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War. Sci. Tech. Vol. 36, No. 6-7, pp. 295-301, 1997.. 1997 IAWQ. Published by Elsevier Science Ltd

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COMBINED PRE-DEGRADATION ANDANAEROBIC DIGESTION FOR THETREATMENT OF A BAKER'S YEASTFACTORY EFFLUENT

M. Van Der Merwe-Botha* and T. J. Britz**

* Krugersdorp Transitional Local Council, Waterpurification Works, PO Box 94,Krugersdorp, 1740, South Africa** Stellenbosch University, Food Science Department, Private Bag Xl, Matieland,7602, South Africa

ABSTRACT

A Chryseomonas luteola strain was isolated from raw baker's yeast factory effluent as the dominant part ofthe microbial community and evaluated for its biodegradative activity, using the raw effluent as substrate.The strain was able to utilise the raw effluent and produce higher concentrations of energetically favourablemetabolites and thereby, could contribute to the first degradation step in an anaerobic biological treatmentprocess. A 3x4x3 factorial design indicated optimal degradation conditions in a specific environmentalframework of 48 h incubation time, COD concentration of 30 gil, pH of 6.0 and temperature of 35OC. The C.luteola strain was thereafter used in a pre-degradation step followed by an anaerobic digestion step in a 5 Ilaboratory-scale hybrid digester. With the use of the pre-degraded effluent, significant improvements werefound in the overall anaerobic digestion performance. These included increased COD (>15%) and TVFA(>50%) removals, especially propionic acid (88%) removal, as well as higher biogas yields (18%). Theresults also showed a prominent improvement in fatty acid utilisation and methanogenesis. The pre•degradation step resulted in better process control and increased stability of the system, even at relativelyhigh organic loading rates (10 kg COD/m3.d). When the raw effluent was not pre-treated (control bioreactor),no improvement in bioreactor efficiency was observed. © 1997 IAWQ. Published by Elsevier Science Ltd

KEYWORDS

Anaerobic digestion; baker's yeast effluent; Chryseomonas luteola; combined treatment; factorial design;natural selection; pre-treatment.

INTRODUCTION

Baker's yeast factory effluents are complex and high-strength waste waters, which are difficult to degrade.Anaerobic digestion has been used in the treatment, but it was clear that only a part of the raw effluent isreceptive to degradation during the digestion process (58% COD removal). It has been suggested that theseparation of the acidogenic and methanogenic phases may be useful if complex substrates are to beconverted anaerobically (Heertjies and van der Meer, 1978). The use of a microbial culture capable ofdegrading a specific part of the raw effluent, before the anaerobic treatment step, may facilitate analternative treatment option for a variety of complex industrial effluents. When considering the fact thatmost factories have a balancing tank, it seems a logical option to use this already active environment as a

295

296 M. VAN DER MERWE-BOTHA and T. 1. BRITZ

pre-digester in combination with the actual anaerobic digestion phase. Spe~ific microbial selection fromsuch a "natural" environment may provide a means to enhance the degradatIon of complex e.ffluents. Theenvironmental conditions of a balancing tank could also be optimised and then be seeded wIth a specificenhanced degrading strain, in order to provide the strain with a competitive advantage. The aim of this studywas to evaluate a combined pre-degradation and anaerobic digestion system for the treatment of a complexbaker's yeast effluent.

MATERIALS AND METHODS

Baker's yeast effluent

The effluent was obtained from a local baker's yeast factory and stored at 40C until required.

Media. enumeration, isolation and characterisation

Nutrient-medium (NA), MRS-medium (MRS) and Yeast-medium (YA) were used for the enumeration andisolation (pH 6.5) of bacterial cultures present in the raw BYE (baker's yeast effluent). Anaerobic colonieswere transferred to peptone yeast glucose (PYG) medium (Gerhardt et al., 1981) for further purification.

Different batches of the effluent from the balancing tank of the baker's yeast factory, were sampled over aperiod of three months. The samples were serially diluted, plated in duplicate and incubated under aerobic,facultative anaerobic and anaerobic conditions at 37°C for 72-96 h. The Harrison disc method (Harrigan andMcCance, 1976) was used for the random statistical selection of representative colonies.

