Treatment of gray water using anaerobic biofilms created on

Process Safety and Environmental Protection 9 2 ( 2 0 1 4 ) 186–192

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Treatment of gray water using anaerobic biofilms created on synthetic and natural fibers H.N. Chanakya, Himanshu Kumar Khuntia ∗ Center for Sustainable Technologies, Indian Institute of Science, Bangalore 560 012, India

a b s t r a c t Gray water treatment and reuse is an immediate option to counter the upcoming water shortages in various parts of world, especially urban areas. Anaerobic treatment of gray water in houses is an alternative low cost, low energy and low sludge generating option that can meet this challenge. Typical problems of fluctuating VFA, low pH and sludge washout at low loading rates with gray water feedstock was overcome in two chambered anaerobic biofilm reactors using natural fibers as the biofilm support. The long term performance of using natural fiber based biofilms at moderate and low organic loading rates (OLR) have been examined. Biofilms raised on natural fibers (coir, ridgegourd) were similar to that of synthetic media (PVC, polyethylene) at lower OLR when operated in pulse fed mode without effluent recirculation and achieved 80–90% COD removal at HRT of 2 d showing a small variability during start-up. Confocal microscopy of the biofilms on natural fibers indicated thinner biofilms, dense cell architecture and low extra cellular polymeric substances (EPS) compared to synthetic supports and this is believed to be key factor in high performance at low OLR and low strength gray water. Natural fibers are thus shown to be an effective biofilm support that withstand fluctuating characteristic of domestic gray water. © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. Keywords: Gray water; Anaerobic biofilms; Extracellular polymeric substance (EPS); Confocal imaging

1.

Introduction

Gray water is that component of domestic wastewater which is not contaminated by human feces and constitutes about two-thirds of the total wastewater generated. As India urbanizes the extent of gray water increases, it typically arises as wastewater from bathrooms, wash basins, washing of clothes (including washing machines), washing dishes in kitchen sinks, etc. Gray water is composed predominantly of carbohydrates (from food), proteins and its partial breakdown products (originating from food and sloughed skin), fats and lipids (including fatty acids liberated during breakdown of food wastes), glycerides, detergents (sodium lauryl sulfate, etc.), soap components (sodium palmitate, sodium hydroxide and sodium carbonate), etc. (Eriksson et al., 2002). In urban areas, e.g. Bangalore, only 810 million liters per day (MLD) of water is supplied against a demand of 1300 MLD. A part of this 500 MLD shortfall is currently being met from very rapidly depleting ground water (GW) resources pumped from 300 m depths. Gray water recycling (67% of the

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current daily water use, c.70l per capita/d, lpcd) will become an effective alternative for secondary uses such as toilet flushing, reducing water consumption by 30% (Burkhard et al., 2000). Out of 70 lpcd of gray water, 32l may be used for toilet flushing and remaining 38l for watering of plants, floor and car washing, etc. Graywater recycling is often practiced in rural India in areas of drought/short supply and is therefore not expected to be a psychological barrier. Food processing wastewater is reused in cattle rearing, kitchen wash water and bath water is recycled to kitchen gardens, other wash water is reused for pour flushing, etc. In spite of this long tradition of sustained gray water reuse this is accepted only for wastewater originating from within one’s own household. Treated water from common pools or mixed sources will therefore become culturally unacceptable. Household level graywater recycling techniques is a better goal to pursue, especially with low energy and carbon footprint. On the other hand treated water arising from a group of houses and combined gray and black water has usually been found to be culturally unacceptable.

