Pergamon PII: S0043-1354(97)00051 - 1

War. Res. Vol. 31, No. 9. pp. 2147-2160, 1997 © 1997 Elsevier Science Ltd. All rights reserved

Printed in Great Britain 0043-1354/97 $17.00 + 0.00

ANAEROBIC TREATMENT OF FISHERY WASTEWATER USING A MARINE SEDIMENT

INOCULUM

ESTRELLA ASPIC1@, M. CRISTINA I~IARTf 2 and M A R L E N E ROECKELI*@

~Depto de lngenieria Quimica, Facultad de Ingenieria, Casilla 53-C, Universidad de Concepci6n, Concepci6n, Chile and 2Depto de Farmacologia, Facultad de Ciencias Biol6gicas, Casilla 152-C,

Universidad de Concepci6n, Concepci6n, Chile

(First received September 1996; accepted in revised form February 1997)

Abstract--The effluent generated by the Chilean fishmeal industry during hydraulic unloading of fish from ships is high in organic load. After recycling and primary treatment to remove fats and proteins, the effluent contains 4-6 kg COD m -~ and high salt content (an average of 1.85 kg SO7 m -3 and 16.2 kg C1- m-3). Marine sediments and fresh pig manure were assayed as anaerobic inocula to purify this saline effluent. The marine inoculum adapted better and faster at 37°C, showing a higher final methanogenic/sulphate-reducing bacteria ratio of 0.0025. Specific methanogenic activity at 37°C was 0.065 kg CH4 COD (kg VSS day) -~, corresponding to 1.3 kg COD (kg VSS) -~ in 20 days. Methane production was inhibited at COD/SO7 ratios lower than 0.5. A 50% inhibition of the activity was found at 0.22 kg H2S m -j, 53 kg Na ÷ m -3 and 10 kg SOl= m -3 respectively; however, at the concentration range in the effluent neither was inhibitory. Kinetic parameters were obtained at 18 and 37°C in mixed flow reactors. At those temperatures,/~ values were 0.267 and 0.479 days -t, while K values for the Chen and Hashimoto model were 2.964 and 1,476, and the growth yield factor (Y) was 0.19 kg VSS (kg COD) -~. The activation energies were estimated as 3.06 kJ mol -~ for #m~x and 27.51 kJ tool -~ for K, showing that the saline wastewater treatment has a lower temperature dependence than the non-saline one. It was concluded that, under these conditions of high organic load, fishery effluent can be anaerobically treated. © 1997 Elsevier Science Ltd

Key words--fish processing effluent, saline substrate, anaerobic treatment, kinetic parameters

INTRODUCTION

More than 25 fish meal processing industries are located nearby Concepci6n, a city in Chile with dense industrial activity. Thus, this area has suffered a strong environmental impact due to the discharge of municipal and industrial wastewaters to its bays. Fish processing plants have discharged an average of 55,900 t of organic load (measured as COD) per year, equivalent to the polluting effect of 1.5 million inhabitants (Roeckel and Asp6, 1991).

A thorough characterization of all the effluents involved in the fish processing was carried out (Becerra et al., 1990), and it was shown that the effluent produced during the unloading with seawater of the fish from the ships generated the largest organic load. The typical unloading rate for Chilean fisheries is 100 t fish h -~ with an organic content of the discharged wastewater that ranges from 13 to 39 kg COD t - ' of unloaded fish.

The effluent volume is usually large, rendering it difficult to treat; its flow ranges from 5 to 10 m 3 t - '

*Author to whom all correspondence should be addressed [e-mail: mroeckel@ merlin.diq.udec.cl].

of unloaded fish and the ships loads range from 100 to 1200 t, As shown elsewhere (Roeckel and Asp6, 1991), recirculation from the wharf to the ships not only reduces this flow to 0 .14m 3 per tonne of unloaded fish but also exhibits additional advantages. Fats and proteins can be removed from recirculation (Marti et al., 1994), thus leaving an effluent ready for biological treatment,

Bacterial anaerobic populations are able to remove high organic loads at low investment and operation costs (Lettinga, 1995), so anaerobic digestion was considered the best alternative as a first step of biological treatment. However, the anaerobic treat- ment of marine wastewaters poses several technical problems, such as sulphide and sodium inhibition (Soto et al., 1991; Visser, 1993; Woolard and Irvine, 1995) and requires careful selection of the appropri- ate anaerobic inoculum.

According to the literature, optimal sodium and sulphate concentrations for anaerobic digestion range from 0.l to 0.2 kg m -3 and 1 x 10 -4 to 1 x 10 -3 kg m -a, respectively, while it is moderately inhibited by concentrations ranging from 3.5 to 5.5kg m -3 and 0.1 kg m -3, respectively (Mosey, 1974), At

2147

2148

Table I. Substrate characterization

Parameters Composition

E. Aspe

pH 4.5 COD (kg 02 m -3) 6.0 Total solids (kg m -3) 39-42.0 Nitrogen (kg m 3) 0.54 Ammonium nitrogen (kg m -3) 0.09 Total volatile solids (kg m -3) 6.5 Volatile suspended solids (kg m -a) 0.18 Total suspended solids (kg m -z) 0.28 Phosphate (kg m 3) 0.0080 Sulphate (kg SO4 m -~) 2.40 Alkalinity (kg CaCO3 m -3) 1.2 Salinity (kg Na + m -3) 7-12

10kg Na + m -3, inhibi t ion for suspended biomass culture has been reported (Henze and Harrem6es , 1983); fur thermore, for g ranular and domest ic digested sludge, sodium ions (added as NaC1) at a concen t ra t ion larger than 4.0-10 kg m -3 produces a 50% inhibi t ion (Field et al,, 1995).

Biological t rea tment processes have been success- fully described by the theory o f con t inuous culture of microorganisms. The biological growth kinetics ob ta ined in these type of culture is based on two fundamenta l relat ionships: growth rate and substra te uti l ization rate. Ra te equat ions are used for the design of reactors (Fogler, 1992), since they not only provide a quant i ta t ive descript ion of the rates of waste uti l izat ion but also in format ion abou t the opera t ional and env i ronmenta l factors (Wiesman, 1988; Pavlostat is and Gi ra ldo-Gomez , 1991).

