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Int. J. Environ. Sci. Tech., 7 (1), 165-174, Winter 2010ISSN: 1735-1472 E. I. Madukasi et al. IRSEN, CEERS, IAU Potentials of phototrophic bacteria in treating pharmaceutical
wastewater
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, 202 Haihe Road Harbin, Nangang district, Harbin 150090, P R China Received 9 May 2009; revised 25 July 2009; accepted 5 November 2009; available online 1 December 2009 AB STRACT: A suspended growth photobioreactor was utilized to treat pharmaceutical wastewater by a wild strain
purple non-sulfur photosynthetic bacterium isolated from the soil. The strain was named Z08 and identified as
Rhodobacter-sphaeroides by 16SrDN. The photobioreactor was illuminated externally with two (40 W) fluorescent
compact light sources on both sides. Its operation pH and temperature were between 6.8 – 7.0 and 20 – 30 ºC,
respectively. Optimum growth of the isolate was obtained after enrichment of the pharmaceutical wastewater with
0.5 % ammonium sulfate and 0.1 % yeast extract under microaerobic optimum light (6000 lx) condition at 5d retention.
Using these optimum conditions, the maximum dry cell weight and chemical oxygen demand percentage removal were
880 mg/L and 80 %. Chemical analysis of the culture after treatment of the enriched and non-enriched wastewater
showed the crude protein content of the biomass to be 54.6 % and 38.0 %, respectively. This study proved that
photosynthetic bacteria could transform complex wastewater that contains recalcitrant organic compounds with a
resultant recovery of useful products.
Keywords: Biotreatment; Microaerobic light; Photosynthetic bacteria; Purple non sulfur bacteria; Static light exposure
reactor
Characteristics of the pharmaceutical industries are BOD content of the wastewater. For instance, the the diversity of their process operations, which gives wastewater for this study has a BOD of 146mg/L and rise to a wide variation in the liquid wastes. There is COD of about 8000 mg/L, resulting in the BOD/COD little similarity between effluents from different factories ratio of about 0.02. Hence, the need to try a novel and individual effluents may differ continually as a biological treatment method which is the application result of process changes. In many cases, these of purple non-sulfur photosynthetic bacteria (PNSB) effluents contain little or no biodegradable organic in degrading pollutants found in the pharmaceutical matters and the pollutant loads in terms of biological oxygen demand (BOD) may be negligible hence higher PNSB are widely distributed in the ocean, lakes, chemical oxygen demand (COD) than BOD rivers, soil and activated sludge also in high . Most substances found in temperature, low-temperature, low-salt, high-salt a pharmaceutical industrial wastewater are structurally environment. They grow in both microaerobic and complex organic chemicals that are resistant to anaerobic light conditions while utilizing various biological degradation substrates as sources of carbon and energy with , thus the need for segregation and collection of ammonium and /or nitrate as a source of nitrogen and particularly toxic materials in a conventional biologicaltreatment of pharmaceutical industrial wastewater may also use sulphide or thiosulphate as an electron donor under photosynthetic conditions Conventional biological treatment methods are usually in appr opr iate for th e tr eatmen t of metabolically the most versatile among all prokaryotes; pharmaceutical wastewaters because of the negligible anaerobically photoautotrophic and hotoheterotrophic in the light or microaerobic light condition Tel./Fax: +8615 8463 85628 . Photosynthetic bacteria B) for wastewater E. I. Madukasi et al. treatment has generally proven to be a cost effective Na MoO : (6). 2g of soil was added to 95 mL sterile system for treating wastewater. This is because PSB medium and incubated anaerobically at 28-35 ºC does not only produce quality effluent, but produces depending on the ambient temperature for about 96hrs substances of commercial interest such as single cell under illumination with a 100W incandescent lamp.
protein (SCP), biopolymers, antimicrobial agents and Anaerobic condition was created by addition of 5ml therapeutic compounds etc ( sterile liquid paraffin on top of the solution in the test tube. Purification of single colonies was achieved by Due to these properties, PNSB have been utilized successive re-streaking on the RCVBN medium by other researchers to treat different types of containing 1.5 % agar and incubated at the same wastewaters such as concentrated latex wastewater condition. The initial pH of the medium was adjusted , odorous swine wastewater to 6.8 by 5M NaOH.