Corresponding author. Tel.: +91 08022933046. E-mail addresses: [email protected], [email protected] (H.K. Khuntia). Received 27 May 2012; Received in revised form 14 November 2012; Accepted 25 December 2012 0957-5820/$ – see front matter © 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.psep.2012.12.004

Process Safety and Environmental Protection 9 2 ( 2 0 1 4 ) 186–192

Primary treatment of gray water using an anaerobic process is an effective water reusing alternative that meets the abovementioned criteria of cost, simplicity, environmental footprint, ruggedness and to large extent cultural acceptability. Such a treatment can convert >95% of BOD into CO2 and CH4 (biogas) with very little undesirable byproducts. Biofilms in anaerobic bioreactors offer long mean cell residence time (MCRT) at lower long hydraulic retention time (HRT) that prevents the washout of microbial consortia as commonly found in suspended growth based anaerobic reactors (Bryers, 1993). However, the overall performance of the biofilm reactor is directly related to the surface area (of biofilm) per unit volume of the support material (Picanco et al., 2001). The formation of biofilms on various inorganic and synthetic support materials like pumice stone, polysulfone, ceramic material, polysterene, sepiolite, PVC, diabase, polyurethane and bentonite has been investigated (Sen and Demirer, 2002; Picanco et al., 2001; McEldowney and Fletcher, 1987; Sanchez et al., 1994). Synthetic biofilm supports while performing well at high loading rates are easily amenable to washout at low feed rates or periods of no feed situations-typical of domestic household situations. Alternative support materials overcoming these difficulties are required. Natural lignocellulosic materials can also be alternatives to the predominant inorganic support material used as a biofilm support. Natural lignocellulosic supports are abundant in developing countries, have lower costs, possess comparable surface area per unit volume, while having high porosity, low specific gravity and higher bacterial adsorption/adhesion (Nabizadeh et al., 2008). Organic materials can perform a dual activity by providing substrate for the bacterial metabolism while the more recalcitrant lingocellulosics can simultaneously function as a support material because of its slower degradation rates (Chanakya et al., 2005). Andersson et al. (2008) studied the rate of biofilm formation and its retention on 20 different support materials that include inorganic, natural and synthetic support materials and found that wood chips had a comparatively faster biofilm formation rate with higher biofilm retention capability. Shao et al. (2009) used rice husk as sole carbon source and bacterial support for the denitrification of waste water. Mshandete et al. (2008) used sisal fiber waste, pumice stone and porous glass beads as the biofilm support to treat sisal leaf waste leachate and found that sisal fibers based biofilm support showed comparatively higher COD reduction. While leaf biomass provided a good biofilm support it had a short half-life of 180 d (Chanakya et al., 1998). Thus organic fibers and various slow to degrade biomass residues can show potential as natural biofilm supports in wastewater treatment over a wide range of OLR, particularly when dealing with low strength wastewaters and low HRT. Typical anaerobic digesters and graywater treatment systems subjected to rapidly varying strength graywater under typical Indian household conditions fail because the easy to decompose food residues within are rapidly converted to VFA, lowering pH (<5.5) and cause methanogen inhibition. In this study we seek technological alternatives to ensure that methanogens are protected against rapid VFA and low pH formation in typical immobilized/biofilm based anaerobic reactors. We attempted a two zone graywater treatment system that contains (a) low pH region in the inner zone (IC, Fig. 1) while protecting the (b). Second zone (OBF, Fig. 1) characterized by predominantly methanogenic activity from possible low pH fluxes and VFA induced inhibition. We believe that rapid VFA build up in inner zone, whenever it occurs, can be released slowly into outer zone, based on the usage pattern thereby

187

Fig. 1 – Sketch of the laboratory scale two zone anaerobic biofilm reactor. (1) Feed inlet, (2) inner chamber (IC), (3) outer chamber (OBF), (4) nylon mesh, (5) outlet. making a reasonably rugged technology in gray water treatment. This strategy avoids active process control needs for small plants. The second aim of this experiment was to test the maximum OLR that a particular bioreactor would be able to withstand without reducing its treatment efficiency as well as to evaluate the performance of the bioreactors at low loading rates of 250 mg/l/day. Finally the efficiency and sustainability of natural fibers as an alternative to the synthetic material as a biofilm support were examined based on their long term performance and support material degradation under anaerobic conditions. Early studies (Chanakya et al., 1998) had indicated biofilms on biomass had a half-life of 180 d and therefore lignin-rich fibrillar materials are being tried to provide a longlife natural material based biofilms support.