Therefore, the goals o f the present work were to study the feasibility of carrying out anaerobic wastewater t rea tment of this type of effluent by assaying and character iz ing two inocula; to determine possible inhibi t ion by Na ÷, SO~ and H2S of the methanogenic activity; and to est imate the kinetic cons tants for C O D removal.

MATERIALS AND METHODS

Wastewater characterization

Wastewater from one fish unloading process was subjected to primary treatment (Marti et al., 1994) and the resulting effluent characterized (Table 1) and used in all the experimental work related to inocula characterization and kinetic parameter estimation. As the effluent composition changes with the freshness of the fish catch and with the unloaded fish volume, different effluent samples were characterized over a period of 6 months. Also, samples were taken from the process line, in order to have a large range for the eventual anaerobic treatment feeding composition. Table 2 shows the maximum range in composition of these wastewaters after primary treatment to remove fats and proteins.

Table 2. Maximum range for fish industry effluents composition after primary treatment

COD (kg m -3) 5-32 Sulphate (kg SO~- m -3) 1.2-2.5 Total dissolved sulphides (kg S m -3) 0.010-0.100 Salinity (kg CI- m -a) 14.6-17.9 NH4 (kg N m -3) 0.039-1.94 COD/SO 4 1.01-22.28

el al.

Analytical method~"

Effluent characterization. Standard methods were used for all measurements: COD, S0£, Na + (ASTM, 1964); suspended volatile solids, alkalinity, ammonium nitrogen, total sulphides (APHA, 1985): fats (Merck, 1983, p. 101) and total Kjeldahl-proteins (Merck, 1983, p. 33). The intermediate alkalinity (1A) was calculated as the difference between the total alkalinity (TA, bicarbonate plus the volatile fatty acids at pH 4.3) and the partial alkalinity (PA, bicarbonate at pH 5.75).

Microbiological assays. Methanogenic bacteria (MB) were identified using a selective medium (Zeikus and Pfenning, 1987), prepared by E-4, E-5 and E-6 dilutions. Each incubation tube was prepared with 6ml of semi-solid culture media gassed with an 80% CO: and 20% H2 mixture and then inoculated with 0.1 ml of sample. Tubes were incubated for 30 days at 18 or 37 C. Methane was determined by gas chromatography. Biogas composition was measured in a Hach Curie Series 100 gas chromatograph equipped with a 2-m long Spherocarb 100/120 column, at 130~C and He at 10psi as carrier gas. Most probable number (MPN) was used for assessing the viable bacterial population count (Postgate, 1969; APHA. 1980).

Sulphate-reducing bacteria (SRB) viable counts were performed using a selective medium for SRB (Sharma and Hobson, 1987) and preparing dilution series according to the MPN method (Postgate, 1969; APHA, 1980). The tubes were inoculated with 0.1 ml of sample and left for t5 days at 18 or 37C. Activity of SRB was estimated by the presence of H2S, confirmed by a microscopic analysis of bacterial frotis treated with 0.2 M NaOH (Sharma and Hobson, 1987).

Inocula adaptation

Two different inocula were tested; the first was obtained from marine bottom soil (marine sediment) and the second from pig manure, Each inocula (450 ml) was added to five different digesters containing 350 ml of Bryant's culture medium (Bryant et al., 1971). The inocula were kept in a batch system, at 18 or 37'C, until the methanogenic microorganisms attained a high growth rate, as shown by more than 30% methane in the biogas. Adaptation to the high-saline substrate was achieved by a stepwise change of Bryant's culture medium by the substrate, at a rate of 1/30 of the volume per day, from 5 to 100%. Thus, batch mode operation was changed to a semi-continuous mode of operation.

Mixed-flow anaerobic' reactors

Twelve digesters from a working volume of 1.5 litres each were run as well-mixed reactors by magnetic stirring. The digesters were fed with substrate plus nutrients in a ratio of 80:7:1 of COD:N:P (Lema and M~ndez, 1988). The pH (8.0) and the temperature (18 or 37°C) were kept constant. The reactors with 2 g I ~ of MLSW (mixed liquor-suspended volatile solids) were initially operated in a batch mode until the MB attained a stationary state (determined by the methane content in the biogas); the second step was carried out at a continuous mode, with daily feeding with saline wastewater at 18 or 37°C. Hydraulic residence times, and hence solids residence times, ranged from 3 to 30 days.

At each hydraulic residence time the continuous mode to a quasi-stationary state was reached, i.e. after a period of more than three turnovers or space times (Fogler, 1992). In all cases, data were collected at the end of an experimental period of approximately 80 days and when the methane volumetric percentage in the biogas had a 10-12% variation. Once this quasi-steady state was reached, the average COD and sulphate concentration, pH, alkalinity, redox potential and hydraulic retention time were registered as steady-state values.

Anaerobic treatment of fishery wastewater 2149

Table 3. Characterization of the sludge used in inhibition assays

VSS (kg m ~) 9.4 Sulphate (kg SO~ m -~) 0.551 Total dissolved sulphides (kg S m -3) 0.077 Salinity (kg NaCI m -3) 14.7 NH~ (kg N m -3) 0.26 pH 6.3

Substrate exhaustion estimation

In order to determine the maximal methane volume (V0) and the COD recalcitrant to anaerobic degradaion (St), two duplicate batch digesters were set and operated during 4 months at 37°C and pH 7.0. After that period, a residual COD value remained constant thereafter and was considered as the recalcitrant or non-biodegradable portion of the organic substrate. The recalcitrant COD content at 18°C was assumed to be the same as that at 37°C based on literature reports (Chen and Hashimoto, 1980).

