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Wastewater treatment and poly-β-hydroxybutyrate production using lighted upflow anaerobic sludge blanket method
JOURNAL
OF BIOSCIENCE
AND BIOENGINEERING
Vol. 87, No. 5, 683-689. 1999
Wastewater Treatment and Poly-b-Hydroxybutyrate Production Using Lighted Upflow Anaerobic Sludge Blanket Method SHIGEKI SAWAYAMA,
* KENICHIRO
TSUKAHARA,
AND
TATSUO YAGISHITA
Biomass Division, National Znstitute for Resources and Environment, AIST, MITI, 14-3 Onogawa, Tsukuba, Zbaraki 305-8569, Japan Received11 December 1998/Accepted26 January 1999 We investigated the performance of a lighted upflow anaerobic sludge blanket (LUASB) reactor for wastewater treatment and poly-ghydroxybutyrate (PHB) production. Phototrophic bacteria were induced from UASB (upflow anaerobic sludge blanket) granules under light conditions (100 $*m-2*s-1). The ammonium and phosphate ion removal efficiencies of the LUASB reactor were higher than those of the UASB reactor. The difference in the results from runs under light and dark conditions suggested that the ammonium and phosphate ion removal efficiencies were improved by increasing the amount of phototrophic bacteria in the LUASB reactor. The average production rate of PHB from the biomass in the effluent from the LUASB reactor was 6.6-14.0 mg.Z-l-reactored-’ using acetate-based media and the average PHB content based on the dry bacterial biomass was 15.1-25.3x. The PI-HI concentration increased by reincubation of the effluent from the LUASB reactor with sodium acetate under light conditions. The UASB granular sludge can decompose a variety of organic substances and in addition the LUASB method can remove ammonium and phosphate ions. The LUASB method thus appears to be appropriate for wastewater treatment and production of phototrophic bacteria and PHB from various wastewaters. [Key words:
poly$-hydroxybutyrate, lighted UASB method, phototrophic bacteria, wastewater treatment]
UASB method. Biodegradable plastics have been studied to reduce environmental pollution due to the huge amount of plastic waste. Extensive studies on bacterial polyhydroxyalkanoates (PHAs) as raw materials for biodegradable plastics have been carried out (8, 9); however, the production cost, without subsidies, is not competitive with that of conventional plastics made from fossil fuels (10). Brand1 et al. reported that a phototrophic bacterium, Rhodospirillum rubrum, produced various PHAs, particularly the PHB type (11). The combination of the production of biodegradable plastics and wastewater treatment using phototrophic bacteria has been proposed to reduce production costs (12). A phototrophic bacterial biomass can be produced during water treatment using a LUASB reactor (7); therefore, PHB could be produced from various wastewaters using the LUASB method. In this study, we investigated the performance of the LUASB reactor during wastewater treatment and poly+hydroxybutyrate (PHB) production on a laboratory scale.
pared with that using the conventional
Environmental pollution due to the production of large amounts of waste and wastewater is an important issue to resolve. Anaerobic digestion of waste and wastewater has the advantages of low levels of sludge production and energy consumption compared with aerobic treatment, and methane production; therefore, this process has been widely studied and used for the treatment of organic wastes and wastewater (1). A conventional contact process for
anaerobic
digestion
cannot
efficiently
treat at a high organic loading rate and is not highly stable. The UASB (upflow anaerobic sludge blanket) concept was proposed to resolve these problems (2, 3). This concept is based on the formation of a well-settling granular sludge, natural agitation caused by gas production and a well-designed gas-solids separator. An UASB reactor, however, cannot efficiently remove nitrogenous compounds and phosphate ion (4). Improvement of phosphate ion removal with the UASB method by the addition of Ca*+ and Mg*+ to the influent was reported (5); however, a biological removal method has not been reported. Biomass production during wastewater treatment could be necessary to simultaneously remove nitrogenous compounds and phosphate ion. Purple non-sulphur phototrophic bacteria used to aerobically and anaerobically decompose organic compounds, and to simultaneously consume nitrogenous and phosphate ions have been studied in wastewater treatment systems; however, phototrophic bacteria can only decompose a limited number of organic substances (6). We reported that a population of phototrophic bacteria were induced from UASB granules under light conditions and proposed the lighted upflow anaerobic sludge blanket (LUASB) method for wastewater treatment (7). There is the possibility of obtaining improved removal of inorganic compounds using the LUASB method com-
MATERIALS
AND METHODS
LUASB reactor Two cylindrical glass reactors (80 x 180mm, Able, Tokyo) used for the LUASB or UASB reactors, each having a volume of 850m1, were filled with 530ml of UASB granules provided by the Ebara Co., Kanagawa. The lower glass surface (10.5cm from the bottom) of the LUASB reactor and the entire glass surface of the UASB reactor were covered with aluminum foil to prevent light penetration (Fig. 1). Reactor operation for water treatment The LUASB and UASB reactors were maintained at 35(tl)‘C and were supplied with synthetic wastewater at a flow rate of 600 ml.d-l (hydraulic retention time=0.9 d) without sterilization. The LUASB reactor was incubated with continuous incandescent illumination of 100 pE.rn-*. s-l.
* Corresponding author. 683
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reactor FIG. 1. Diagram of a laboratory-scale LUASB reactor. The reactor was maintained at 35( + I)‘C with continuous incandescent illumination of 100 pE . m-2. SK’ and was supplied with organic acid, starch and peptone media.