2.

Materials and methods

2.1.

Experimental setup

Laboratory scale anaerobic biofilm reactors were fabricated using polyethylene containers as shown in Fig. 1. The total volume of each bioreactor was 5l .The volume of the inner and the outer chambers were 1 and 4l, respectively measured by its geometry. This size chosen based on the availability of containers for fabricating such digesters and ease of scaleup. The inner chamber and the outer chamber of each reactor were packed with one type of support material (Table 1). Various support materials like PVC spiral, coconut husk fiber (coir), ridgegourd fiber (RG), bioflow (BF) made of polyethylene (PE) were used as biofilm support material in the experiment. The anaerobic biofilm reactor used in this study comprised of an inlet for gray water, an inner chamber (IC) that acts as a sludge collection tank and acidogenic zone, an outer chamber with biofilm support (OBF) that served predominantly as methanogenic zone and an outlet for the treated gray water. The gray water enters the inner chamber by gravity and displaces the existing partially treated water on this chamber into the outer biofilm chamber in an upflow mode. Two replicate reactors each of PVC spirals, BF, coir, RG and control with no packing material were operated. All the bioreactors were operated at room temperature which varied between 18 and 26 ◦ C without the addition of buffers and with no recirculation and therefore emulated potential use in a typical urban

188

Inf - PVC spiral

COD, mg/L

55 0.233 0.275 35/30 55 48.8 70.35 29 Convoluted spiral Cylindrical Cylindrical Cone with trapezoidal fins 3.2 3.5 3.5 3.5 4

Diameter

Shape of biofilm support material

350 179.1 145.75 320 0

Inf - BF

Bioflow

2000

1000

0 100

0

200 Days

300

400

Fig. 2 – Change in COD for PVC spiral and bioflow (BF) bioreactors. household. To start the reactors, effluent from a biomass based biogas plant operated at the Centre for Sustainable Technologies, Indian Institute of Science was used as inoculum. The start up period of all the bioreactors was 73 d after which all the reactors were operated continuously for 335 d. The feeding of untreated waste water into the bioreactors was carried out manually and envisaged to flow in a plug flow mode. All the bioreactors were operated initially for 42 d as shown in (Figs. 2 and 3) with a wastewater of COD of 1500 mg/l at HRT of 2 days that and an OLR of 750 mg COD/l/day. The OLR was increased in steps of 250 mg/l/day to evaluate its conversion efficiency.

2.2.

300 100 119 191 0

PVC spiral

Analytical methods

All the bioreactors were operated with synthetic gray water having chemical compounds that are expected to be found in domestic gray water. Synthetic gray water was prepared using arrowroot starch, commercial detergent, liquid hand wash and urea in the dry weight ratio of 1:0.6:0.5:0.2 respectively maintaining a C:N ratio of 15:1. The soluble COD of the synthetic gray water was 25–40% of the total COD. The untreated (Inf) and treated gray water samples of all the bioreactors were monitored on a daily basis for chemical oxygen demand (COD), total suspended solids (TSS), total dissolved solids (TDS), pH by Standard (1975). The pectin, lignin, cellulose and hemicellulose content of the organic fiber were determined based on the methods described by (Chesson, 1978). The gas composition was determined by a gas chromatograph with Porapaq-Q column using a thermal conductivity detector and hydrogen as carrier at 80 ◦ C and the volatile fatty acid (VFA) was determined in a gas chromatograph fitted with an EC 1000 column using a flame ionization detector maintained at 200 ◦ C with nitrogen as a carrier gas. 2000

Inf - Coir & Control

Coir

Inf - RG

RG

Control

150 20 30 85 0

1500

COD mg/L

Quantity used in inner chamber (g)

Quantity used in outer chamber (g)

Total surface area per unit volume (m2 /m3 )

Working volume of reactors (l)

Length

3000

1000

PVC spiral Ridge gourd fiber Coir fiber Bioflow Control

500

Biofilm support material

Table 1 – Biofilm support materials used in the inner and outer chambers of the bioreactors used in the study.