Inhibition assays of the methanogenic actirity

The inoculum was started with a mixture of 1 litre of fresh marine sediment, 1.5 litres of Bryant's culture media (Bryant et al., 1971) and 0.5 litres of unloaded effluent previously subjected to primary treatment, i.e. precipitation of protein and fat with ferric chloride, as described elsewhere (Marti et al., 1994). Three days later, methane production was detected and adaptation to the saline substrate was induced by daily substitutions of the mixture by 0.2 litres of effluent until methane content in the gas reached 55%. The resultant sludge (volatile suspended solids (VSS) adjusted to 2 g 1 -~) was used to inoculate 16 reactors (effective volume of 100 ml) containing a volatile acids mixture (Soto et al,, 1993a) and to which either soidum as NaCI, sulphate as Na:SO4 or sulphide as Na2S

was added. Initial concentrations of sodium, sulphate and sulphide in the inoculum were considered as reference values and are shown in Table 3. The amount of these inhibitors added to the substrate were far higher than the reference values, so that the latter were negligible. Pure nitrogen was used for air purging.

RESULTS AND DISCUSSION

Bacterial adaptation and growth

As shown in Table 1 the subst ra te ' s salinity and sulphate content are quite high, since the effluent is mainly seawater; however, as can be seen in Fig. 1 mar ine inoculum adap ta t ion to this medium in the ba tch mode was achieved, as shown by a stable methane conten t of approximate ly 45% in the biogas. The ra ther low methane content in the biogas produced by the pig manure inoculum may be a t t r ibuted to an increase in the SRB popula t ion compet ing for substrates such as hydrogen and acetate (Oreland and Polcin, 1982). The subst ra te anaerobic digestion by the pig manure inoculum steadily increased up to the tenth day, bu t thereafter suffered a fast decline to zero, p robab ly due to salt toxicity levels (Na ÷) or sulphide inhibi t ion (Hil ton G. and Archer , 1988).

It has been shown tha t high sodium concent ra t ions produce inhibi t ion to MB in non-adap ted anaerobic systems (Soto et al., 1993b; Feijoo et al., 1995; Omil et al., 1995) and specifically to aceticlastic M B (Rinzema et al., 1988; Omil et al., 1995). Omil et al. (1995) studied the inhibi tory effect of sodium on M B

60,

50.

o o J~

40- ¢.-

..c

30. 0

~ 20.

0

IV

g 10.

0

45 50 Time (d)

Fig. 1. Time-course of methane volumetric percentage in the biogas produced by batch anaerobic degradation at 37°C carried out by a pig manure inoculum CA) and a marine sediment inoculum (O).

2150 E. Asp6 et al.

11

i 10.

°°' 9"

-6 8. ib

1,,i 0 to 7

Marine Sediment 1|.

"6

u

0 7'

Pig Manure

: -- . . . . . .

0 lb 2b 3b 40 0 3b Time(d) Time(d)

Fig. 2. Bacterial growth at 37~C of a marine sediment inoculum and a pig manure inoculum assessed by the most probable number method (MPN): methanogenic bacteria (O); sulphate-reducing bacteria (11).

4 0

from a digester treating saline wastewater from a seafood processing factory. Volatile fatty acids or acetic acid were used as substrates, and these authors reported a 20% inhibition in methanogenic activity by the addition of 17.5kg Na + m -3, although a non-limiting hydrogen concentration for methane production was present when volatile fatty acids were used as substrate. The sodium concentration range in the fishing effluents used as substrate in this work is within the inhibiting concentrations reported by these authors, so this might explain the decline in methanogenic activity observed in the pig manure inocula.

On the other hand, the SRB population in both inocula exhibited a steady increase, while the methanogenic population increased slightly up to the tenth day and thereafter remained relatively stable and low, as shown in Fig. 2. In the presence of sulphate, competition between sulphate-reducers and the anaerobic bacteria involved in methanogenesis can occur at a number of different levels in the stepwise degradation process. Because of their diverse substrate range, SRB species can compete for substrates against fermentative, syntrophic obligate hydrogen-producing acetogens, homoacetogenic and MB (Colleran et al . , 1995). Besides, free energy values predict that sulphate-reducers should outcompete methanogens for both hydrogen and acetate; furthermore, SRB have a higher affinity for hydrogen than methanogens (Gupta et al. , 1994) and lower threshold values for hydrogen and acetate than methanogens (Colleran et al . , 1995).

According to the literature (Hilton and Archer, 1988; Rinzema and Lettinga, 1988), the inhibition in MB growth is due to H2S production by the SRB; moreover, for a variety of substrates the literature

reports different levels of inhibition of the acetoclastic MB activity by H:S (Colleran et al. , 1995). It has been suggested (Soto et al . , 1991; Visser, 1993) that inhibition by H2S (produced by the reduction of sulphate) is produced at a COD/SO£ ratio lower than 8-10 and that SRB outcompetes the MB due to the presence of SOg. In this regard, the SRB population did not grow as well in the marine sediment inoculum as in the pig manure inoculum, and the MB/SRB ratio decreased at a faster rate in the pig manure inoculum as compared to the marine sediment inoculum, as shown in Fig. 2.

The marine sediment adapted better and faster to the saline substrate; thus, after 40 days a higher ratio of MB/SRB was observed. The initial ratios were 0.10 and 0.0426 for marine sediment and pig manure inocula, respectively, and their ratios after the 40 day period were 0.0025 and 0.000125, respectively. Although it may be presumed that the marine MB and SRB populations were already adapted to a saline environment and their ratio was already stabilized in seawater, the SRB population outcom- peted the MB population when they were exposed to high concentrations of organic matter, in agreement with research carried out with intertidal sediments that demonstrated a strong dominance of the SRB (Winfrey and Ward, 1983). Hence, the marine sediment inoculum was chosen to carry out the substrate anaerobic degradation experiments.