Both the LUASB and UASB reactors were supplied with three kinds of synthetic wastewater: (i) 2.5 g.l-’ sodium acetate (Wake Pure Chemical Industries, Osaka), 1.25 g. 1-l sodium lactate (Wako), 1.25 g.l-’ sodium propionate (Wako); (ii) 4 g.l-* starch soluble (Wako); and (iii) 4 g .I-’ trypticase peptone (containing 13.6 wt% nitrogen and 0.85 wt% phosphorus, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). The following chemicals were also added to all the media used: 200 mg . 1-l NH&l, 16 mg . I-l KH2P04, 25 mg .I-’ CaClz. 2Hz0, 25 mg . I- * MgClz .6HrO, 30 mg . I-’ Fe-EDTA, 5 mg .I-’ CoClZ.6Hz0, 5mg.l-I MnC12.4H20 and 3OOmg.I-’ yeast extract (containing 10.2 wt% nitrogen and 0.99 wt% phosphorus). The organic acid medium was supplied to the LUASB reactor from day 1 to 60, the starch medium from day 61 to 98, and the peptone medium was from day 99 to 134. When the TOC concentration in the effluent was stable, the reactor was considered to have reached a steady-state. The LUASB reactor was placed in the dark for 24 h on operational days 1-19, 31, 37, 38, 45, 52, 60, 89, 90, 97, 98, 123, 124, 133, and 134. Effluent samples were collected at room temperature for 3 h and then centrifuged (5500 x g for 5 min) before analyses. Any phototrophic bacteria attached on the inside glass surface of the LUASB reactor were removed once a week to keep it transparent. Reactor operation for PHB production The LUASB reactor was maintained at 35(-+1)“C with continuous incandescent illumination of 100 pE.rnp2.ss1, and synthetic wastewater was supplied at a flow rate of 530ml.d-r TABLE Influents MO Ml M2 M3 M4 M5 M6 M7 The following 6H20, 30mg.l-r
Sodium acetate (g.l-‘1 4.7 4.7 4.7 4.7 4.7 4.1 0 0
(hydraulic retention time= 1.0 d) without sterilization. The LUASB reactor was supplied with eight kinds of synthetic wastewater whose compositions are shown in Table 1 (MO to M7). Effluent samples were collected at room temperature for 24 h and then centrifuged (5500 x g for 5 min) before the PHB analyses. Any phototrophic bacteria attached on the inside glass surface of the LUASB reactor were removed once a day from operational day 159 to keep it transparent. Two-step incubation for PHB production An effluent sample from the LUASB reactor using the M7 medium as influent was mixed with sodium acetate (170 mg. 1-l) and then poured into a conical flask (500 ml). The air in the flask was purged with nitrogen gas and then the flask was maintained at 35( + l)“C with continuous incandescent illumination of 100 pE . m-2. s - l for 24 h. After the two-step incubation, the bacterial biomass was collected by centrifugation (5500 x g for 5 min) and the PHB concentration was analyzed. Analyses The concentrations of total carbon, inorganic carbon and total organic carbon (TOC) were determined using a TOC meter (TOC-SOOOA, Shimadzu, Kyoto). The NH*+ concentration in the effluent was determined by calorimetry with Nessler reagent (13) or an ammonium electrode (Model 95-12, Orion, Beverly, MA, USA). The N02- and POd3- concentrations were colorimetrically determined (13). The N03- concentration in the effluent was determined using a nitrate electrode (Model 93-07, Orion). After degradation and oxidation with NaOH and K2S208, the total nitrogen (TN) concentration was determined by the ultraviolet absorption method (13). After degradation with K2S208, the total phosphorus (TP) concentration was calorimetrically determined (13). The dissolved oxygen concentration in the reactor was monitored using an oxygen electrode (CSP-2, Able). The biogas yield from the bioreactor was monitored by displacement of a saturated sodium chloride solution. The biogas composition was determined by gas chromatography (Model GC-8A and GC-12A, Shimadzu) with a WG-100 column (GL Sciences, Tokyo) at 50°C and a Porapak Q column (Shinwakakou, Kyoto) at 90°C. Bacteriochlorophyll was extracted with an acetonemethanol solution and its concentration was measured using a Spectrophotometer 120A (Shimadzu) (14), and the absorption spectrum of the effluent was analyzed using a Spectrophotometer 1600PC (Shimadzu). The PHB concentration in the bacterial biomass was determined by the method of Braunegg et al. (15). PHB was depolymerized and methylesterized at 100°C for 3.5 h in a methanol-H2SOrchloroform solution, and then the ,B-
1. Compositions of synthetic wastewaters supplied to the LUASB reactor Starch k%.,-‘) 0 0 0 0 0 0 3.4 3.0
NH&l (mg.l-I) 2.6 10 50 0 0 0 0 0
Yeast extract (mg.l-r) 0 0 0 100 300 0 300 300
Trypticase peptone (mg . I- r) 0 0 0 0 0 300 0 300
Supplied days 172-182 183-190 191-197 198-204 205-211 212-217 218-225 226-239
chemicals were also added to all the media from MO to M7: 16 mg. 1-r KH2P04, 25 mg. I-- 1 Car&. 2H20, 25 mg. 1-r MgC12. Fe-EDTA, 5 mg.l-r CoC12.6H20, and 5 mg.l-r MnC12.4H20.
PHB PRODUCTION
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Operational days I I ’ Starch Organic acid medium medium
Peptone medium
FIG. 2. Changes in bacteriochlorophyll concentration and optical density (865 nm) of the effluent from the LUASB reactor. The reactors were operated at 35°C with a hydraulic retention time of 0.9 d under continuous illumination of 100 ,eE. m-z. s-r. Compositions of the synthetic wastewater are described in the text. Symbols and line: 0, bacteriochlorophyll concentration of the effluent; ----, optical density at 865 nm; n , operational day when the LUASB reactor was placed in the dark for 24 h. ,
hydroxybutylmethylester produced was measured using a gas chromatograph (GC) (5890, Hewlett-Packard, Wilmington, DE, USA) with a CBPlO-S-25-050 column (Shimadzu). The initial oven temperature of the CC was held at 110°C for 2 min, then increased to 260°C at 10”C.minl. n-Dodecane was used as the internal standard. A GC-MS analysis of j-hydroxybutylmethylester was conducted using an HP-5890II/HP-5971 (HewlettPackard). RESULTS
> r
:
Bacteriochlorophyll in the effluent from LUASB reactor The color of the liquid in the lighted area and granules in the upper surface area of the LUASB reactor turned red under light conditions. The LUASB reactor was placed under light conditions from day 20 and bacteriochlorophyll was detected in the effluent after day 23
s 3* 1000 g 2
, I
1
; i
0 20
40
60
80
100
120
140
Operational days ’ Organic acid ’ Starch ’ Peptone ’ medium medium medium FIG. 3. Change in TOC concentration of the effluent from the LUASB reactor. Symbols and line: 0, TOC concentration of the effluent; ----, TOC concentration of the influent; n , operational day when the LUASB reactor was placed in the dark for 24 h.
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20
20
40
60 80 100 120 140 Operational days I I Organic acid ’ Starch ’ Peptone medium medium medium
FIG. 4. Change in NH4+ concentration of the effluent from the LUASB reactor. Symbols and line: 0, NH4+ concentration of the effluent; mm--,NH4+ concentration of the influent; w , operational day when the LUASB reactor was placed in the dark for 24 h.
(Fig. 2). Bacteriochlorophyll was not detected in the effluent from the UASB reactor throughout the entire incubation period. The bacteriochlorophyll concentration in the effluent from the LUASB reactor under dark conditions was lower than that under light conditions (Fig. 2). The bacteriochlorophyll concentration in the effluent from the LUASB reactor using the peptone medium was higher than those using the organic acid and starch media. The absorption maxima of the effluent sample using the starch medium were 378, 484, 528, 590, 808, and 866nm. The change in optical density of the effluent from the LUASB reactor at 865 nm corresponded well with that of the bacteriochlorophyll concentration (Fig. 2). Water treatment performance of LUASB reactor The TOC concentration in the effluent from the LUASB reactor was less than 60mg C.l-r under light conditions (Fig. 3). The TOC concentration in the effluent from the LUASB reactor increased under dark conditions comTABLE
40
60 80 100 120 140 Operational days I I Organic acid Starch Peptone medium medium medium FIG. 5. Change in POd3- concentration of the effluent from the LUASB reactor. Symbols and line: 0, POd3- concentration of the effluent; ----, P04)- concentration of the influent; n , operational day when the LUASB reactor was placed in the dark for 24 h.
pared with under light conditions. The TOC removal efficiency of the LUASB reactor was 94-97X under light conditions using the organic acid and starch media as the influent, and this range of efficiency was the same as that of the UASB reactor (Table 2). The NH4+ concentration in the effluent from the LUASB reactor with the starch medium was lower than that with the organic acid medium, and that with the peptone medium was higher than that with the organic acid and starch media (Fig. 4). The POd3- concentrations in the effluent from the LUASB reactor with the organic acid and starch media were lower than that with the peptone medium (Fig. 5). The NH4+ and POA3- concentrations in the effluent from the LUASB reactor increased under dark conditions compared with those under light conditions. The average TN and TP removal efficiencies of the LUASB reactor using the organic acid, starch and peptone media were higher than those of the UASB reactor (Table 2). The concentrations of nitrate and nitrite ions in the effluent from the LUASB and UASB reactors were less
3. Average PHB and bacterial biomass yields in the effluent from the LUASB reactor
Bacterial biomass PHB PHB content on PHB production Bacteriochlorophyll concentration dry biomass base rate Influents concentration (mg . Ir) (mg.l-r) (mg I-r-reactor. d-l) (mg . I- r) (%I Ml 59.8” 14.0 23.9 (11.7)b (2.4) (4.8) 10.3 20.8 M2 50.8 (11.5) (1.5) (4.2) 13.2 25.3 M3 51.8 (2.4) (1 .a ( 6.8) 15.1 10.5 M4 111.5 16.4 (15.9) (2.3) (3.5) (1.4) 20.7 14.0 M5 107.2 21.9 (17.4) (5.5) (6.1) (3.5) 2.3 M6 88.5 (z?.i (3y (16.1) (1.8) M7 92.7 3.9 (31.3) (3.5) The compositions of the media are listed in Table 1. B Average value of effluent. Average values were calculated from measurements taken 2 d after changing the medium. b Standard deviation.