Size in mm (mean)

Process Safety and Environmental Protection 9 2 ( 2 0 1 4 ) 186–192

0 0

100

200

Days

300

400

Fig. 3 – Change in COD for ridge-gourd (RG), control and coir bioreactors.

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Process Safety and Environmental Protection 9 2 ( 2 0 1 4 ) 186–192

9.0

Biofilm analysis

8.0

7.0

pH

In order to do image analysis of anaerobic biofilm was carried out with the help of CLSM (Confocal Laser Scanning Microscopy), LAS-AF, ImageJ to visualize the presence and distribution of anaerobic bacteria and EPS in a matured biofilm grown on synthetic and natural support and explain the underlying basis for the improved performance. In order to stain the biofilm, it was scraped from the surface of inorganic support media whereas the biofilm formed on the surface of organic media were difficult to scrap so they were stained directly without removing the biofilm. The biofilms were stained with various fluorescent dyes (Molecular Probes, Bengaluru, India). The biofilms were washed thrice with distilled water and 200 ␮l of each dye were added in sequential manner starting with Concanavalin A, followed by Lectin SBA and Syto9 at an interval of 10 min each. After addition of each dye the biofilm were washed thrice with distilled water to remove the excess dye. The imaging and analysis of biofilms were based on the modified methods described by Staudt et al. (2004) and Chen et al. (2007). The lectins chosen were based on their specificity for different compounds that are likely to be present in the bacterial biofilm. Lectin SBA from Glycine max (soybean) conjugated to Alexa Fluor 647 was used to target terminal ␣- and ␤-N-acetyl galactosamine and galactopyranosyl residues (dye 1) where as Alexa Fluor 350 conjugate of succinylated Concanavalin A (dye 2) was used to stain ␣mannopyranosyl and ␣-glucopyranosyl residue. SYTO 9 (dye 3) was used to stain both the bacteria (live and dead) in the biofilm. Stock solutions of the dyes were prepared as per the guidelines stated by the dye supplier. The imaging of the biofilms was carried out using Leica SP5-AOBS confocal laser scanning microscope (CLSM). The CLSM was equipped with an upright microscope having a numerical aperture of 1.0. The excitation of the dye 2 and dye 3 was done with Ar laser at 458 nm and 488 nm that fluoresce blue and green respectively where as a He/Ne laser was used to excite dye 1 at 633 nm. The stained biofilms were placed in between two glass cover slips and the imaging was carried out using a water immersion objective of 20×. The 3D image of the biofilm was analyzed using ImageJ and LAS-AF Lite software. Each biofilm stack of 5 ␮m thickness was split into individual RGB channels and a constant threshold value was set based on average threshold values of 20 individual stacks. To measure biofilm thickness on synthetic support, coir and RG fibers, their thickness were measured under a precalibrated light microscope using a stage micrometer before and after the biofilms were scraped from the surface of support media. The mean biofilm thickness was determined as the difference in thickness before and after washing it with distilled water along with gentle brushing of the fiber surface. A soft brush was used to remove the biofilm without dislodging the fiber structure. The dry biofilm density is reported (gravimetrically) as dry weight of biofilm material recovered in distilled water per unit surface area of support material scraped.