The following tests were also performed: bright field microscopy; catalase; oxidase; endospore formation;haemolysis on blood agar; growth on MacConkey agar; as well as the oxidation/fermentation in Hugh•Leifson's medium. The isolates were characterised using combinations of the API 20E, 20NE, 50CH,20Strep, Staph and ATB 32A, as well as API-ZYM for constitutive enzyme characterisation. Theidentification of each culture was confirmed using standard identification systems (Holdeman et al., 1977;Krieg and Holt, 1984; Sneath et aI., 1986) as well as a comparison with reference strains, where possible.

Strain selection on BYE-substrate

First, second and the final natural selections. All the original 326 isolates were re-grown on the BYE•substrate to determine their metabolite formation potential, and were on this basis systematically tested andselected.

Biodegradation studies

Biodegradation of the raw BYE-substrate for the selected strains was done in batch studies and six hourly(for 60 h) evaluated using the following parameters: bacterial growth; pH; COD reduction; total volatilefatty acids (TVFA), ethanol, 2,3-buthanediol and acetoin production; as well as the formation of carbondioxide (C02) and hydrogen (H2). A control was included for each duplicate experimental set.

Batch cultures of the five most suitable selected strains were subjected to individual and combinations of thedifferent environmental factors. These included: temperature (20, 25, 30, 35°C); time (0, 24, 48, 72 h); pH(5.0, 5.5, 6.0) an~ COD concentration (l0, 20, 30 gil), These variables were selected as they are consideredto have a strong mfluence on the metabolism of the bacterial community present in anaerobic digesters andrepresent a range of conditions generally found during the anaerobic treatment of industrial effluents.Biodegradation of the raw BYE-substrate by the five bacteria was again evaluated using the previouslymentioned biodegradation parameters. Samples were taken every 24 h for 72 h and analysed forbiodegradative activities.

Statistical desi~n

Treatment of yeast factory effluent 297

A 3x4x3 factorial design at four temperatures (20, 25, 30, 35OC) was used to evaluate the quantitative effectsof the variables, individually and in combination, on the five bacterial strains. The effects and interactions oftime (T), pH (P) and COD concentration (C) on the microbial responses were calculated using the Yates'salgorithm (Box et aI., 1978). The main effect (T or P or C) is defined as the influence of a specific variableon a response, averaged over the span of the other variables. The detection of a positive two or threefactorial interaction value, implies an enhancement of the final response when the two or three variables areincreased simultaneously.

Anaerobic di~estion

Laboratory-scale anaerobic digesters with working volumes of five liters (Britz and Van der Merwe, 1993)were used at hydraulic retention times (HRT) of 3 days, combined with the microbial pre-treatment.

Analytical methods

All analyses were done according to Standard Methods (APHA, 1985). The VFAs, 2,3-buthanediol, acetoin,ethanol, CO2 and H2 were determined gas chromatographically. The BYE was diluted to the required CODconcentrations (l0, 20 or 30 gil). The pH of the raw effluent was adjusted to the required pH of 5.0,5.5 or6.0. and this BYE-medium was used as growth medium for the bacteria.

Table 1. General composition of the baker's yeast factory effluent

COMPOUNDS AVE. VALUE COMPOUNDS AVE. VALUE

pH 5.4 Zinc (mgll) 0.15COD (gil) 32.8 Iron (mgll) 9.13Volatile fatty acids (gil) 3.1 Manganese (mgll) 0.03Acetic acid (gil) 1.5 Copper (mgll) 36.00Propionic acid (gil) 0.81 Digestible Nitrogen (%) 5.39iso-Butyric acid (gil) 0.34 Energy value (mJ/kg) 6.30n-Bytyric acid (gil) 0.28 Crude protein (%) 1.40iso-Valeric acid (gil) 0.026 Bypass protein (%) 60.00n-Valeric acid (gil) 0.142 Dry matter (%) 0.10Caproic acid (gil) 0.018 Salt (%) 0.86Total solids (gil) 43.2 Cobalt (mg/kg) 0.00Volatile Solids (gil) 30.5 Iodine (mg/kg) 0.00Non-volatile solids 13.1 Vitamin E (iu/kg) 0.00P04-P (gil) 0.106 Thiamine (mg/kg) 0.00S04 (gil) 6.1 Riboflavin (mg/kg) 0.00Nitrogen (gil) 1.4 Niacin (mg/kg) 0.00Alkalinity (gil as CaC03) 2.5 Pantothenic acid (mg/kg) 0.00Folic acid (mcg/kg) 60.00 Choline (mg/kg) 0.00