(& (k:, (K) (Z) (& (Z) (6)
(lag (ly
NH.,+ concentration (mg N./-r) 23.9
(1.8)
0.3 (0.5)
(oqbo (oy coq; 3.8 (4.2)
VOL.
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1500
9 8 ;; 1000 s 5 z 22 x 8
5oo 0
20 I
40
Organic acid medium
60 80 100 Operational days ’
Starch medium
’
120
Peptone
140 I
medium
FIG. 6. Change in biogas and methane yield produced from the LUASB reactor. Symbols: 0, biogas yield; 0, methane yield; n , operational day when the LUASB reactor was placed in the dark for 24h.
than 0.1 mg N-I-l throughout the entire incubation period. The dissolved oxygen concentration in the LUASB reactor was O.Omg.l-’ throughout the entire incubation period. The average methane yield from the LUASB reactor was lower than that of the UASB reactor (Table 2). The gas and methane yields from the LUASB reactor under dark conditions were higher than those under light conditions (Fig. 6). The averPHB production using LUASB reactor age PHB concentration and the PHB contents based on the dry biomass in the effluent from the LUASB reactor using the acetate-based media (Ml-M5) as influent were higher than those using the starch-based media (M6-M7) (Table 3). The PHB concentration in the effluent decreased due to the increase in the NH&l concentration (Fig. 7). The average production rate of PHB of the bacterial biomass in the effluent was 6.6-14.0 mg . I-l-reactor. d-l using the acetate-based media and the average PHB content based on the dry biomass was 15.1-25.3x (Table 3). The yeast extract and trypticase peptone promoted phototrophic bacterial growth but not the PHB production in the bacterial biomass. The GCMass analysis confirmed that the sample peak, which showed the same retention time as the methyl-esterized P-hydroxybutyrate on GC, was that of the P-hydroxybutylmethylester (data not shown). Two-step incubation for PHB production After reincubation of the effluent from the LUASB reactor TABLE
4.
Operational days 1 I / I I 1 ’ Ml ’ M2 ’ M3 ’ M4 ’ MS’ M6 ’ M7 FIG 7. Change in concentrations of PHB, ammonium, and bacteriochlorophyll in the effluent from the LUASB reactor (top). Change in PHB content based on bacterial biomass and bacterial biomass yield (bottom). The reactors were operated at 35°C with a hydraulic retention time of 1.0 d under continuous illumination of 100 PE. mm2.s-1. The detailed compositions of the media (Ml-M7) are described in the text. Symbols and line: l , PHB concentration; ----, bacteriochlorophyll concentration; A, ammonium concentration; n , PHB content based on bacterial biomass; 0, bacterial biomass concentration.
with sodium acetate for 24 h, the bacteriochlorophyll concentration and the average PHB concentration in the culture increased from 3.5mg.l-’ to 7.3 rng.l-l and from 16.7 mg. 1-l to 99.6 mg. I-‘, respectively (Table 4). The average PHB content based on the dry bacterial biomass also increased from 9.6 to 32%. DISCUSSION Detection of bacteriochlorophyll in the effluent from the LUASB reactor under light conditions means that phototrophic bacteria grew in the reactor. The absorption maxima of a living suspension of Rhodobacter capsulatus were reported to be 377, 482, 514, 593, 809, and 866 nm (16); therefore, purple non-sulphur bacteria could be induced from the UASB granules in the
Average PHB and bacterial biomass yields in the effluent from the LUASB reactor and in the culture after two-step incubation of the effluent
Bacterial biomass Bacteriochlorophyll PHB PHB production PHB content on NH4+ concentration concentration concentration rate dry biomass base concentration (mg-1-l) (mg.l-I) (rng.,~‘) (mg . I- l-reactor. d-l) (mg N./-l) (%) Effluent from LUASB reactor 176a 3.5 16.7 10.7 9.6 0.1 (lllb (0.4) ( 2.2) ( 1.4) ( 1.8) u-w Reincubated culture 319 7.3 99.6 63.6 32.0 0.0 (32.8) (20.9) (11.2) (73) (0.5) (0.0) The effluent sample from the LUASB reactor using M7 as the influent was mixed with sodium acetate (170 mg .I-‘) and then maintained in a conical flask at 35(+- l)OC with a continuous incandescent light illumination of 100 ,uE.rnm2. s-l for 24 h. The composition of the medium is described in the text. B Average value. Average values were calculated from measurements of 3 data points. b Standard deviation.
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LUASB reactor under light conditions and released from the LUASB reactor. Phototrophic bacteria have been reported to simultaneously remove nitrogenous compounds and phosphate ion (6, 17-19). The difference in the ammonium and phosphate removal efficiencies between the LUASB and UASB reactors suggests that the growth of the phototrophic bacteria in the LUASB reactor improved the removal efficiencies compared with the UASB reactor. This microbial flora in the LUASB reactor efficiently removed the ammonium form of nitrogen. Many purple non-sulphur photosynthetic bacteria are known to assimilate nitrate (20); therefore, this LUASB system could be applicable to the treatment of wastewaters containing ammonium and/or nitrate. The present results suggest that a medium with organic nitrogenous compounds (yeast extract and trypticase peptone) promoted phototrophic bacterial growth more effectively than that with inorganic nitrogenous compounds only. Increases of the TOC, NH4+ and POd3- concentrations in the effluent from the LUASB reactor under dark conditions were observed and the methane yield from the LUASB reactor was lower than that of the UASB reactor. These results also suggest consumption of organic compounds, NH4+ and POd3- by the phototrophic bacteria in the LUASB reactor. Dissolved oxygen was not detected in the LUASB reactor; therefore, metabolism of an oxygenic photosynthetic microorganism in the LUASB reactor was unlikely. The phosphate removal behavior observed in this study coincides with the report that phototrophic bacteria produce significant amounts of inorganic polyphosphates under anaerobic light conditions and not under anaerobic or aerobic dark conditions (21, 22). Phosphate removal was not stable during the present experiments compared with TOC and ammonium removal. This instability could be due to the uptake and storage of phosphate ions, and subsequent release from the phototrophic bacteria and the UASB granules. The PHA inclusion bodies are generally accumulated by the bacteria during metabolic stress caused by nutrient-limiting conditions in the presence of excess carbon and may account for up to 80% of the purple nonsulphur bacterial biomass (8, 11, 23). These reports are in good agreement with our results that ammonium-limiting conditions promoted PHB production in the effluent from the LUASB reactor in the present study. Acetate was thought to be a suitable carbon source for polyester accumulation in R. rubrum (24) and the acetate-based media were also suitable for PHB production using the LUASB reactor. The promotion of PHA accumulation by the subsequent transfer of the phototrophic bacterial cells to fresh medium without any nitrogen source was reported (25). Considering that the nitrogen concentration of wastewater is changeable and that the PHB production rate increases by reincubation of the effluent from the LUASB reactor with acetate, the two-step incubation could be suitable for PHB production from wastewater using the LUASB reactor. The present study suggests that the LUASB method is useful for the production of phototrophic bacterial biomass, PHAs, and other materials as well as wastewater treatment. The recovered bacterial biomass could also be used as fertilizer (26). Further investigation into the optimization of operating conditions and reactor design, and the utilization of the bacterial biomass produced are necessary to develop the system for large-scale applica-