), tuna condensate (, oil-containing sewage wastewater and latex rubber sheet wastewater Th e isolate was ch ar acter ized usin g both morphological and physiological properties and studied the influence of photosynthesis on calcite accor din g to Bergey's man ual of systematic bacteriology (nd Bergey's The main aims of this study are to isolate, identify manual of determinative bacteriology and test the ability of wild strain PNSB as well as todetermine the optimum conditions for this isolate to be Isolation and identification of bacteriochlorophylls most effective in treating pharmaceutical wastewater.
Bacteriochlorophylls were isolated, characterized There could be another advantage in utilizing this PNSB and identified as described by Oe to treat pharmaceutical wastewater because the . The isolated bacteria spectra were obtained with biomass of PNSB has been reported to be a source of an Aminco DW-200 UV-Visible spectrometer in the split SCP production and could be used as an alternative to manure, fish feed, or agricultural supplement as it isrich in protein and vitamins Cellular fatty acids analysis Ph otosyn thetic isolates wer e cultur ed on harvested biomass from the tr eatment will be bactotryptic soy broth agar (Difco) and incubated at analytically evaluated to ascertain it's suitability as a 28 oC for 48h, the cellular fatty acids contents were determined using the MIDI procedure (MIDI, Inc, New This research was carried out between March 2008 York Del). Identification of the isolates were based on and March 2009 at the School of Municipal and a comparison of fatty acid profiles using the fatty acid Environmental Engineering Department of Harbin profiles in tryptic soy broth agar anaerobe database Institute of Technology, China.
MATERIALS AND METHODS
Bacterium and culture The inoculums were prepared by growing the cells PNSB isolation in a modified Sistrom's minimal malate medium (pH 6.8) A purple non-sulfur bacterium named Z08 and microaerobically in a reciprocating thermostatic shaker isolated from soil was used to treat pharmaceutical (unitron, Infors AG, Germany) at 200 rpm for 48 h with a wastewater. The bacterium was isolated with Sistrom temperature range of 28-30 ºC. Ammonium sulfate (0.99 minimal medium (RCVBN) consisting g/L) and malate (4.02 g/L) were used as sources of of (in g/L) DL-malate: (4). MgSO : (0.12). (NH ) SO : nitrogen and carbon, respectively. Sterilization of the (1). CaCl : (0.075). KH PO : (0.3). Na EDTA: (0.020).
medium, photobioreactor and wastewater-test-media VB (0.001). Nicotinic acid: (0.001). Biotin: (0.015). Trace were accomplished by autoclaving at 121ºC for 15min, element solution, 1mL. The trace element solution inoculation into the medium or bioreactor was as 10 % contained (in mg/L) Zn SO .7H O: (20). MnCl .4H O: v/v inoculums from a culture growing in the modified (6). H BO : (60). CoCl .6H O: (40). CuCl .2H O: (2).
RCVBN medium. For use as an inoculums, cell 165-174, Winter 2010 suspension was adjusted to the desired optical density optimized wastewater (SOW); i.e. the wastewater was (0.500) at 660 nm using the sterile RCVBN medium as supplemented with 0.5 % (NH ) SO and 0.1% yeast diluents. The sterile RCVBN medium was also used as extract, grown under light intensities of 500, 1000, 2000, 4000, 6000 and 8000 lumens, respectively. Thesevariations were obtained by adjusting the distances between light sources and the reactor tubes. In all these, Wastewater from a pharmaceutical company located the temperature variation was same (20-31ºC).
in Harbin, Heilongjiang province, China was collectedat different times from the company's holding tank, each collection was analyzed for COD, BOD, suspended A test tube of 100 mL with cellophane tapered edge solids (SS), total dissolved solids (TDS) and pH. The to prevent volatilization was used as a bioreactor. Three properties of the wastewater varied at each collection treatments were set as follows: (SOW) as a control time, hence representative wastewater sample was without inoculums; SOW plus 10 % inoculums and centrifuged at 4000 rpm for 10 min followed by SW plus 10 % inoculums. The photobioreactor autoclaving at 121ºC for 15 min to achieve sterile temperature was between 20-31ºC depending on the condition prior to use as the growth medium. It is ambient temperature. Illumination was with 40 W imperative that autoclaving could cause volatilization compact fluorescent lamps at the desired lumens on of fatty acids and H S as the Teflon screw cap was at both sides of the bioreactor. Cell growth, pH and COD that stage loosely closed and this apparently would were monitored at the end of each retention time of 3, 5 affect both the COD and BOD values. For this reason, and 7 days of cultivation, respectively. Microaerobic the COD and BOD values reported in this study were condition was achieved by creating a head space in determined after centrifugation and autoclaving of the the PBR, no flushing with either argon or nitrogen gases r aw wastewater. The or igin al phar maceutical and the cultivation condition was static-light exposure.