Calcium (mgll) 903.5 Pyridoxine (mg/kg) 0.00

Magnesium (mgll) 540 Biotin (mcg/kg) 0.00

Potassium (mgll) 4.48 Sulfur (%) 0.00

Sodium (mgll) 390

298 M. VAN DER MERWE-BOTHA and T. J. BRITZ

RESULTS AND DISCUSSION

Substrate

The general composition of the raw baker's yeast effluent is given in Table 1 (Van Der Merwe and Britz,1993).

Enumeration

The average viable bacterial counts from the raw effluent varied from 6.5 x 105 to 1.8 x. 1.07

cfull with theaerobic and the MRS counts generally being higher. The level of viable counts was surprIsmg as the baker'syeast effluent is produced during a microbial fermentation and no easily metabolisable carbon so~ces

remained in the raw effluent after the yeast fermentation process. The counts, even on the poorer medIUm,suggest that environmental pressure had already forced the selection of a competitive microbial communitythat could compete and grow under the conditions in the balancing tank.

Bacterial strain selection

First and second natural selection. For this selection, all the originally isolated strains (Van Der Merwe andBritz, 1994) were re-grown on the BYE-substrate. Forty-two strains were then selected that were able toproduce "acidogenic phase" metabolites on the BYE-substrate. Secondly, 11 strains producing the highestconcentrations of "acidogenic phase" metabolites were selected from the 42 strains.

Final strain selection. From these 11 strains, five strains were selected as the most effective degraders ofBYE-substrate under the conditions used in this study. All five strains produced higher concentrations ofTVFA, acetate and CO2, The selected strains included Fusobacterium mortiferum (N28), Enterobacteragglomerans (B), Chryseomonas luteola (D), an unidentified Gram-negative rod (G2A) and Klebsiellaoxytoca (A).

Biodegradation data

The data (influence of temperature (20, 25, 30, 35°C); time (0-24h = T I' 0-48h = T2' 0-72h = T3); pH (5.0·5.5 = PI' 5.5-6.0 = P2) and COD concentration (10-20 gil = C1, 10-30 gil =C2»were statistical evaluatedusing the factorial design and TVFA production as the most representative indicator of biodegradation.TVFA reflects a major part of the metabolite production by the bacterial strains and plays an important rolein the anaerobic digestion process (Van Lier et al., 1993). This article will only address the data obtained forChryseomonas luteola in more detail, due to the space restriction.

The statistical data, obtained after the five strains were subjected to individual and environmentalcombinations in the factorial design, as well as the quantitative TVFA production and TVFA yields, aresummarised in Table 2. Although the data sets of the statistical and quantitative results showed generalsimilarities, differences were observed in the order of strain performance. The statistical data, showing themost positive interactions, indicated that C. luteola was influenced the most strongly (+335.00), followed byF. mortiferum (+193.50), the Gram-negative rod (+148.00), E. agglomerans (+121.00) and K. oxytoca(+119.75). The results in terms of TVFA yields, indicate the best VFA production by K. oxytoca (3.70),followed by C. luteola (2.67), the Gram-negative rod (2.57), F. mortiferum (1.57) and E. agglomerans(1.17).

The statistical effects and the qualitative TVFA data can obviously not be directly compared. The statisticaldata from the factorial design represents the effects of environmental changes, while the quantitative TVFAdata set gives the direct experimental data. However, when comparing these data sets, the increased TVFAproduction from inoculation time to the end of the specific experimental phase (TVFA in mgll - given inbrackets) can be observed: K. oxytoca (307 to 1136 mgll); E. agglomerans (899 to 1054 mgll); C. luteola

Treatment of yeast factory effluent 299

634 to 1690 mg/l); F. mortiferum (685 to 1075 mgll) and the Gram-negative strain (660 to 1697 mg/l)Table 2).