J. BIOSCI. BIOENG..
tion. A mechanism that allows methane fermentation and the growth of phototrophic bacteria to proceed in one reactor is ecologically interesting. The advantages of this LUASB reactor system over the UASB reactor are the improvement of the nitrogen and phosphate removal efficiencies, and the production of phototrophic bacterial biomass and PHAs. The disadvantages of this LUASB reactor system are the necessities of the light supply, the separation of suspended bacterial biomass in the effluent, and the treatment of the excess ohototronhic bacterial biomass if it cannot effectively use for s&h as PHB production. ACKNOWLEDGMENTS We are grateful to Ms. Tae Kimura and Ms. Yukiko Fukuda for their technical assistance. REFERENCES 1. Famdn, K. F., Conrad, J. R., Srivastava, V. J., Jerger, D. E., and Chynoweth, D. P.: Anaerobic processes. J. Wat. Poll. Control Fed., 55, 623-632 (1983). 2. Lettinga, G., van Velsen, A. F. M., Hobma, S. W., de Zeeuw, W., and Klapwijk, A.: Use of upflow sludge blanket (USB) reactor concept for biological wastewater treatment, especially for anaerobic treatment. Biotechnol. Bioeng., 22, 699-734 (1980). 3. Lettinga, G. and Pal, L. H.: Advanced reactor design, operation and economy. Wat. Sci. Tech., 18, 99-108 (1986). 4. Lettinga, G. and van Haandel, A. C.: Anaerobic digestion for energy production and environmental protection, p. 817-839. In Johansson, T. B., Kelly, H., Reddy, A. K. N., and Williams, R. H. (ed.), Renewable energy sources for fuels and electricity. Island Press, Washington, D.C. (1993). 5. Chang, Y. and Nishio, N.: Removal of PO4 and NH4 from wastewaters by UASB methanogenic fermentation. J. Ferment. Bioeng., 77,450-452 (1994). 6. Sasikala, C. and Ramana, C. V.: Biotechnological potentials of anoxygenic phototrophic bacteria. 1. Production of single-cell protein, vitamins, ubiquinones, hormones, and enzymes and use in waste treatment, p. 173-226. In Neidleman, S. L. and Laskin, A. I. (ed.), Advances in applied microbiology, vol. 41. Academic Press, San Diego (1995). 7. Sawayama, S., Yagjshita, T., and Tsukahara, K.: Lighted upflow anaerobic sludge blanket. J. Biosci. Bioeng., 87, 258260 (1999). 8.
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Anderson, A. J. and Dawes, E. A.: Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbial. Rev., 54, 450-472 (1990). Saaikala, C. and Ramana, C. V.: Biotechnological potentials of anoxygenic phototrophic bacteria. II. Biopolyesters, biopestitide, biofuel, and biofertilizer, p. 227-278. In Neidleman, S. L. and Laskin, A. I. (ed.), Advances in applied microbiology, vol. 41. Academic Press, San Diego (1995). Fukuda, K.: A current situation of biodegradable plastics and biotechnology, p. 22-31. In Doi, Y. (ed.), Biodegradable plastics hand book. NTS Ltd., Tokyo (1995). Brand& H., Knee, E. J., Jr., Fuller, R. C., Gross, R. A., and Lenz, R. W.: Ability of the phototrophic bacterium Rhodospirillum rubrum to produce various poly(P-hydroxyalkanoates): potential sources for biodegradable polyesters. Int. J. Biol. Macromol., 11, 49-55 (1989). Shhni, Y., Yamaguchi, M., Knsuhayashi, N., Hibi, K., Ueyama, K., and Hashimoto, K.: Production of biodegradable COpolymers by a fed-batch culture of photosynthetic bacteria, p. 263-265. In Teo, W. K., Yap, M. G. S., and Oh, S. K. W. (ed.), Better living through innovative biochemical engineering. Continental Press Pte. Ltd., Singapore(1994). APHA: Standard methods for the examination of water and wastewater, 17th edition, p. (4) 117-178. American Public Health Association, Washington, D.C. (1989).
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14. Cohen-Bazire, G., Sistrom, W. R., and Stanier, R. Y.: Kinetic studies of pigment synthesis by non-sulfur purple bacteria. J. Cell. Comp. Physiol., 49, 25-68 (1957). 15. Braunegg, G., Sonnleitner, B., and Lafferty, R. M.: A rapid gas chromatographic method for the determination of poly-phydroxybutyric acid in microbial biomass. Eur. J. Appl, Microbial. Biotechnol., 6, 29-37 (1978). 16. Pfeanig, N. and Trtiper, H. G.: The family Chromatiaceae, p. 3200-3221. In Balows, A., Trtiper, H. G., Dworkin, M., Harder, W., and Schleifer, K. (ed.), The prokaryotes, 2nd edition. Springer-Verlag, New York (1991). 17. Sawada, H. and Rogers, P. L.: Photosynthetic bacteria in waste treatment. J. Ferment. Technol., 55, 297-310 (1977). 18. Sudo, H., Yamada, A., Kokatsu, K., Nakamura, N., and Matsunaga, T.: Development of a phosphate-removal system using a marine photosynthetic bacterium Chromatium sp. Appl. Microbial. Biotechnol., 47, 78-82 (1997). 19. Sawayama, S., Rae, K. K., and Hall, D. 0.: Immobilization of Rhodobacter capsulatus on cellulose beads and water treatment using a photobioreactor. J. Ferment. Bioeng., 86, 517-520 (1998). 20. Sabaty, M., Gans, P., and Vermeglio, A.: Inhibition of nitrate reduction by light and oxygen in Rhodobacter sphaeroides forma sp. denitrflcans. Arch. Microbial., 159, 153-159 (1993).
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21. Carr, N. G. and Sandhu, G. R.: Endogenous metabolism of polyphosphates in two photosynthetic micro-organisms. Biothem. J., 99, 29-30 (1966). 22. Hi&&i, A., Yanase, A., and Kftamura, H.: Polyphosphate accumulation by Rhodobacter sphaeroides grown under different environmental conditions with special emphasis on the effect of external phosphate concentrations. Bull. Jpn. Sot. Microb. Ecol., 6, 25-32 (1991). 23. Dierstein, R. and Drews, G.: Nitrogen-limited continuous culture of Rhodopseudomonas capsulatu growing photosynthetically or heterotrophically under low oxygen tensions. Arch. Microbiol., 99, 117-128 (1974). 24. Brand& H., Gross, R. A., Lenz, R. W., Lloyd, R., and Fuller, R. C.: The accumulation of poly (3-hydroxyalkanoates) in Rhodobacter sphaeroides. Arch. Microbial., 155, 337-340 (1991). 25. Liebergesell, M., Hustede, E., Timm, A., Steinbtichel, A., Fuller, R. C., Leaz, R. W., and Schlegel, H. G.: Formation of poly(3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria. Arch. Microbial., 155, 415-421 (1991). 26. Kobayashi, M. and T&an, Y.T.: Treatment of industrial waste solutions and production of useful by-products using a photosynthetic bacterial method. Wat. Res., 7, 1219-1224 (1973).