Figure 3 shows the average methane production and illustrates COD degradation of industrial wastewater in all 12 digesters inoculated with identical marine sediment. Also shown is the methane content in the biogas and the standard deviation from the average value. The maximal standard deviation was 9%, indicating the process reproducibility and

Anaerobic treatment of fishery wastewater 2151

the inoculum stability. Methane production and COD reduction reached a stable level after 15 days which remained after 30 days of operation. The specific methanogenic activity at 37°C was 0.065 kg CH4 COD (kg VSS day) -t, corresponding to 1.3 kg COD (kg VSS) -~ in 20 days. According to Monroy (1995), inocula from natural sources (lake sediment, fresh pig manure, septic tank sludge) exhibit low specific methanogenic activity, which ranges from 0.01 to 0.2 kg COD (kg VSS) -~, while granular sludge and anaerobic filter biofilms specific methanogenic activity ranges from 0.4 to 1.5 kg COD (kg VSS)-~; thus, the marine inoculum specific methanogenic activity is within the usual range for natural source inocula.

Figure 4 shows that, at up to 30 days of operation of the batch digesters, the viable count ratio for MB and SRB was practically constant, while the COD/SO~ ratio increased. These results were expected, as SO~ consumption by SRB is thermo- dynamically and kinetically favored over COD consumption by MB (Visser, 1993). The alkalinity and the pH increased in this period from 1.2 to 3.0 kg CaCO3 m -3 and from 4.5 to 8.5, respectively. Although the COD/SO~ ratio exhibited values well below 10, no evidence of sulphate toxic effect on the MB/SRB ratio was observed; this result was later confirmed by experiments described below.

Effect o f inhibitors

Figure 5 shows methane production variation at different sodium concentrations; as shown, at up to

150h of reaction, there were no appreciable differences between methane production capacity and therefore the methanogenic activity was estimated after that period. At 20.8 kg Na m -3 there was a 47% drop in the methanogenic activity, and at 60 kg Na m -3 a 50% inhibition was observed. Considering that sodium concentration in fishery effluents usually ranges from 7 to 12 kg m -3, a 15-25% inhibition of the methanogenesis should have been present from the start.

Although adaptation of methane bacteria to high levels of salt has not been reliably demonstrated (Field et al., 1995), some experiments with these kind of effluents have shown that adaptation is possible if the etfluent's saline concentration is similar to the inoculum sludge salinity (Soto et al., 1993b). Thus, bacterial adaptation might occur after a long-term operation of a fixed-bed reactor fed with fish unloading effluents.

Figure 6A shows methane batch production by marine sediment inocula at different initial sulphate concentrations at pH 8.0, i.e. a pH close to the one reached at steady state in mixed flow reactors. Clearly, after 150 h of operation, the methanogenic activity was affected by the sulphate initial content. The activity of the reference (inoculum without the addition of any possible inhibitor and with a basal sulphate concentration of 0.1 kg SO; m -3) was slightly smaller than activities for 0.6 kg SO2 m-3; however, this difference may be attributed to the experimental error of the method. Figure 6B shows the effect of increasing

7O

- 50

c 40

3 o

, o

1.4

1.2

X

o: 1.o

~ 0.8

~ 0.6

~ 0.4

U 0.2 ,<

0 . 0

7

5 ~ c

i, 2f,

: l : I

0 10 20 30 40 T ime(d )

Fig. 3. Methane volumetric percentage in the biogas (&), accumulated methane production (O) and the effluent COD content ([]) versus time. Mean values were obtained from 12 identical anaerobic batch

reactors at 37°C. Vertical bars represent standard errors.

2152 E. Asp6 et al.

5 "

o~ .~ /+. v

.2 t .

c~ 3"

8 u 2~

14

~ 0,5 ,- ,

z o. 3[

3£

m

- 0,2

-0.1

0

T i m e ( d )

Fig. 4. Time-course of operation parameters: methanogenic/sulphate-reducing bacterial ratio (©) and COD/SO; ratio (0) of batch reactors at 37cC.

sulphate concentrations on the methanogenic ac- tivity at pH 7.0.

Sulphate in effluents causes inhibition problems during its anaerobic treatment due to sulphide generation by the biological reduction of S04; the

un-ionized form of sulphide of free H:S is toxic to bacteria (Visser e t al . , 1993). The limit of sulphide concentration that causes inhibition for granular sludge is 0.1 kg S m-3; while 0.14 kg S m -~ causes a 50% decrease of the COD conversion rates (Rinzema

100 -

80.

i 60. o~

40"

70- ~ 20. O. i i i i i i

20 40 60 80 tO0 120 60- Sodium conCentralionlkg/m 3 }

50 0

E -~ 4 0 >

t -

O 30-

20-

10-

0 - 0 16o z6o

Time(h)

[No +]

1.15 kg n~3 (tel.)

3.10 kgnT 3

20.8 kgnT 3

119.2 k g n,T3

360

Fig. 5. Time-course of the volumetric methane production by marine inoculum at different initial sodium concentrations. Insert: methanogenic activity at different sodium concentrations as compared to a

reference.

120.

100.

E " ; 8o.

60,

20-

Anaerobic treatment of fishery wastewater 2153

A : Sulphotle inhibition at pH=8

7; 1~0

g ~

o 5o

[so~ ] 120, ii..~ a Sulphote mhtbdi~olpH=)O

2.0 kg~ 3

lOO. o.~ kg,~ 3

[so~l

~ . 8 0 Sulphate ¢oncentrotion {kgl

10.1 k g r ~ 3 ~ . 40-

soJ kg~ ) 20-

0 " - . i i i i i i

100 %50 200 250 300 350 400 450 50 100 1S0 200 250 300 350 400 Timelh) Time(h)

Fig. 6. Time-course of the volumetric methane production by marine inoculum at different initial sulphate concentrations: (A) at pH 8 and (B) at pH 7. Inserts: methanogenic activity at different sulphate

concentrations as compared to a reference.

and Lettinga, 1988) and 0.25 kg S m -3 causes a 50% inhibit ion of the methanogenic activity (Field et al., 1995). On the other hand, sulphate is relatively non-toxic, 3.3 kg S m -3 being the 50% inhibitory SO4 concentrat ion for aceticlastic bacteria (Field et al., 1995). The equilibrium between the undissociated hydrogen sulphide (free H2S), the bisulphide ion and the sulphide ion is very much controlled by the medium pH. At neutral pH, the H2S/HS-/S = ratio is 1.1/1/10 -5 and shifts towards the dissociated forms (pK~ = 7.04, pK2 = 11.96) at increasing pH values (Van Gemerden and De Wit, 1986); thus, at the working pH of 8.0, inhibition by H2S should be less than at pH 7.0.