wastewater without any pretreatment (centrifugation/ The dissolved oxygen (DO) levels of all treatment were autoclaving) had COD and BOD values of 9450 and between 0.7-1.63 mg/L.
197 mg/L, respectively.
Optimization of wastewater composition and The incident light intensity was measured by a TES- 1330A digital light meter, while the functional groups Effect of added nitrogen source, ammonium or nitrate were determined with GC-FTIR. Organic compounds were added to the sterilized wastewater (SW) medium in the wastewater were identified qualitatively using at varied concentrations of 0, 0.1, 0.2, 0.3 and 0.4 %.
gas chromatography coupled with mass spectrometry The wastewater pH of 6.6 was not adjusted as the (GC-MS). Instrumentation was a Hewlett packard (HP) optimum pH for growing most bacteria is in the range 5890 Series II gas chromatograph, interfaced with a HP Chem-Station data system and linked to a HP 5972 mass Ammonium being the best nitrogen source was tested selective detector operated in scan mode. Results are again at higher concentrations of 0 0.5, 0.75, 1.0 and reported as lists of those compounds reliably and 1.5% respectively. In most cases SW medium was used tentatively identified. It meant that the organic under microaerobic light (3500 lx) for 72 h.
compounds identification were carried out by computer SW medium supplemen ted with optimal matching against a HP Wiley 275 library of 275,000 mass concentration (0.5 %) of ammonium was used as the spectra combined with expert interpretation and that base control to evaluate the effect of added yeast extract the identified compounds were not confirmed against (YE). Yeast extract is a complex natural material with their standard compounds because of the inability to high vitamin B level; it was tested for its effect on growth get the standard compounds during the time of the at the following concentrations; 0, 0.01, 0.05, 0.1, 0.2, 0.3 %. All the cultivations were grown three consecutive For the determination of the parameters, the cell times for 72 hrs under microaerobic light conditions.
suspensions were centrifuged at 4000 rpm for 20 min Th e effect of ligh t in ten sity var iation was and cell pellets were used for the determination of dry investigated by growing the isolate in sterilized cell weight and crude protein. The supernatant was E. I. Madukasi et al. Phototrophic bacteria in pharmaceutical wastewater used for COD, organic compounds, SS and pH the wastewater. All experiments were conducted in determinations. The bacterial cell concentration was triplicate and mean value reported.
determined by optical density at 660 nm (i.e.OD ) using a UV-VIS spectrophotometer (Shimadzu UV-120).
RESULTS AND DISCUSSION
The cell dry weights were determined by centrifuging Identification of the bacterium 10 mL aliquots of culture at 4000 rpm for 20 min and the The isolate was a non-motile, gram negative, oval cell pellets were washed properly with distilled water rod, 0.25 µm wide and 1.50 µm long and reproducing then filtered using 0.45 µm pore size, 47mm in diameter by binary fission, internal photosynthesis membrane membrane filter paper to remove salts and non-cellular appeared as lamella. After growth under anaerobic materials. Each loaded filter was dried at 105 ºC until photoheterotrophic conditions, cell suspensions were the weight was invariant (about 72 h). The dry weight red an d the absorption spectra of living cells of the blank filter was subtracted from that of the loaded suspension showed maxima at 370, 570, 800, 827 and filter to obtain the dry cell weight (DCW). The OD 852 as shown in e two main peaks at 800 and value was converted to DCW concentration via proper 850 nm, which are closely related to bacteriochlorophyll calibration, (where 1.0 OD approximately equals 0.6g a and b (characteristics of photobacteria pigment) as dry cell/L.).
