diminished in the fifth week biofilm (3.0 × 102 counts/mL) under anaerobic conditions. While in aerobic environments P. aerouginosa maintained their counts from the fourth- to fifth-week-old biofilms (2.5 × 102 to 3.0 × 102 counts/mL) (Figure 9(b)). Thus, a decline of HPC/mL of pathogenic microbes after the sixth and fifth weeks of the biofilm under aerobic

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Figure 10. SEM survey of stone surface and biofilm development: (a) before colonization of the stone surface, (b) numerous rod-shaped cells and few cocci appear on the surface in six-week-old biofilm and (c) abundant bacilli and cocci cover the surface in nine-week-old biofilm under aerobic conditions. While (d) indicates some bacilli and cocci in six-week biofilm and (e) bacilli become arranged in long string after nine weeks of biofilm formation under anaerobic conditions.

and anaerobic conditions, respectively, can be correlated to a decrease in the weight (dry and wet) and OD of the biofilm at a similar time frame of seven and five weeks. The removal of organic matter (sludge COD and BOD reduction), changes in pH, DO, nutrients and SEM micrographs until ninth weeks illustrate the presence of unculterable bacteria (autotrophs) and those microbes (both hetrotrophs and autotrophs) that were not considered in the present investigation. 3.3.4. SEM of biofilm Most of the recent understanding concerning monitoring of biofilm growth is due to the advances in imaging studies mainly the SEM. Biofilm formation, composition, allocation and the relationship to solid surfaces can be revealed by SEM.[69] Presently, preliminary attachment of bacteria on media monitored by light microscopy showed maximum adhesions and growth of bacilli of different sizes

and few cocci in three-week-old biofilm. When various different sizes and shapes appeared, then SEM micrographs of media were taken from different angles in order to study the biofilm absolutely. Figure 10(a) shows the rough appearance of the surface topography of stone when investigated by SEM. It has a granular, porous and uneven nature providing a sufficient place for progression of the biofilm. This roughness of the substratum increases the surface area, aiding colonization, and enables glycocalyx to affix more easily by providing anchoring points, and protects the microbes from fluid dynamic shear forces. The adhesion of bacteria in the six-week-old biofilm has a large community of bacilli as compared with sparsely distributed cocci under the aerobic condition as in Figure 10(b). On the other hand, Figure 10(c) represents the maturation phase of bacterial growth on the surface of media after nine weeks of succession. Under the anaerobic condition, comparatively a smaller number of bacilli and cocci appeared under 5000 × resolution in six-week-old biofilm (Figure 10(d)).

Environmental Technology Figure 10(e) shows the detachment phase in which the bacilli have lined up in a long string like appearance surrounding the stone matrix after nine weeks. There was no remarkable change in the surface morphology of stone media during biofilm development.

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4. Conclusions This research was an attempt to evaluate the biodegradative efficiencies of free living and biofilm-forming microbes over a period of two months. Two different types of reactors, i.e. attached (packed with stone media and sludge) and suspended growth (containing only sludge) reactors, were designed and operated under aerobic and anaerobic conditions. Physico-chemical analysis (COD, BOD, DO, etc.) of the sludge indicates higher metabolic competence in the biofilm-forming microbes in aerobic attached growth reactors and free living microbes in suspended growth reactors under anaerobic conditions. Among nutrients, significant reductions in the concentrations of nitrites, nitrates and phosphates from sludge were achieved in aerobic attached growth reactors, while sulphates showed a remarkable reduction in both aerobic and anaerobic suspended growth reactors. The decrease in plate count of pathogenic indicators in the biofilm and MPN index of indicators, i.e. E. coli and fecal coliforms in the sludge, represents a shift from pathogens to unculturable microbes and deterioration of organic contaminants in the sludge. Monitoring of the growth of the biofilm by weight, OD, HPC/mL and SEM revealed the six-week-old biofilm to be physiologically active and free of pathogenic indicators almost in both aerobic and anaerobic environments. SEM of stone media surface colonized by biofilms illustrated no significant change under both conditions, thus it can be used as a filter media in the fixed biofilm reactors for wastewater treatment. These observations allow an improved understanding of the characteristics of microbial activities in biofilm systems under aerobic and anaerobic conditions, and will help in designing, retrofitting and operating robust biofilm processes for efficient wastewater treatment.

[4] [5] [6] [7] [8] [9]

[10] [11]

[12] [13] [14] [15]

[16] [17]

Acknowledgements This research project was funded by higher education commission of Pakistan (HEC) under Pak–US joint academic & research programme (2009–2010). Authors sincerely acknowledge Dr Steven Hinder (Materials Science and Engineering, University of Surrey, UK) for his cooperation in XPS analysis of stone media during the research visit of Miss Iffat Naz under International Research Support Programme of HEC, Pakistan.

[18] [19] [20]

Disclosure statement No potential conflict of interest was reported by the authors.

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Physiological activities associated with biofilm growth in attached and suspended growth bioreactors under aerobic and anaerobic conditions.

This research work evaluated the biofilm succession on stone media and compared the biochemical changes of sludge in attached and suspended biological...
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