Table 4 summarizes values of the methanogenic activity and its relation to the COD/SO; ratio at each sulphate concentration. As shown, there was no appreciable inhibition of the methanogenic activity, even for COD/SO7 values well below 10; in fact, the activity for a COD/SO; ratio of 1.8 was almost the same as the reference activity. Activity values and the corresponding COD/SO; ratio, and the effect of the COD/SO; ratio on the methanogenic activity, were as follows: a 56% inhibition was found at a

COD/SO4 ratio of 0.37 and activity was almost completely inhibited at ratios smaller than 0.37. By interpolating the data, a 50% inhibition of the methanogenic activity was found for a COD/SO~ ratio of approximately 0.43. These inhibitory ratio values are considerably smaller than those reported in the literature (M6ndez et al., 1989; Visser, 1993) and might be explained by the previous adaptat ion of the inoculum to the saline substrate.

It can also be shown that the intrinsic SO~ concentration affects the degree of inhibition of the methanogenic activity, as for low sulphate concen- trations (0.6 or 2.1 kg m -3) no inhibition was observed even at COD/SO; ~ ratios lower than 10 (6.1 and 1.8). As sulphate was added as sodium sulphate, only the joint effect of sodium and sulphate could be determined from the aforementioned experiences; but, as shown in Table 4, the intrinsic sodium toxicity was also determined by extrapolating the degree of inhibition produced by the same concentration of sodium added as NaC1. The metbanogenic activity at a sulphate concentration of 2.1kg SO~ m 3 was similar to the reference; nevertheless, the sodium content should produce a

Table 4. Influence of the sulphate concentration on the methanogenic activity of marine sediment inoculum Methanogenic Inhibition Na + added Inhibition activity [SO;'] by [SO;] COD~/SO; as sulphate by Na ~ Na/SO; (kg COD (kg VSS day)-') (kg m -a) (%) (kg kg ~) (kg m =3) (%) (kg kg ~) 0.096 0.1 ~ ~0 34 1.15 ~0 11.5 0.102 0.6 t0 6.1 1.39 ~0 2.31 0.100 2.1 ~0 1.8 2.1 ~5 1.0 0.045 10.1 56 0.37 5.9 20 0.59 0.0126 50.1 88 0.07 25.1 40 0.5 =Acetic acid, propionic acid and butyric acid equal to 3.75 kg COD m-L bDctermined from Fig. 5. ~Reference.

2154

100- i

90-

80-

E. Asp6 et al.

70- E

60. E ~ 5 0 .

4o.

:~ 30.

20- i

10-

O ~ o

A: Sulphide inhd)ltmn ol pH.8

loo [S "] 100.

~, 60 90. 0.020 k 9 m -3

~ o (tef.) 80. o , , , ,

o 1 l 3 ~. s __~ 70.

g ® 60.

o so.

0.22 kg,,r] ~ ~0.

5.02 kg m'3 Z 30- 1.02 kg rn'3

20.

10.

ibo 260 36o 4~o ~ o Time(h)

B S u l P h i d e mhlb~tlol~ a! ph i7

,.o[ / o oo... (ref.)

20 • ~ o l 2 . . . ; /

0 I 2 3 1. 5

0.22 k g ~ 3

1.02 kgn~ 3 5.02 kgrff 3

50 100 150 200 250 300 350 400 Time(h)

Fig. 7. Time-course of the volumetric methane production by marine inoculum at different initial sulphide concentrations: (A) at pH 8 and (B) at pH 7. Inserts: methanogenic activity at different sulphide

concentrations as compared to a reference.

reduction of approximately 5% of the activity, as shown in Fig. 5. This fact has not been reported in the literature and might be ascribed to the experimental error range. By comparing the inhibitory effects of sulphate at 10.1 and 50.1 kg SO; m -3 and of sodium at 5.9 and 25.1 kg m -3 at similar Na+/SO~ ratios (0.59 and 0.50, respectively), it can be concluded that the sulphate inhibitory effect is larger than the sodium one.

Figure 7A shows the toxic effect of different total sulphide concentrations at pH8.0 on the displaced methane volume and the corresponding methanogenic activity. Methanogenic activity de- creased more than 50% at low initial sulphide concentrations (0.22 kg S m 3); these results are in agreement with those reported in the literature (Hilton B. L. and Oleszkiewicz, 1988; Field et al . ,

1995). Figure 7B shows the effect of increasing sulphide concentrations on the methanogenic ac- tivity at pH 7.0; as shown, a lower inhibition was observed at pH 8.0 than at pH 7.0, due to the shift in equilibrium towards the sulphide ionized forms.

It may be assumed that the sulphide concen- tration in the reactor was maintained almost constant, in spite of the interference occurring as a result of the sulphate reduction reaction going on at the same time. In fact, if the reference's sulphate content had been totally converted to sulphide by reduction at pH 8, it would have given rise to 0.034kg S m 3, and the interference occurring as a result of the sulphate reduction reaction to sulphide should be approximately 15% when 0.2 kg S m -3 was added and negligible when

1.0 and 5.0kg S m -3 were used as inhibitory concentrations.

It has been reported (Hilton B. L. and Oleszkiewicz, 1988) that hydrogen sulphide is not only toxic for MB but also produces a similar effect on SRB. At certain H2S concentrations this effect could explain why SRB do not outcompete methanogenic activity and why sulphide (from the reduction of sulphate) does not inhibit at COD/ SO~ ratios larger than 8.0 (Soto et al . , 1993b; M6ndez et al . , 1989).