well as carotenoid, are characteristics of PNSB Crude protein was expressed as percentage total nitrogen (% N) multiplied by 6.25. TDS, SS, BOD, COD chromatography of fatty acid methyl ester) analysis and pH were measured according to standard methods the PNSB isolate was identified as being closely related . It is worthy to mention that the COD to Rhodobacter sphaeroides strains with similarity values quoted in this study were measured after indices of 0.85 ± 0.05. This also identifies the isolate centrifugation and sterilization of the wastewater by as Rhodobacter sphaeroides autoclaving. Trace metals were measured using a Perkin and it is named Rhodobacter Elmer Optima 5300DV ICP aided by a WTW microwave spheroides z08. The isolate grew with organic digester (WTW is a supplier name that deals on compounds or thiosulphate in minimal/basal medium electrochemistry instruments just like Hach or YSI).
with either (NH ) SO or NaNO as a nitrogen source Organic acids (butyric, propionic and acetic acids) and anaerobically or microaerobically in the light.
alcohols were determined using gas chromatography(GC-14B, Shimadzu, Tokyo, Japan) equipped with a flame ionization detector (FID). Samples were injected The results of characterization of pharmaceutical into a 15 m long capillary column with an internal wastewater are shown in According to diameter of 0.53 mm. Nitrogen was used as the carrier , the wastewater contains recalcitrant organic gas with a flow rate of 20 mL/min. The temperature of compounds such as octadecane, heptacosane and injector and detector was set at 220 and 230 ºC, octacosane which are resistant to biodegradation, also respectively. The oven temperature was initially set at compounds like benzothiazole and enzenepropanamine 110 ºC, increased from 110 to 200 ºC at a rate of 8 ºC/min are all toxic and lethal in nature. These identifications and held at 200 ºC for 5 min. Liquid samples were were made by GC-MS spectrometry and it is supported centrifuged (4000 rpm for 10 min) and filtered (0.45µm by GC-FTIR where three peaks were evident at membrane) prior to being injected into GC for analysis.
1304 cm-1 and 1600-1300 cm-1 again suggesting Biomass productivity was calculated from the total the presence of aliphatic amino salts and aromatic volume of wastewater used and the total weight of compounds respectively. All these compounds are biomass produced in the process after each retention resistant to biodegradation and accumulate in the time. The pH was measured using a digital pH-meter environment with a resultant negative effect on the (Metrohm 620). Centrifugation was carried out in a food web.lso shows that the wastewater is thermostatic Sigma centrifuge (B. Braun, Melsungen, high in COD than BOD, hence low value of BOD to Germany). Since the properties of the wastewater varied COD ratio (0.02). This again depicts th at the at each collection time, representative sample was used wastewater contains high level of non biodegradable for the analyses of the organic compounds, functional organic substances ) group, total nitrogen (TN) and organic acids present in and difficult to treat with conventional biological ),s i et a 174, Winter 2010 Table 1: List of organic compounds tentatively identified in the pharmaceutical wastewater Peak No. Name of compound Molecular ion peak (m/z) Fragment ion (m/z) 1,2-Benzenedicarboxylic acid 1-Butanamine Benzenepropanamine Desmethyldoxepin Methylpent-4-enylamine Nitro-L-arginine Ta ble 2: Chara cteristics of the u ntr eated and tr eated pharmaceutical wastewater (all in mg/L except pH) Total suspended solids Total dissolved solids Wa ve le nght (nm ) Fig. 1: In vivo spectrum of the isolate (pure culture) treatment methods. These organic compounds found wastewater into some acceptable organic compounds.