6.0

5.0

Inf- PVC spiral

4.0 0

PVC spiral

100

Inf- BF

200

BF

300

400

Days

Fig. 4 – Change in pH for PVC spiral and bioflow (BF) bioreactors. of COD between 300 and 400 mg/l whereas control bioreactor produced an effluent with COD above 400 mg/l. PVC spiral bioreactor produced an effluent of COD below 200 mg/l and BF bioreactor between 200 and 300 mg/l. The pH of the treated and untreated graywater discharged by bioreactors having PVC spiral, BF, RG, Coir and control is shown in Figs. 4 and 5, respectively. There was a fall in pH among bioreactors using BF, Coir and control reaching up to 5.5, whereas PVC spiral and RG bioreactors showed a pH above 6. At this stage in order to avoid low pH based inhibition of methanogenic activity the OLR for BF, coir and control bioreactors were reduced to 500 mg/l/day. However, the OLR of PVC spiral and RG bioreactors was maintained at 750 mg/l/day as they had a consistent COD removal of >80% with a stable pH. In order to verify the maximum OLR that PVC spiral would be able to withstand the OLR was increased to 1000 mg/l/day and 1250 mg/l/day after 66 d and 135 d, respectively. At an OLR of 1000 mg/l/day PVC spiral bioreactor continued to produce an effluent with COD below 200 mg/l at a pH of 6–6.5 and its performance fell at an OLR of 1250 mg/l/day with the pH falling down rapidly to levels below 5. Therefore the OLR for PVC spiral bioreactor was reduced and maintained at 750 mg/l/day. In order to validate the concept of a two chamber bioreactor having firstly a low pH based inner chamber followed by a high pH based outer chamber, the change in pH flux was measured on an hourly interval for all the bioreactors by removing samples from the inner and outer chamber as shown in Fig. 6. Coir and RG fiber are natural materials that undergo bacterial decomposition (Antheunisse, 1979); therefore the composition and retention of fiber in anaerobic reactors would be vital for the attachment and persistence of the biofilm. Table 2 shows the composition of Coir and RG before and after 9.0 8.0

Inf - Coir & Control Coir

7.0

Control

Inf - RG

RG

pH

2.3.

6.0

3.

Results

Continuous operation of bioreactors at different OLR was initiated after a start up period of 76 d. Figs. 2 and 3 show the COD reductions obtained with PVC spiral, BF, RG, coir and control bioreactors. At an OLR of 750 mg/l/day reactors with coir and RG had poor COD removal producing an effluent

5.0 4.0 0

100

200

300

400

Days

Fig. 5 – Change in pH for ridge-gourd (RG), control and coir bioreactors.

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Table 2 – Composition of fiber material before and after 408 d of use as biofilm support. Type of Fiber Coir – initial composition Coir – composition after 408 days Coir fraction lost (%) Ridge gourd – initial composition Ridge gourd – composition after 408 days Ridge gourd fraction lost (%) a

Pectinsa %

Weight (g TS) 149 45.3 69.6 120 7.8 93.5

4.4 0 100 20.2 0 100

Hemi-cellulose % 18.3 5.4 70.7 20.9 1.3 93.7

Cellulose % 15.1 5.2 65.7 14.9 1.3 91.4

Lignin % 62.2 19.5 68.7 44 3.7 91.6

Water soluble and oxalate soluble pectin in primary material. As EPS is embedded in hollow parts of the fiber in the digesters, its weight was also recorded in this fraction but not reported in this table.

Table 3 – Comparison of the physical properties of biofilm formed on various support media. Parameters Mean biofilm thickness (mm) Total biofilm volume (cm3 ) available in the reactor Total dry biofilm (g) available in the reactor Total wet biofilm (g) available in the reactor Mean biofilm density (mg dry/cm3 ) Total surface area of the support material (m2 ) available in the reactor after 400 d Dry wt. of biofilm (g/m2 ) of available support material

undergoing anaerobic degradation for 408 days. Biofilm structures are heterogeneous and differ based on substratum on which the biofilm is initiated. However at lower loading rates biofilms undergo rapid withering and washout from the surfaces therefore it was vital to verify the presence and structure of biofilm on all supports after operation at low OLR’s. Table 3 shows a comparison of the biofilms formed on various support media and Fig. 7 elucidates the biofilm structure with the help of the CLSM imaging formed on BF (A), PVC-spiral (B), RG (C) and coir (D)

Discussion

4.