Ta~le. 2. Summary ~f o.ptimal TVFA production conditions at the different temperatures as indicated by thestatIstical and quantitative evaluation data (Responses were calculated using the Yates's algorithm of Box et

al., 1978)

K. oxytoca E. agglomerans C. luteola F. mortiferum Gram-negativerod

Main effects T lxC2xP2 TlxC2xP2 T2xC2xP l TlxC2xP2 T1xC2xPl(25 & 30°C) (20 & 25°C) (25 & 35°C) (25 & 35°C) (25 & 30°C)

Two-factor T1xC2xP2 T2xC2xP2 TiT3XC2xPlIP2 TlIT3XC2xP1 T1xC2xPlIP2interactions (22,25 & (22, 25 & 30°C) (30°C) (25 & 30°C) (35°C)

30°C)

Three factor TlxC lxP2 T/T2xC2xP2 T2xC2xPl T1xC2xPlIP2 T1xC2xPlIP2interactions (25°C) (30 °C) (35°C) (35 °C) (35°C)

Optimal TlxC1xP2 TlxC2xP2 T2xC2xPl TlxC2xP l TlxC2xP2interactions (25°C) (30 °C) (35°C) (35 °C) (35°C)

Responsesat three +119.75 +121.00 +355.00 +193.50 148.50factor level

TVFA 307-1136 899-1054 634-1690 685-1690 660-1697(mg/l)(* - **)

TVFAyield 3.70 1.17 2.67 1.57 2.57* TVFA substrate concentration** TVFA product concentrationTemperature (20,25,30 and 35°C); time (0-24h = Tl , 0-48h = T2, 0-72h = T3); pH (5.0-5.5 = Pi> 5.5-6.0 =P2) and COD concentration (10-20 gil = Cl , 10-30 gil =C2)

Pre-treatment of the BYE-substrate with Chryseomonas luteola

In general, pre-degradation of the raw effluent resulted in an increase in the pH, TVFAs, acetate, alkalinityand P04-P (Table 3). The results from this study on BYE-substrate indicate that an increase inbiodegradation of the complex compounds and even detoxification (by S04 reduction), was taking place inthe raw BYE-substrate within the 48 h period. The composition of the pre-treated BYE seems to indicatethat it would be a more acceptable substrate for anaerobic digestion and methane production.

Control and experimental dil:ester performance

The average compositions of the raw BYE used and that of the BYE-substrate after pre-degradation withChryseomonas luteola, are given in Table 3 and the efficiencies of the digesters are given in Table 4. Bothdigesters were operated, under the same conditions, and the efficiency parameters were monitored to ensurethat stable state conditions persisted. For the operation of the control and experimental digesters, at organicloading rates (OLR) of 9.49 and 9.78 kg COD/m3.d respectively, HRT of 3.0 d and substrate COD

300 M. VAN DER MERWE-BOTHA and T. J. BRITZ

concentrations of 28 520 and 29 440 mg/l respectively, COD removal efficiencies of about 58 and 7~%respectively were obtained over a period of 16 d. The control digester was found t~ p~rfo~ fll1l'lyeffectively during the control period, however, poor removal of the TVFAs, especially prOpIOnIC aCId, and arelatively low biogas yield was observed.

Table 3. Average composition of the raw BYE used as substrate in the pre-degradation step and the pre•treated BYE-substrate used for the anaerobic digestion process (±SD)

Parameters

pHCOD (mgll)TVFA(mgll)Acetic acid (mgll)Propionic acid (mgll)iso-Butyric acid (mgll)n-Butyric acid (mgll)iso-Valerie acid (mgll)n-Valeric acid (mgll)S04 (mgll)P04-P (mgll)Alkalinity (mg/l as CaC03)