OF BIOSCIENCE
AND BIOENGINEERING
Vol. 87, No. 5, 683-689. 1999
Wastewater Treatment and Poly-b-Hydroxybutyrate Production Using Lighted Upflow Anaerobic Sludge Blanket Method SHIGEKI SAWAYAMA,
* KENICHIRO
TSUKAHARA,
AND
TATSUO YAGISHITA
Biomass Division, National Znstitute for Resources and Environment, AIST, MITI, 14-3 Onogawa, Tsukuba, Zbaraki 305-8569, Japan Received11 December 1998/Accepted26 January 1999 We investigated the performance of a lighted upflow anaerobic sludge blanket (LUASB) reactor for wastewater treatment and poly-ghydroxybutyrate (PHB) production. Phototrophic bacteria were induced from UASB (upflow anaerobic sludge blanket) granules under light conditions (100 $*m-2*s-1). The ammonium and phosphate ion removal efficiencies of the LUASB reactor were higher than those of the UASB reactor. The difference in the results from runs under light and dark conditions suggested that the ammonium and phosphate ion removal efficiencies were improved by increasing the amount of phototrophic bacteria in the LUASB reactor. The average production rate of PHB from the biomass in the effluent from the LUASB reactor was 6.6-14.0 mg.Z-l-reactored-’ using acetate-based media and the average PHB content based on the dry bacterial biomass was 15.1-25.3x. The PI-HI concentration increased by reincubation of the effluent from the LUASB reactor with sodium acetate under light conditions. The UASB granular sludge can decompose a variety of organic substances and in addition the LUASB method can remove ammonium and phosphate ions. The LUASB method thus appears to be appropriate for wastewater treatment and production of phototrophic bacteria and PHB from various wastewaters. [Key words:
poly$-hydroxybutyrate, lighted UASB method, phototrophic bacteria, wastewater treatment]
UASB method. Biodegradable plastics have been studied to reduce environmental pollution due to the huge amount of plastic waste. Extensive studies on bacterial polyhydroxyalkanoates (PHAs) as raw materials for biodegradable plastics have been carried out (8, 9); however, the production cost, without subsidies, is not competitive with that of conventional plastics made from fossil fuels (10). Brand1 et al. reported that a phototrophic bacterium, Rhodospirillum rubrum, produced various PHAs, particularly the PHB type (11). The combination of the production of biodegradable plastics and wastewater treatment using phototrophic bacteria has been proposed to reduce production costs (12). A phototrophic bacterial biomass can be produced during water treatment using a LUASB reactor (7); therefore, PHB could be produced from various wastewaters using the LUASB method. In this study, we investigated the performance of the LUASB reactor during wastewater treatment and poly+hydroxybutyrate (PHB) production on a laboratory scale.
pared with that using the conventional
Environmental pollution due to the production of large amounts of waste and wastewater is an important issue to resolve. Anaerobic digestion of waste and wastewater has the advantages of low levels of sludge production and energy consumption compared with aerobic treatment, and methane production; therefore, this process has been widely studied and used for the treatment of organic wastes and wastewater (1). A conventional contact process for
anaerobic
digestion
cannot
efficiently
treat at a high organic loading rate and is not highly stable. The UASB (upflow anaerobic sludge blanket) concept was proposed to resolve these problems (2, 3). This concept is based on the formation of a well-settling granular sludge, natural agitation caused by gas production and a well-designed gas-solids separator. An UASB reactor, however, cannot efficiently remove nitrogenous compounds and phosphate ion (4). Improvement of phosphate ion removal with the UASB method by the addition of Ca*+ and Mg*+ to the influent was reported (5); however, a biological removal method has not been reported. Biomass production during wastewater treatment could be necessary to simultaneously remove nitrogenous compounds and phosphate ion. Purple non-sulphur phototrophic bacteria used to aerobically and anaerobically decompose organic compounds, and to simultaneously consume nitrogenous and phosphate ions have been studied in wastewater treatment systems; however, phototrophic bacteria can only decompose a limited number of organic substances (6). We reported that a population of phototrophic bacteria were induced from UASB granules under light conditions and proposed the lighted upflow anaerobic sludge blanket (LUASB) method for wastewater treatment (7). There is the possibility of obtaining improved removal of inorganic compounds using the LUASB method com-
MATERIALS
AND METHODS
LUASB reactor Two cylindrical glass reactors (80 x 180mm, Able, Tokyo) used for the LUASB or UASB reactors, each having a volume of 850m1, were filled with 530ml of UASB granules provided by the Ebara Co., Kanagawa. The lower glass surface (10.5cm from the bottom) of the LUASB reactor and the entire glass surface of the UASB reactor were covered with aluminum foil to prevent light penetration (Fig. 1). Reactor operation for water treatment The LUASB and UASB reactors were maintained at 35(tl)‘C and were supplied with synthetic wastewater at a flow rate of 600 ml.d-l (hydraulic retention time=0.9 d) without sterilization. The LUASB reactor was incubated with continuous incandescent illumination of 100 pE.rn-*. s-l.
* Corresponding author. 683
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reactor FIG. 1. Diagram of a laboratory-scale LUASB reactor. The reactor was maintained at 35( + I)‘C with continuous incandescent illumination of 100 pE . m-2. SK’ and was supplied with organic acid, starch and peptone media.