in the pharmaceutical wastewater could be vital part of This finding is in support of everyday life, but most of them are of environmental which stated that complete biodegradation of concern because of their toxicity, persistence and recalcitrant organic compounds to harmless end tendency for bioaccumulation ( products such as CO and H O or methane may not Some studies have demonstrated inhibition of enzyme always occur; instead biotransformation to different activities with differ en t pollutan ts, in cludin g organic compound is possible. Those recalcitrant and hydrocarbons, heavy metals and surfactants xenobiotic compounds pose problems in conventional wastewater treatment, due to their resistance to biodegradation. This also shows that the PNSB used contributed to the difficulty in conventional biological for this treatment is candidate for bioremediation since treatment of pharmaceutical wastewater, while being it could transform some toxic and recalcitrant organic the major consideration for using PNSB to treat this wastewater. The GC-MS fragmentation analyses of the pharmaceutical wastewater after treatment of the treated oil containing sewage wastewater using optimized wastewater indicate that the PNSB photosynthetic bacteria; they recorded successful was able to either ameliorate or transform those transformation of C 10- C 26 (hydrocarbon) by the recalcitrant and xenobiotic materials found in the initial E. I. Madukasi et al. Table 3: List of organic compounds tentatively identified in treated optimized pharmaceutical wastewater Name of compound Molecular ion peak (m/z) Fragment ion (m/z) 2-Butenoic acid, 2 methyloxy-3methyl ester. 1-2Benzenedicarboxylic acid Fig. 2: GC-FT IR spectra of the pharmaceutical untreated wastewater (Fluka library supplied by Perkin-Elmer) Effects of nitrogen source The test water sample has a TN value of about concentrations; it gave maximum growth 530 mg/L (about 0.05 % nitrogen by the weight of of 672.0 mg/L DCW at 5000 mg/L while further the BOD) which did not conform with the biological increment retards the growth. Therefore, in order to wastewater treatment of carbon to nitrogen ratio of treat this pharmaceutical wastewater, additional 5 % nitrogen by the weight of the BOD ( nutrients need to be added to the wastewater to bearing in mind the load of recalcitrant organic stimulate increase in the growth of the microbes.
pollutants. Hence, the need for additional nutrients This procedure is in accordance with the methods in the form of either (NH ) SO or NaNO .
which have been commonly used in wastewater shows that (NH ) SO produced the highest dry cell weight (592.3 mg/L) at 4000 mg/L against NaNO with . This experiment also confirmed that the isolate (318.2 mg/L) at the same concentration. Ammonium being the best nutrient was tried further at higher 174, Winter 2010 Effects of yeast extract with a COD reduction of about 75 % (initial COD value The addition of yeast extract on ammonium of 2500 to final value of 604 mg/L, respectively) and supplemented wastewater further increased the cell the cell growth was 820.5 mg/L DCW. While a growth of the PNSB from OD660 1.03 – 1.502 in the temperature range of 20-30 ºC depending on the ambient pharmaceutical wastewater test medium. It recorded temperature was maintained. This finding could be 0.997g/L DCW at 1.0 g/L concentration. This attributed to the fact that the PNSB thrives better in an cultivation was carried out over 96 h under microaerobic intense light.
light conditions. High concentration had no significanteffect Although yeast extract contain many B Optimization of wastewater treatment conditions vitamins, it may also act as a source of protein and its hows the experiment carried out to infer the use depends on the economic evaluation of the effective retention time for the treatment. The isolate grew up to 377.4mg/L in SW in 5 d and almost doublethe amount in SOW (688.3mg/L) at the same 5 d Effects of light intensity retention). Alsoows that the COD In order to optimize the treatment conditions, the reduction was higher in SOW at 5d than SW (64.8 and incident light intensity were varied between 500 to 8000 43.0 %, respectively), although at 7d, the COD and dry lumens. The best growth was observed at 6000lx ) cell weight were slightly higher than at 5d, but 5d was Fig. 3: Effect of nitrogen sources and their concentrations on the growth of the PNSB in pharmaceutical wastewater under microaerobic light conditions for 72 h; (a) Source of nitrogen; (b) Added (NH ) SO on bacterial growth Light intens ity (Lx) Fig. 4: Effect of added yeast extract and the concentration on the growth of PNSB in pharmaceutical wastewater under Fig. 5: Effect of light intensity variation on % COD removal microaerobic condition for 72h with (NH ) SO = 5 g/L and cell growth (mg/L). C = % COD removal, B = Cell chosen as the optimal retention time for the treatment compensated for by lower energy consumption over for economic reasons. The SW with lower COD reduction could be attributed to the inhibition of the The growth kinetics of the PNSB in batch culture cell activity by the recalcitrant pollutants in the (data not shown), depict that the bacteria maintained wastewater, while the sharp reduction of COD speedy growth after 19 h of cultivation and reached in the SOW with inoculums shows that the bacteria saturation after 5d. From the result, the specific growth activity was increased with additional nutrient hence rate was calculated as 0.015 h-1 and 0.011 h-1 for SOW able to metabolize most of the pollutants faster before and SW, respectively. While the process productivity the onset of inhibition. There was no COD reduction based on the five days optimal growth is 0.082 g/L/day or cell growth in SOW without inoculation which with a biomass yield of 12.8 % for the optimized indicates that no mineralization of pollutants occurred wastewater based on the chemical oxygen demand.