Table 2 shows typical values of SO~ and S- concentrations and COD/SO~ ratios in the pre- treated effluent from fishing factories. According to the present results, it can be assumed that the methanogenic activity will not be inhibited by sulphur at these values. Furthermore, the primary treatment of the effluent involves flocculation with ferric chloride (Martl et al. , 1994) and it has been shown that SRB are limited by the concentration of soluble iron ions, since the latter precipitate with sulphide (Van Gemerden and De Wit, 1986). Therefore, the eflluent's pretreatment should lower the amount of H2S and prevent its toxic effect in the reactor.

A lower methanogenic activity when using the industrial substrate as compared to methanogenic activity using a standard acid mix- ture was observed. This may be due to the fact that the industrial effluent must undergo hydrolysis and acidogenesis prior to methanogenesis. In the overall process the kinetic parameters of the acidogenic and methanogenic steps can be in the same order of magnitude, while the hydrolysis step can be rate limiting, thus explaining the

Anaerobic treatment of fishery wastewater 2155

observed differences; moreover, ammonia inhi- bition could occur due to protein degradation (Henze and Harrem6es, 1983). These effects are presently under study.

Kinetic parameters estimation

Operation of the mixed-flow reactors was fol- lowed at hydraulic residence times (HRTs) of 3, 5, 10, 15, 20 and 30 days and of 5, 15, 20 and 30 days at 18 and 37°C, respectively. Figure 8 shows the reactor behaviour and quasi-steady-state attain- ment assessed by methane content after at least three space time periods had elapsed. The redox potential stayed below - 3 0 0 m V , thus ensuring methanogenic bacterial growth and activity (Switzembaum, 1990). The assessment of the intermediate alkalinity was less than 30% of the

total alkalinity, indicating the operational stability of the reactors,

The concentration of substrate recalcitrant (S,) to anaerobic degradation at 37°C was 1.36kg COD 02 m -3, which represents approximately 30% of the total substrate concentration. The growth yield factor was 0.19 kg VSS (kg COD) -~ at both temperatures, as determined experimentally from batch experiments.

The effect of the growth limiting substrate (S) concentration on the rate of microbial specific growth (#) in anaerobic digestion has been de- scribed by various mathematical models. These models are based on the assumption that there is a linear relationship between the VSS concentration and either substrate or COD consumption; that a perfectly well mixed continuous flow system with-

A: 3 cloys of HRT B : 5 doys of HRT

• ~ ~.~u-~'"-.~ 't~'-.n~ ,o SO I~0

50 • 40 ~ 40

. 2 0 ' { ! , o l k ,o 10 : ~ o ~ ~ - . . . . . . . , .o - . ' , , . . , o

0 20 40 $0 |0 0 20 40 60. |0

C : 10 days of HRT D: 15 days of HRT

o- :t 4O

, 0

• 2 0 2O

, ' 1 ! i i

20 40 eo Io i0 io io ~ 0 0 80

E: 20 days of HRT F= 30 days of HRT 7o 7o

, o

= 3 0

'o ° , ~ _ , ,,,

I'IMEId) I'IMEIdl

Fig. 8. Time-course of methane volumetric percentage in the biogas produced by methanogenic bacteria from marine sediment inoculum and quasi-steady-state achievement at 18°C ([::1) and 37°C (A): (A) for a 3-day hydraulic residence time (HRT), (B) for 5-day HRT, (C) for 10-day HRT, (D) for 15-day HRT,

(E) for 20-day HRT and (F) for 30-day HRT.

W R 3 1 , 9 - B

2156 E. Asp6 et al.

Table 5. Kinetic models used in anaerobic treatment (adapted from Pavlostatis and Giraldo-Gomez, 1991y Specific growth Substrate Steady-state mass balance for

Model rate consumption rate mixed-flow reactor

First order

Monod

Contois

Chen and Hashimoto

k S dS So + bO ~ = ~d~--~ - b - ~7 = k s S = 1 + k----ff

_ ~ _ d S ,u~.~XS b K,(1 + bO) It - K~ + S b - d~ = Y(K, + S) - Y S = o ( i t ~ _ b) _ 1

dS I ~ X S b S B Y ( I + bO) _ ~_Y_z~__ b . . . . .

I ~ - B X + S dt Y ( B X + S ) Y if0 = 0(p,~ - b) - I +BY(I +bO)

t t~xS - b d S Itma.XS b S K(I + bO) I ~ = B Y ( S o - S ) + S - - d T = K X + Y S - Y ~ = O ( t ~ + K - I ) ( l + b O )

or

~maxS I~ = K(So - S ) + S - b

~k, maximum specific substrate utilization rate (day-0; S, biodegradable substrate concentration (kg m-a), in the Chen and Hashimoto model calculated as the difference between total substrate concentration (Sto) in the influent and the recalcitrant substrate concentration (St); So, influent biodegradable substrate concentration (kg m-~), in the Chen and Hashimoto model calculated as the difference between the influent total substrate concentration (Sto) and the recalcitrant substrate concentration (S,); b, specific microorganism decay rate (day-0;/~a~, bacterial maximum specific growth rate (day-~); K~ = half-saturation constant (kg substrate m-0 for the Monod model (Wiesman, 1988); B, Contois parameter (mass of consumed substrate per mass of produced cells, kg kg -)) (Contois, 1959); K, dimensionless parameter, equal to YB in the Chen and Hashimoto model (Chen and Hasbimoto, 1980).

out solids recycle is used; that the influent is devoid of microorganisms; and that a constant ratio between cell mass production and substrate consumption exists. Table 5 shows the expressions for the first-order, Monod, Contois, and Chen and Hashi- moto models; these expressions were adapted from Pavlostatis and Giraldo-Gomez (1991).

In these models, the specific growth rate is described by the following general equation:

1 dX ;' = ~ - d 7 (1)

where # is the bacterial specific growth rate (day-~), X is the microorganism concentration (kg m-3), and t is time (days).