Both the LUASB and UASB reactors were supplied with three kinds of synthetic wastewater: (i) 2.5 g.l-’ sodium acetate (Wake Pure Chemical Industries, Osaka), 1.25 g. 1-l sodium lactate (Wako), 1.25 g.l-’ sodium propionate (Wako); (ii) 4 g.l-* starch soluble (Wako); and (iii) 4 g .I-’ trypticase peptone (containing 13.6 wt% nitrogen and 0.85 wt% phosphorus, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA). The following chemicals were also added to all the media used: 200 mg . 1-l NH&l, 16 mg . I-l KH2P04, 25 mg .I-’ CaClz. 2Hz0, 25 mg . I- * MgClz .6HrO, 30 mg . I-’ Fe-EDTA, 5 mg .I-’ CoClZ.6Hz0, 5mg.l-I MnC12.4H20 and 3OOmg.I-’ yeast extract (containing 10.2 wt% nitrogen and 0.99 wt% phosphorus). The organic acid medium was supplied to the LUASB reactor from day 1 to 60, the starch medium from day 61 to 98, and the peptone medium was from day 99 to 134. When the TOC concentration in the effluent was stable, the reactor was considered to have reached a steady-state. The LUASB reactor was placed in the dark for 24 h on operational days 1-19, 31, 37, 38, 45, 52, 60, 89, 90, 97, 98, 123, 124, 133, and 134. Effluent samples were collected at room temperature for 3 h and then centrifuged (5500 x g for 5 min) before analyses. Any phototrophic bacteria attached on the inside glass surface of the LUASB reactor were removed once a week to keep it transparent. Reactor operation for PHB production The LUASB reactor was maintained at 35(-+1)“C with continuous incandescent illumination of 100 pE.rnp2.ss1, and synthetic wastewater was supplied at a flow rate of 530ml.d-r TABLE Influents MO Ml M2 M3 M4 M5 M6 M7 The following 6H20, 30mg.l-r
Sodium acetate (g.l-‘1 4.7 4.7 4.7 4.7 4.7 4.1 0 0
(hydraulic retention time= 1.0 d) without sterilization. The LUASB reactor was supplied with eight kinds of synthetic wastewater whose compositions are shown in Table 1 (MO to M7). Effluent samples were collected at room temperature for 24 h and then centrifuged (5500 x g for 5 min) before the PHB analyses. Any phototrophic bacteria attached on the inside glass surface of the LUASB reactor were removed once a day from operational day 159 to keep it transparent. Two-step incubation for PHB production An effluent sample from the LUASB reactor using the M7 medium as influent was mixed with sodium acetate (170 mg. 1-l) and then poured into a conical flask (500 ml). The air in the flask was purged with nitrogen gas and then the flask was maintained at 35( + l)“C with continuous incandescent illumination of 100 pE . m-2. s - l for 24 h. After the two-step incubation, the bacterial biomass was collected by centrifugation (5500 x g for 5 min) and the PHB concentration was analyzed. Analyses The concentrations of total carbon, inorganic carbon and total organic carbon (TOC) were determined using a TOC meter (TOC-SOOOA, Shimadzu, Kyoto). The NH*+ concentration in the effluent was determined by calorimetry with Nessler reagent (13) or an ammonium electrode (Model 95-12, Orion, Beverly, MA, USA). The N02- and POd3- concentrations were colorimetrically determined (13). The N03- concentration in the effluent was determined using a nitrate electrode (Model 93-07, Orion). After degradation and oxidation with NaOH and K2S208, the total nitrogen (TN) concentration was determined by the ultraviolet absorption method (13). After degradation with K2S208, the total phosphorus (TP) concentration was calorimetrically determined (13). The dissolved oxygen concentration in the reactor was monitored using an oxygen electrode (CSP-2, Able). The biogas yield from the bioreactor was monitored by displacement of a saturated sodium chloride solution. The biogas composition was determined by gas chromatography (Model GC-8A and GC-12A, Shimadzu) with a WG-100 column (GL Sciences, Tokyo) at 50°C and a Porapak Q column (Shinwakakou, Kyoto) at 90°C. Bacteriochlorophyll was extracted with an acetonemethanol solution and its concentration was measured using a Spectrophotometer 120A (Shimadzu) (14), and the absorption spectrum of the effluent was analyzed using a Spectrophotometer 1600PC (Shimadzu). The PHB concentration in the bacterial biomass was determined by the method of Braunegg et al. (15). PHB was depolymerized and methylesterized at 100°C for 3.5 h in a methanol-H2SOrchloroform solution, and then the ,B-
1. Compositions of synthetic wastewaters supplied to the LUASB reactor Starch k%.,-‘) 0 0 0 0 0 0 3.4 3.0
NH&l (mg.l-I) 2.6 10 50 0 0 0 0 0
Yeast extract (mg.l-r) 0 0 0 100 300 0 300 300
Trypticase peptone (mg . I- r) 0 0 0 0 0 300 0 300
Supplied days 172-182 183-190 191-197 198-204 205-211 212-217 218-225 226-239
chemicals were also added to all the media from MO to M7: 16 mg. 1-r KH2P04, 25 mg. I-- 1 Car&. 2H20, 25 mg. 1-r MgC12. Fe-EDTA, 5 mg.l-r CoC12.6H20, and 5 mg.l-r MnC12.4H20.
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Operational days I I ’ Starch Organic acid medium medium
Peptone medium
FIG. 2. Changes in bacteriochlorophyll concentration and optical density (865 nm) of the effluent from the LUASB reactor. The reactors were operated at 35°C with a hydraulic retention time of 0.9 d under continuous illumination of 100 ,eE. m-z. s-r. Compositions of the synthetic wastewater are described in the text. Symbols and line: 0, bacteriochlorophyll concentration of the effluent; ----, optical density at 865 nm; n , operational day when the LUASB reactor was placed in the dark for 24 h. ,
hydroxybutylmethylester produced was measured using a gas chromatograph (GC) (5890, Hewlett-Packard, Wilmington, DE, USA) with a CBPlO-S-25-050 column (Shimadzu). The initial oven temperature of the CC was held at 110°C for 2 min, then increased to 260°C at 10”C.minl. n-Dodecane was used as the internal standard. A GC-MS analysis of j-hydroxybutylmethylester was conducted using an HP-5890II/HP-5971 (HewlettPackard). RESULTS
> r
:
Bacteriochlorophyll in the effluent from LUASB reactor The color of the liquid in the lighted area and granules in the upper surface area of the LUASB reactor turned red under light conditions. The LUASB reactor was placed under light conditions from day 20 and bacteriochlorophyll was detected in the effluent after day 23
s 3* 1000 g 2
, I
1
; i
0 20
40
60
80
100
120
140
Operational days ’ Organic acid ’ Starch ’ Peptone ’ medium medium medium FIG. 3. Change in TOC concentration of the effluent from the LUASB reactor. Symbols and line: 0, TOC concentration of the effluent; ----, TOC concentration of the influent; n , operational day when the LUASB reactor was placed in the dark for 24 h.
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20
20
40
60 80 100 120 140 Operational days I I Organic acid ’ Starch ’ Peptone medium medium medium
FIG. 4. Change in NH4+ concentration of the effluent from the LUASB reactor. Symbols and line: 0, NH4+ concentration of the effluent; mm--,NH4+ concentration of the influent; w , operational day when the LUASB reactor was placed in the dark for 24 h.
(Fig. 2). Bacteriochlorophyll was not detected in the effluent from the UASB reactor throughout the entire incubation period. The bacteriochlorophyll concentration in the effluent from the LUASB reactor under dark conditions was lower than that under light conditions (Fig. 2). The bacteriochlorophyll concentration in the effluent from the LUASB reactor using the peptone medium was higher than those using the organic acid and starch media. The absorption maxima of the effluent sample using the starch medium were 378, 484, 528, 590, 808, and 866nm. The change in optical density of the effluent from the LUASB reactor at 865 nm corresponded well with that of the bacteriochlorophyll concentration (Fig. 2). Water treatment performance of LUASB reactor The TOC concentration in the effluent from the LUASB reactor was less than 60mg C.l-r under light conditions (Fig. 3). The TOC concentration in the effluent from the LUASB reactor increased under dark conditions comTABLE
40
60 80 100 120 140 Operational days I I Organic acid Starch Peptone medium medium medium FIG. 5. Change in POd3- concentration of the effluent from the LUASB reactor. Symbols and line: 0, POd3- concentration of the effluent; ----, P04)- concentration of the influent; n , operational day when the LUASB reactor was placed in the dark for 24 h.
pared with under light conditions. The TOC removal efficiency of the LUASB reactor was 94-97X under light conditions using the organic acid and starch media as the influent, and this range of efficiency was the same as that of the UASB reactor (Table 2). The NH4+ concentration in the effluent from the LUASB reactor with the starch medium was lower than that with the organic acid medium, and that with the peptone medium was higher than that with the organic acid and starch media (Fig. 4). The POd3- concentrations in the effluent from the LUASB reactor with the organic acid and starch media were lower than that with the peptone medium (Fig. 5). The NH4+ and POA3- concentrations in the effluent from the LUASB reactor increased under dark conditions compared with those under light conditions. The average TN and TP removal efficiencies of the LUASB reactor using the organic acid, starch and peptone media were higher than those of the UASB reactor (Table 2). The concentrations of nitrate and nitrite ions in the effluent from the LUASB and UASB reactors were less
3. Average PHB and bacterial biomass yields in the effluent from the LUASB reactor
Bacterial biomass PHB PHB content on PHB production Bacteriochlorophyll concentration dry biomass base rate Influents concentration (mg . Ir) (mg.l-r) (mg I-r-reactor. d-l) (mg . I- r) (%I Ml 59.8” 14.0 23.9 (11.7)b (2.4) (4.8) 10.3 20.8 M2 50.8 (11.5) (1.5) (4.2) 13.2 25.3 M3 51.8 (2.4) (1 .a ( 6.8) 15.1 10.5 M4 111.5 16.4 (15.9) (2.3) (3.5) (1.4) 20.7 14.0 M5 107.2 21.9 (17.4) (5.5) (6.1) (3.5) 2.3 M6 88.5 (z?.i (3y (16.1) (1.8) M7 92.7 3.9 (31.3) (3.5) The compositions of the media are listed in Table 1. B Average value of effluent. Average values were calculated from measurements taken 2 d after changing the medium. b Standard deviation.