by photo oxidation as COD is the amount of oxygen The protein content of the culture after treatment in necessary to oxidize both the organic and inorganic both th e supplemen ted an d non -supplemented matters to CO , H O and NH wastewater is well above the required protein content The conditions chosen for the treatment of the for it to be acceptable as a protein source for either fish pharmaceutical wastewater were therefore as follows: feed or animal feed (. Therefore, there is supplemented wastewater was with 0.5 % ammonium a great potential for the culture to be used as a SCP.
sulphate and 0.1% yeast extract with a final pH of 6.6- However, the process productivity was low (0.082 g/L/ 7.0, incubation was without shaking at 20-30 ºC for 5 d day). Photosynthetic bacteria typically have lower with an incident light intensity of 6000 lx using process productivity than heterotrophic bacteria fluorescent compact light also the non-supplemented because th e cell den sities ach ieved ar e low wastewater. The choice of fluorescent compact lamp . As for the yield, normal for illumination of the PBR was as a result that greater heterotrophs growing aerobically could convert 50 % proportion of its power is converted to usable light of organic carbon into biomass, whereas with anaerobic while smaller proportion is converted to heat as against conditions only 5 % is converted into biomass incandescent lamp which is heat driven and energy For this microaerobic condition the yield is about draining lamp. Although the initial cost of fluorescent 12 % which is much favorable than complete anaerobic lamp is higher than the incandescent lamp but condition. Also, in this present study, the bacteria did fluorescent's longer life reduces lamp replacement cost.
not thrive well in a complete anaerobic light condition, Typically a fluorescent lamp will last between 10 – 20 for in stan ce, after about 2weeks cultivation times as long as an equivalent incandescent lamp when anaerobically, its optical density was (OD = 0.442 ± operated several hours at a time. Therefore, the higher 0.0014). Whereas, at microaerobic light, it recorded initial cost of a fluorescent lamp is usually more than greater optical density values in less than 2 weeks of Fig. 6: Cell growth and COD removal as a function of time (at 3500lx, initial COD = 2500mg/L); (a) Biomass in mg/L DCW; (b) % COD Removal; SW = non-supplemented wastewater; SOW = supplemented wastewater ; (N H ) SO = 5 g/L a nd 1)s,i et a 174, Winter 2010 cultivation (OD = 1.234 ± 0.0020). This suggested Choorit, W.; Thanahoset, P.; Thongpradistha, J.; Sasaki, K.; th at the isolated PNSB could th rive better in Noparatnaraporn, N.; (2002). Identification and cultivationof photosynthetic ba cter ia in wastewater from a microaerobic light than anaerobic light condition.
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Madukasi, E. I., Research officer, The Federal Institute of Industrial Research, Oshodi, Lagos Nigeria and a Ph.D. Candidate, School of
Municipal and Environmental Engineering, Harbin Institute of Technology, China. Email: [email protected]
Dai, X., M.Sc. Studen t, Schoo l of Mun icipal and Environmental Enginee ring, Harbin Ins titute o f Techno logy, Ch ina.
Email: [email protected]
He, C., M. Sc. Stud ent, School of Municip al and E nvironm ental En gineeri ng, Harb in Inst itute of Techno logy, Ch ina.
Email: [email protected]
Zhou, J., M.S c. Stude nt, Sch ool of M unicipal and E nvironme ntal En gineerin g, Harb in Insti tute of Techno logy, Ch ina.
Email: [email protected]
How to cite this article: (Harvard style)
Madukasi, E. I.; Dai, X.; He, C.; Zhou, J., (2010). Potentials of phototrophic bacteria in treating pharmaceutical wastewater. Int. J.
Environ. Sci. Tech., 7 (1), 165-174.

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