A passive, low energy anaerobic treatment and recycling system for graywater can greatly reduce water-footprint of current urban life-styles in developing countries. Earlier studies at CST (unpublished) using single chambered bioreactor with gray water could not establish stable operation due to rapid fall in pH at OLR >500 mg/l/d. We therefore sought a two chamber configuration to isolate the low pH pockets from the biofilms, overcome low pH inhibition and enable sustained operation. The eight reactors with four different supports and a reactor without any support were fed with increasing OLR of graywater in steps of 250 mg/l/d till rapid fall in the pH of the bioreactors were noticed (pH < 5.5). While PVC spirals and RG could tolerate an OLR between 750 and 1250 mg/l/d for short

9

Control PVC spiral Coir RG BF

8

pH

7 6 5

Chamber 1 (inner

4 0

10

Chamber 2 (outer)

20

30

40

50

hours

Fig. 6 – Hourly change in pH in the inner chamber (0–24 h) and outer chamber (25–48 h) for the bioreactors.

PVC spiral

Bio-flow

Coir

0.91 888.34 9.94 635.21 11.10 0.97 10.18

0.74 539.39 2.73 174.12 5.05 0.72 3.74

0.06 189.43 3.55 226.84 18.70 4.02 0.88

RG 0.12 575.99 4.13 263.96 7.10 4.39 0.94

periods, steady state operation without sharp fall in pH could be achieved only between 750 and 875 mg/l/d with the above two support materials and this feedstock (Figs. 2 and 3). BF, coir and control reactors on the other hand could accept an OLR of about 500 mg/l/d under similar circumstances suggesting insignificant differences between the types of supports used to overcome this problem. Typical CSTR (suspended growth) normally tolerate OLR up to 1000 mg/l/d under similar conditions and the causes needed further explanation (Chan et al., 2009). The (low) pH front could be traced to the rapid transformation of graywater immediately after feeding, taking between 2 and 3 h to form a large and persistent (24 h) low-pH pocket or an unmixed zone in the first chamber. However, after 24 h, when fresh gray water is fed to the first chamber, this front or zone of low pH graywater is displaced into the second chamber filled with biofilms support. Subsequent microbiological transformations restore the pH to neutral conditions (Fig. 6). VFA levels (C2 –C4 ) were found to be low (below detectable) and COD in this region had no discernible trends (60–70% removal in <6 h, not presented). It thus is clear that the low pH phenomenon is not the typical VFA induced – common in anaerobic digestion. Rapid release of CO2 , long chain fatty acids, failure of syntrophic organisms, etc. could be possible causes and need further investigations. Such a phenomenon has not been reported before. Split feeding (typical in domestic use) offered a simple solution to this problem where the COD removal increased to 80–90% in all the bioreactors with acceptable pH (Figs. 2 and 3). The basis of longevity of the fiber based biofilms supports was assessed both in physico-chemical terms and microscopy (Table 2). About 6.5 and 30.4% (RG/Coir) of the original dry weight of added support material remained after 408 d suggesting a half life close to a year and a useful life of about 2 years unlike leaf biomass with 180 d (Chanakya et al., 1998). It was also seen that although a significant part of the coir or RG was digested from the inside, a thin tubular shell persisted over which the biofilms continued to function – although highly splintered (Fig. 7). Between 8.3 and 31% of the lignin remained as the fiber support and a large part of the remaining support (possibly the early colonizing material on fiber) was now predominantly hot water soluble material (80–95%) and