(& (k:, (K) (Z) (& (Z) (6)
(lag (ly
NH.,+ concentration (mg N./-r) 23.9
(1.8)
0.3 (0.5)
(oqbo (oy coq; 3.8 (4.2)
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9 8 ;; 1000 s 5 z 22 x 8
5oo 0
20 I
40
Organic acid medium
60 80 100 Operational days ’
Starch medium
’
120
Peptone
140 I
medium
FIG. 6. Change in biogas and methane yield produced from the LUASB reactor. Symbols: 0, biogas yield; 0, methane yield; n , operational day when the LUASB reactor was placed in the dark for 24h.
than 0.1 mg N-I-l throughout the entire incubation period. The dissolved oxygen concentration in the LUASB reactor was O.Omg.l-’ throughout the entire incubation period. The average methane yield from the LUASB reactor was lower than that of the UASB reactor (Table 2). The gas and methane yields from the LUASB reactor under dark conditions were higher than those under light conditions (Fig. 6). The averPHB production using LUASB reactor age PHB concentration and the PHB contents based on the dry biomass in the effluent from the LUASB reactor using the acetate-based media (Ml-M5) as influent were higher than those using the starch-based media (M6-M7) (Table 3). The PHB concentration in the effluent decreased due to the increase in the NH&l concentration (Fig. 7). The average production rate of PHB of the bacterial biomass in the effluent was 6.6-14.0 mg . I-l-reactor. d-l using the acetate-based media and the average PHB content based on the dry biomass was 15.1-25.3x (Table 3). The yeast extract and trypticase peptone promoted phototrophic bacterial growth but not the PHB production in the bacterial biomass. The GCMass analysis confirmed that the sample peak, which showed the same retention time as the methyl-esterized P-hydroxybutyrate on GC, was that of the P-hydroxybutylmethylester (data not shown). Two-step incubation for PHB production After reincubation of the effluent from the LUASB reactor TABLE
4.
Operational days 1 I / I I 1 ’ Ml ’ M2 ’ M3 ’ M4 ’ MS’ M6 ’ M7 FIG 7. Change in concentrations of PHB, ammonium, and bacteriochlorophyll in the effluent from the LUASB reactor (top). Change in PHB content based on bacterial biomass and bacterial biomass yield (bottom). The reactors were operated at 35°C with a hydraulic retention time of 1.0 d under continuous illumination of 100 PE. mm2.s-1. The detailed compositions of the media (Ml-M7) are described in the text. Symbols and line: l , PHB concentration; ----, bacteriochlorophyll concentration; A, ammonium concentration; n , PHB content based on bacterial biomass; 0, bacterial biomass concentration.
with sodium acetate for 24 h, the bacteriochlorophyll concentration and the average PHB concentration in the culture increased from 3.5mg.l-’ to 7.3 rng.l-l and from 16.7 mg. 1-l to 99.6 mg. I-‘, respectively (Table 4). The average PHB content based on the dry bacterial biomass also increased from 9.6 to 32%. DISCUSSION Detection of bacteriochlorophyll in the effluent from the LUASB reactor under light conditions means that phototrophic bacteria grew in the reactor. The absorption maxima of a living suspension of Rhodobacter capsulatus were reported to be 377, 482, 514, 593, 809, and 866 nm (16); therefore, purple non-sulphur bacteria could be induced from the UASB granules in the
Average PHB and bacterial biomass yields in the effluent from the LUASB reactor and in the culture after two-step incubation of the effluent
Bacterial biomass Bacteriochlorophyll PHB PHB production PHB content on NH4+ concentration concentration concentration rate dry biomass base concentration (mg-1-l) (mg.l-I) (rng.,~‘) (mg . I- l-reactor. d-l) (mg N./-l) (%) Effluent from LUASB reactor 176a 3.5 16.7 10.7 9.6 0.1 (lllb (0.4) ( 2.2) ( 1.4) ( 1.8) u-w Reincubated culture 319 7.3 99.6 63.6 32.0 0.0 (32.8) (20.9) (11.2) (73) (0.5) (0.0) The effluent sample from the LUASB reactor using M7 as the influent was mixed with sodium acetate (170 mg .I-‘) and then maintained in a conical flask at 35(+- l)OC with a continuous incandescent light illumination of 100 ,uE.rnm2. s-l for 24 h. The composition of the medium is described in the text. B Average value. Average values were calculated from measurements of 3 data points. b Standard deviation.
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LUASB reactor under light conditions and released from the LUASB reactor. Phototrophic bacteria have been reported to simultaneously remove nitrogenous compounds and phosphate ion (6, 17-19). The difference in the ammonium and phosphate removal efficiencies between the LUASB and UASB reactors suggests that the growth of the phototrophic bacteria in the LUASB reactor improved the removal efficiencies compared with the UASB reactor. This microbial flora in the LUASB reactor efficiently removed the ammonium form of nitrogen. Many purple non-sulphur photosynthetic bacteria are known to assimilate nitrate (20); therefore, this LUASB system could be applicable to the treatment of wastewaters containing ammonium and/or nitrate. The present results suggest that a medium with organic nitrogenous compounds (yeast extract and trypticase peptone) promoted phototrophic bacterial growth more effectively than that with inorganic nitrogenous compounds only. Increases of the TOC, NH4+ and POd3- concentrations in the effluent from the LUASB reactor under dark conditions were observed and the methane yield from the LUASB reactor was lower than that of the UASB reactor. These results also suggest consumption of organic compounds, NH4+ and POd3- by the phototrophic bacteria in the LUASB reactor. Dissolved oxygen was not detected in the LUASB reactor; therefore, metabolism of an oxygenic photosynthetic microorganism in the LUASB reactor was unlikely. The phosphate removal behavior observed in this study coincides with the report that phototrophic bacteria produce significant amounts of inorganic polyphosphates under anaerobic light conditions and not under anaerobic or aerobic dark conditions (21, 22). Phosphate removal was not stable during the present experiments compared with TOC and ammonium removal. This instability could be due to the uptake and storage of phosphate ions, and subsequent release from the phototrophic bacteria and the UASB granules. The PHA inclusion bodies are generally accumulated by the bacteria during metabolic stress caused by nutrient-limiting conditions in the presence of excess carbon and may account for up to 80% of the purple nonsulphur bacterial biomass (8, 11, 23). These reports are in good agreement with our results that ammonium-limiting conditions promoted PHB production in the effluent from the LUASB reactor in the present study. Acetate was thought to be a suitable carbon source for polyester accumulation in R. rubrum (24) and the acetate-based media were also suitable for PHB production using the LUASB reactor. The promotion of PHA accumulation by the subsequent transfer of the phototrophic bacterial cells to fresh medium without any nitrogen source was reported (25). Considering that the nitrogen concentration of wastewater is changeable and that the PHB production rate increases by reincubation of the effluent from the LUASB reactor with acetate, the two-step incubation could be suitable for PHB production from wastewater using the LUASB reactor. The present study suggests that the LUASB method is useful for the production of phototrophic bacterial biomass, PHAs, and other materials as well as wastewater treatment. The recovered bacterial biomass could also be used as fertilizer (26). Further investigation into the optimization of operating conditions and reactor design, and the utilization of the bacterial biomass produced are necessary to develop the system for large-scale applica-