Table 5 also shows the substrate utilization rate ( d S / d t ) by applying a constant growth yield factor (Y) to the Monod, Contois and Chen and Hashimoto models:

dX [ X - X0] dt

Y = [So - S] - dS (2)

dt

where Y is the growth yield coefficient (mass of new ceils formed per mass of consumed substrate, kg kg -)) and X0 = 0 (influent biomass concentration, kg VSS m-3).

Then, solving for d S / d t and replacing equation (1) in equation (2)

dX dS dt ~X (3) dt Y Y

which is shown in the second column of Table 5.

By applying a mass balance for the biomass at steady-state operation of a continuous culture in a mixed-flow reactor and by neglecting the influent microorganism concentration, the following equation is obtained:

1 /~ = ~ (4)

where 0 is space time (days), defined as in chemical reactor design (Fogler, 1992).

Values for the concentration of the growth-limiting substrate (S) in the effluent were estimated by applying the model equations at steady-state oper- ation conditions. The third column of Table 5 was obtained by replacing the specific growth rate (/l) by 1/0 in equation (4) and solving for the substrate concentration (S) in the reactor.

All the above-mentioned models were used to fit the experimental data by rearranging the equations to linear ones. As shown in Table 6, the only model that fitted the experimental data was the Chen and Hashimoto (1980) kinetic model; the other models gave negative values for the biological constants. The fitting by the Chen and Hashimoto model was possible by assuming that the endogenous respiration or cell maintenance (represented by the specific microorganism decay ratio) was zero and that a portion of the substrate (recalcitrant, St) was not consumed by the microorganisms.

The resulting values for /tm~x were 0.267 and 0.479 day -~ and for Kwere 2.964 and 1.476 at 18 and 37°C, respectively. These values are within the range of those reported by Chen and Hashimoto for anaerobic digestion (Chen and Hashimoto, 1980). Minimum 0 values, corresponding to the average washout times at 18 and 37°C, were 3.75 and 2.09

A n a e r o b i c t r e a t m e n t o f f i s h e r y w a s t e w a t e r

T a b l e 6. E x p e r i m e n t a l d a t a f i t t ing w i t h d i f fe ren t k ine t i c m o d e l s

2 1 5 7

E q u a t i o n r" #m~ K~ K B b

At 37 C: M o n o d w i t h o u t m a i n t e n a n c e 0 .9647 - 0 . 0 3 1 3 8 - 4 . 3 5 3 9 - - - - - - W i t h o u t M o n o d w i t h m a i n t e n a n c e S 0 .9647 - 0 . 0 3 1 3 8 - 4 . 3 5 3 9 - - - - 0 s u b t r a c t i n g M o n o d wi th m a i n t e n a n c e b 0 .9647 - 0 . 0 3 1 3 8 - 4 . 3 5 3 9 - - - - 0 r e c a l c i t r a n t subs t r a t e M o n o d w i t h o u t m a i n t e n a n c e 0 .9431 - 1 . 0 6 6 0 - 9 . 4 2 5 0 - - - - - - S u b t r a c t i n g M o n o d w i t h m a i n t e n a n c e ~ 0 .9431 - 1 . 0 6 6 0 - 9 . 4 2 5 0 - - - - 0 r e c a l c i t r a n t M o n o d wi th m a i n t e n a n c e ~ 0 .7330 0 .5527 1.1435 - - - - 0 .1017 subs t r a t e ~ C o n t o i s w i t h o u t m a i n t e n a n c e 0 .9654 - 0 . 1 0 0 3 - - - 2 . 2 9 6 2 - 1 2 . 0 8 5 - - C o n t o i s w i t h m a i n t e n a n c e ~ 0 .9654 0 .8997 - - - 2 .1062 - 11.085 0 C o n t o i s w i t h m a i n t e n a n c e ~ 0 .8793 0 .9196 - - 1 .2820 6 .7474 0 .2856 C h e n a n d H a s h i m o t o

w i t h o u t m a i n t e n a n c e 0 .9431 0 .4793 - - 1.4761 7 .7690 - - C h e n a n d H a s h i m o t o

wi th m a i n t e n a n c e ~ 0 .9431 1.4793 - - 1.6661 8 .7690 0 At 18 C: M o n o d w i t h o u t m a i n t e n a n c e 0 .9122 - 0 . 1 3 1 2 - 4 . 5 8 7 8 . . . . . W i t h o u t M o n o d w i t h m a i n t e n a n c e ~ 0 .9122 - 0 . 1 3 1 2 - 4 . 5 8 7 8 - - - - 0 s u b t r a c t i n g M o n o d w i t h m a i n t e n a n c e b 0 .9669 - 0 . 0 3 3 8 - 4 . 5 8 7 8 - - - - 0 r e c a l c i t r a n t subs t r a t e M o n o d w i t h o u t m a i n t e n a n c e 0 .9122 - 0 . 1 3 6 2 - 4 . 5 8 7 8 - - - - - - S u b t r a c t i n g M o n o d w i t h m a i n t e n a n c e ~ 0 .9122 - 0 . 1 3 6 2 - 4 . 5 8 7 8 - - - - 0 r eca l c i t r an t M o n o d w i t h m a i n t e n a n c e b 0 .9122 - 0 . 1 3 6 2 - 4 . 5 8 7 8 - - - - 0 subs t r a t e c C o n t o i s w i t h o u t m a i n t e n a n c e 0 .9647 - 2 . 9 9 4 8 - - - 9 4 . 4 3 - 4 9 7 - - C o n t o i s w i t h m a i n t e n a n c e ~ 0 .9647 - 2 . 9 9 4 8 - - - 9 4 . 4 3 - 4 9 7 0 C o n t o i s w i th m a i n t e n a n c e h 0 .9647 - 2 .9948 - - - 94 .43 - 497 0 C h e n a n d H a s h i m o t o

w i t h o u t m a i n t e n a n c e 0 .9122 0 . 2 6 7 6 - - 2 .964 15.6 --- C h e n a n d H a s h i m o t o

w i t h m a i n t e n a n c e ~ 0 .9431 0 . 2 6 7 6 - - 2 .964 15.6 0

~Vary ing b (>_0) so t h a t r : a p p r o a c h e s 1.000. W a r y i n g b (>_0) so t h a t /~m~, r e sembles t h a t o f C h e n a n d H a s h i m o t o (1980) . ~Al though M o n o d ' s m o d e l does n o t c o n s i d e r s u b t r a c t i o n o f t he r e c a l c i t r a n t subs t r a t e , th is poss ib i l i ty was a l so exp lo red .

days, respectively. Figure 9 shows the predicted and observed reduction in residual biodegradable COD (kg COD m -~ day -~) under steady-state conditions as the HRT was increased.