Process Safety and Environmental Protection 9 2 ( 2 0 1 4 ) 186–192

191

Fig. 7 – CLSM image of biofilm clockwise (A) BF, (B) PVC spiral, (C) coir, (D) ridge gourd. Red, blue and green colors represent dye 1, dye 2 and dye 3 respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) has not been reported earlier and needs further research. Biofilm formation on synthetic supports has been reported to occur in three stages; bacterial adhesion to surface, secretion of EPS to form an immobilization matrix embedding the bacteria, continuous erosion of the top layer of biofilm in its matured state (Sutherland, 2001; McEldowney and Fletcher, 1987; Bryers, 1993). Such results were observed in the images of biofilm formed on BF and PVC surfaces (Fig. 7A and B). For biofilms formed on natural fibers these are not apparent. Biomass is rapidly colonized by anaerobic bacteria taking between 10 and 20 d in leaves (Chanakya et al., 1998). Initial degradation of fiber appears to create holes on the surface and making bacterial access to colonization of the hollow fiber (Fig. 7C and D) and both the inner and outer surfaces are colonized. The biofilm density (g TS/m2 ) of different support materials (Table 3) showed about 10 and 4 times more biofilm on PVC and BF, respectively, compared to coir and ridgegourd although COD degradation levels under these circumstances have remained somewhat similar. Methanogens are known to adhere to degrading plant cellulosics and are not easily dislodged (Chanakya et al., 1998). Following such attachment there is very little need for extracellular polysaccharides to be secreted to entrap large numbers of methanogens as is found on inorganic supports. We thus hypothesize that with lesser COD diverted toward

EPS formation, natural supports are colonized early by anaerobic microflora needs further research. As the gray water discharged from house is expected to have a COD between 200 and 1000 mg/l therefore all the bioreactors were later operated at a COD of 500–700 mg/l corresponding to an OLR of 250–350 mg/l/day. At low COD/OLR, our results show that natural supports have lower biofilm retention capability in comparison to inorganic support (Table 3). However the CLSM images (Fig. 7) show lower EPS and higher bacteria for organic supports in comparison to the inorganic support. This might explain why the organic support had a similar performance in comparison to inorganic supports at lower loading rates. Elsewhere ridge gourd (Luffa cylindrica) fiber employed as bacterial support in aerobic domestic wastewater treatment achieved high COD removal efficiency (92.8%, OLR 250 mg/l/d, HRT = 1 d; Marín et al., 2009). Anaerobic filter-anaerobic hybrid reactor with biofilm supported on reticulated polyurethane foam (RPF) gave a 71% gray water treatment efficiency (HRT = 12 h, 13 ◦ C; Elmitwalli et al., 2001). A two stage anaerobic–aerobic graywater treatment system with PVC rings (HRT = 1 d) gave 64% COD removal (Ghunmi et al., 2009). Anaerobic biofilms formed on leafy material were used to treat coffee processing wastewater at OLR = 2–10 g COD/l/day coupled with long periods of non feeding with a half-life of 180 d (Chanakya et al., 1998; Mohan et al., 2005).

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5.

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Conclusion

In this long term experiment using graywater as feedstock, it was found that most support materials performed satisfactorily at the required OLR of 500–750 mg COD/l/d. At higher feed rates a pH front develops that cannot be attributed to acetic or propionic acids accumulation and therefore needs to be examined further. RG and coir fibers have a half-life of about a year and biofilms on them could survive about 2 years although much of the lignaceous materials would have disappeared. Biofilms on natural materials have lesser extent of EPS compared to synthetic supports and hence form denser biofilms. A two chambered bioreactor with intermittent feeding overcomes the pH front developing in single-stage graywater reactors.

Acknowledgements This research was funded by Department of Science and Technology (DST), New Delhi, India through the Water Technology Initiative (WTI). Authors are thankful to Dr.Michel Torrijos, LBE-ENSAM-INRA, Narbonne, for providing Bioflow and IMARIS team (IPC, IISc) for their technical help in CLSM imaging of biofilm.

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