Raw BYE

5.528520 ± 1 227.6

1 037.9 ± 125.6681.2 ± 129.7

285.6 ± 9.626.8 ± 2.134.1 ± 1.69.5 ± 1.30.6 ± 1.5

616.7 ± 147.21 170 ± 42.9

2438.5 ± 304.8

Pre-degraded BYE

6.0± 0.229440 ± 658.01131.7±560.91 296.6 ± 270.5

399.2 ± 164.627.4 ± 2.3

65.9 ± 59.411.5 ± 4.03.5 ± 6.8

500.0 ± 200.01 226.7 ± 58.9

2541.7 ± 188.2

Overall, the pre-degradation of the raw BYE-substrate with Chryseomonas luteola had a definite positiveeffect on the anaerobic digestion process (Table 4), when compared to the performance during the controlperiod. Up to 90% acetate, 87% propionate, 92% butyrate and 100% valerate removals were obtained at theend of the experimental study. The final digester effluent pH varied from 7.44 to 8.24, indicating good pHconditions for optimal methane production. These results are a clear indication that pre-degradation of theraw BYE-substrate with the C. luteola strain resulted in more efficient digestion. The results also indicatedthe stabili3ation of an active VFA-utilising population in the anaerobic digester. The data clearly show thatthe breakdown of the influent substrate is more complete when pre-degradation is applied. It also seemspossible that some of the inhibitory factors were neutralised during the pre-degradation, thus enabling theanaerobic digester community to perform more efficiently.

During the study, pre-degradation of the BYE-substrate, combined with the anaerobic digestion process,resulted in an overall increase in the CH4 content of the biogas. Undoubtedly, these results present a furtherconfirmation of the stable and more efficient anaerobic digestion that was prevalent at the end of this study.It seems possible that the degradation of the complex compounds during the pre-degradation stepaccelerated the process of methanogenesis. Relatively high CH4 yields (0.33 m3/kg CODremovOO> wereobserved for the control digester. However, for the experimental digester a lower yield was obtained. Theincreased CH4 yields are comparatively slow to reach stable state conditions and better yields wouldprobably have been reached if the experiment had been operated over a longer time period.

CONCLUSIONS

The baker's yeast industry is responsible for the production of high-strength and complex effluents whichmust be treated before disposal. These effluents consist mainly of complex compounds, since no easilydegradable compounds remain after the yeast fermentation process. During this study, microbial strains wereselected from their natural environment, raw baker's yeast effluent, and successfully used in a combined pre•degradation and anaerobic treatment process.

Treatment of yeast factory effluent 301

The data obtained with the 3x4x3 factorial design showed that the manipulation of the most importantenvironmental factors (temperature, pH, time and COD concentration) had a prominent effect on metaboliteformation on all statistical levels. It is clear that environmental frameworks can be constructed to enhancethe degradation of the complex compounds in the raw effluent, resulting in an increased metaboliteproduction by the naturally selected strains from the baker's yeast effluent. It is important to note that theselected strains for baker's yeast effluent degradation, are only as effective as the set environmentalconditions.

Application of Chryseomonas luteola in a pre-digester, would involve a process system operated at 350Cover a hydraulic retention time (HRT) of 24 h to 48 h, with a substrate feed of 30 000 mg/l COD. One veryimportant parameter that should be maintained is a substrate pH of 5.5 for optimal TVFA production. It isclear that considerable biotic biodegradation potential exists for industrial effluents, pre-treated by naturallyselected microbes. However, larger scale studies must be done and evaluated to demonstrate this potential.Application of the C. luteola in the pre-degradation step resulted in an increase in COD removal, VFAremoval and CH4 production in the combined process. These higher efficiency parameters were related toenhanced metabolite production, especially acetate, in the presence of the C. luteola strain. No improvementin digester efficiency was observed when the baker's yeast effluent was not pre-treated (control digester).

The results from this study show that specific strain selection has tremendous potential to become part of thetechnological treatment solutions to minimise environmental pollution. This study clearly indicated that themanipulation of various environmental factors in biologically controlled systems, such as anaerobicdigesters, could further enhance the biodegradative efficiency of a natural microbial population and thatchemical post-treatment may act as a final polishing step in the overall treatment process.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the financial support of the Foundation for Research Development,Welkom Yeast (Pty) and National Chemical Products (Pty), and research support of the University of theOrange Free State.

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