J. BIOSCI. BIOENG..
tion. A mechanism that allows methane fermentation and the growth of phototrophic bacteria to proceed in one reactor is ecologically interesting. The advantages of this LUASB reactor system over the UASB reactor are the improvement of the nitrogen and phosphate removal efficiencies, and the production of phototrophic bacterial biomass and PHAs. The disadvantages of this LUASB reactor system are the necessities of the light supply, the separation of suspended bacterial biomass in the effluent, and the treatment of the excess ohototronhic bacterial biomass if it cannot effectively use for s&h as PHB production. ACKNOWLEDGMENTS We are grateful to Ms. Tae Kimura and Ms. Yukiko Fukuda for their technical assistance. REFERENCES 1. Famdn, K. F., Conrad, J. R., Srivastava, V. J., Jerger, D. E., and Chynoweth, D. P.: Anaerobic processes. J. Wat. Poll. Control Fed., 55, 623-632 (1983). 2. Lettinga, G., van Velsen, A. F. M., Hobma, S. W., de Zeeuw, W., and Klapwijk, A.: Use of upflow sludge blanket (USB) reactor concept for biological wastewater treatment, especially for anaerobic treatment. Biotechnol. Bioeng., 22, 699-734 (1980). 3. Lettinga, G. and Pal, L. H.: Advanced reactor design, operation and economy. Wat. Sci. Tech., 18, 99-108 (1986). 4. Lettinga, G. and van Haandel, A. C.: Anaerobic digestion for energy production and environmental protection, p. 817-839. In Johansson, T. B., Kelly, H., Reddy, A. K. N., and Williams, R. H. (ed.), Renewable energy sources for fuels and electricity. Island Press, Washington, D.C. (1993). 5. Chang, Y. and Nishio, N.: Removal of PO4 and NH4 from wastewaters by UASB methanogenic fermentation. J. Ferment. Bioeng., 77,450-452 (1994). 6. Sasikala, C. and Ramana, C. V.: Biotechnological potentials of anoxygenic phototrophic bacteria. 1. Production of single-cell protein, vitamins, ubiquinones, hormones, and enzymes and use in waste treatment, p. 173-226. In Neidleman, S. L. and Laskin, A. I. (ed.), Advances in applied microbiology, vol. 41. Academic Press, San Diego (1995). 7. Sawayama, S., Yagjshita, T., and Tsukahara, K.: Lighted upflow anaerobic sludge blanket. J. Biosci. Bioeng., 87, 258260 (1999). 8.
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Anderson, A. J. and Dawes, E. A.: Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbial. Rev., 54, 450-472 (1990). Saaikala, C. and Ramana, C. V.: Biotechnological potentials of anoxygenic phototrophic bacteria. II. Biopolyesters, biopestitide, biofuel, and biofertilizer, p. 227-278. In Neidleman, S. L. and Laskin, A. I. (ed.), Advances in applied microbiology, vol. 41. Academic Press, San Diego (1995). Fukuda, K.: A current situation of biodegradable plastics and biotechnology, p. 22-31. In Doi, Y. (ed.), Biodegradable plastics hand book. NTS Ltd., Tokyo (1995). Brand& H., Knee, E. J., Jr., Fuller, R. C., Gross, R. A., and Lenz, R. W.: Ability of the phototrophic bacterium Rhodospirillum rubrum to produce various poly(P-hydroxyalkanoates): potential sources for biodegradable polyesters. Int. J. Biol. Macromol., 11, 49-55 (1989). Shhni, Y., Yamaguchi, M., Knsuhayashi, N., Hibi, K., Ueyama, K., and Hashimoto, K.: Production of biodegradable COpolymers by a fed-batch culture of photosynthetic bacteria, p. 263-265. In Teo, W. K., Yap, M. G. S., and Oh, S. K. W. (ed.), Better living through innovative biochemical engineering. Continental Press Pte. Ltd., Singapore(1994). APHA: Standard methods for the examination of water and wastewater, 17th edition, p. (4) 117-178. American Public Health Association, Washington, D.C. (1989).
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14. Cohen-Bazire, G., Sistrom, W. R., and Stanier, R. Y.: Kinetic studies of pigment synthesis by non-sulfur purple bacteria. J. Cell. Comp. Physiol., 49, 25-68 (1957). 15. Braunegg, G., Sonnleitner, B., and Lafferty, R. M.: A rapid gas chromatographic method for the determination of poly-phydroxybutyric acid in microbial biomass. Eur. J. Appl, Microbial. Biotechnol., 6, 29-37 (1978). 16. Pfeanig, N. and Trtiper, H. G.: The family Chromatiaceae, p. 3200-3221. In Balows, A., Trtiper, H. G., Dworkin, M., Harder, W., and Schleifer, K. (ed.), The prokaryotes, 2nd edition. Springer-Verlag, New York (1991). 17. Sawada, H. and Rogers, P. L.: Photosynthetic bacteria in waste treatment. J. Ferment. Technol., 55, 297-310 (1977). 18. Sudo, H., Yamada, A., Kokatsu, K., Nakamura, N., and Matsunaga, T.: Development of a phosphate-removal system using a marine photosynthetic bacterium Chromatium sp. Appl. Microbial. Biotechnol., 47, 78-82 (1997). 19. Sawayama, S., Rae, K. K., and Hall, D. 0.: Immobilization of Rhodobacter capsulatus on cellulose beads and water treatment using a photobioreactor. J. Ferment. Bioeng., 86, 517-520 (1998). 20. Sabaty, M., Gans, P., and Vermeglio, A.: Inhibition of nitrate reduction by light and oxygen in Rhodobacter sphaeroides forma sp. denitrflcans. Arch. Microbial., 159, 153-159 (1993).
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21. Carr, N. G. and Sandhu, G. R.: Endogenous metabolism of polyphosphates in two photosynthetic micro-organisms. Biothem. J., 99, 29-30 (1966). 22. Hi&&i, A., Yanase, A., and Kftamura, H.: Polyphosphate accumulation by Rhodobacter sphaeroides grown under different environmental conditions with special emphasis on the effect of external phosphate concentrations. Bull. Jpn. Sot. Microb. Ecol., 6, 25-32 (1991). 23. Dierstein, R. and Drews, G.: Nitrogen-limited continuous culture of Rhodopseudomonas capsulatu growing photosynthetically or heterotrophically under low oxygen tensions. Arch. Microbiol., 99, 117-128 (1974). 24. Brand& H., Gross, R. A., Lenz, R. W., Lloyd, R., and Fuller, R. C.: The accumulation of poly (3-hydroxyalkanoates) in Rhodobacter sphaeroides. Arch. Microbial., 155, 337-340 (1991). 25. Liebergesell, M., Hustede, E., Timm, A., Steinbtichel, A., Fuller, R. C., Leaz, R. W., and Schlegel, H. G.: Formation of poly(3-hydroxyalkanoates) by phototrophic and chemolithotrophic bacteria. Arch. Microbial., 155, 415-421 (1991). 26. Kobayashi, M. and T&an, Y.T.: Treatment of industrial waste solutions and production of useful by-products using a photosynthetic bacterial method. Wat. Res., 7, 1219-1224 (1973).