The Contois and the Chen and Hashimoto models have been used quite extensively to account for the effect of the influent substrate concentration on the effluent quality (Pavlostatis and Giraldo- Gomez, 1991); however, the Contois model is essentially similar to the Monod model, except that K, is considered as a variable. Although none of the models consider individual reaction rates of the digestion process (hydrolysis, fermentation, anaero-

bic oxidation, aceticlastic methanogenesis and re- ductive methanogenesis), it must be pointed out that a large number of studies have assumed methanogenesis to be a one-step process. A summary of kinetic data using one-step processes has been compiled by Henze and Harremoes (1983); based on an extensive literature review, they proposed kinetic values representatives of the acid phase and of the methane phase of the digestion. Thus, for the overall and for the methanogenic step, they reported a #m~x value of 0.4 day t at 35°C, in accordance with the above-mentioned kinetic parameter estimation. In addition, the

1.2-

O UI

UI

1 . 0 -

0.8-

0.6-

0.4-

0.2-

0 I I I ! / !

5 10 15 20 25 30 TRH |d)

Fig. 9. Predicted values using the Chen and Hashimoto (1980) equation ( ) and experimental substrate concentrations in the effluent at different hydraulic residence times, at 37°C (O) and at 18~C (×) ,

2158 E. Asp6 et al.

Table 7. Comparison of the activation energy for mesophiles

Temperature range E.

Basis Rate (C) (kJ mol ~) Ref.

Biodegr. partic, organics Hydrolysis rate constant (day ~) 20-25 100.59 Gujer and Zehnder (1983) Butyrate~acetate ,u (h ~) 35-55 89.26 Lier (1993) Methanobacteriun arboriphilus CH4 activity 20-35 80 .46 Lubberding and Stares (1995) Methanothrix soehngenii CH, activity 25-38 71 .33 Lubberding and Stares (1995) Anaerobic fixed film reactor Specific substrate utilization rate 10-30 71.26 Henze and Harrem6es (1983) Digesting sludge (adapted) CH, activity 15-25 65 .34 Lubberding and Stams (1995) Sewage sludge /~m~ 20-35 52.62 Chen and Hashimoto (1980) Digesting sludge CH~ activity 15-30 50 .75 Lubberding and Stams (1995) (not adapted) Biodegr. partic, organics Hydrolysis rate constant (day ~) 20-30 50.69 Gujer and Zehnder (1983) Granular sludge Acetotrophic activity 36~6 44.91 Lier (1993)

(g acetate COD g-~ VSS day -~) Acetate--,methane ~ (M. thrix) (h ~) 35 55 43~22 Lier (1993) Propionate~acetate u (h ~ ) 35-55 41,98 Lier (1993) H2/CO2~methane It (M. sarcine) (h -~) 35-55 40.25 Lier (1993) Sewage sludge K (inverse relationship) 20-35 32.49 Chen and Hashimoto (1980) Anaerobic fixed film reactor Organic load rate (OLR) 18 37 (T > 10) 30.88 Lema et al. (1992)

(kg COD day ~) Lake sediments (adapted) CH4 activity 15-30 28 .23 Lubberding and Stams (1995) Eseherichia coli #m~ 26-37 16.20 Pirt (1975) Klebsiella aerogenes ~mox 20-40 14.23 Pirt (1975) Aspergillus nidulans ~amax 20-37 14.00 Pirt (1975) Marine sediment ,Um,x 18--37 23.06 The present work Marine sediment K (inverse relationship) 18-37 27.51 The present work

growth yield coefficient at 35°C reported by Henze and Harremoes (1983) is 0.18 kg VSS (kg COD) -~, which is very close to the value obtained in the present experimental work.

Experimental values show that p increases and K decreases with an increase in temperature, suggesting higher substrate utilization rates as the temperature is raised. Temperature is one of the main environ- mental factors affecting the reaction rate in anaerobic processes (Henze and Harremoes, 1983); thus, it will determine the H R T and hence the reactor size. According to Moser (1981) the constant rates are dependent on the temperature and the water activity (material moisture). By assuming the same water activity for cells and reacting volumes, an Arrhenius type o f relationship between kinetic growth constants and temperature can be applied (Moser, 1981):

E, k = k0 exp - ~-~ (5)

where k is a kinetic constant, k0 is a frequency factor, E, is the activation energy (J mol-~), R is a gas constant, 8.31 (J mol -~ K - j ) and T is the reaction temperature (K).

The activation energy for the saline substrate anaerobic digestion between 18 and 37°C was estimated using equation (5). The same equation was used for calculating activation energies from data reported in the literature for other processes carried out in the mesophilic range (references in Table 6) as a way of comparison with the activation energy for the process described in this work. Equation (5) was also used to describe the temperature dependence of the methanogenic activity and the substrate con- sumption rate by assuming that the yield factors are constant; that for both temperatures other environ-

mental conditions remained constant; and that the methanogenic activity and the substrate consumption rate are only dependent on the temperature.

As shown in Table 7, the saline wastewater digestion has a lower temperature dependence than most reported rate values but a similar dependence as that of the one reported for adapted lake sediment inoculum; thus, i f the temperature is raised from 20 to 30°C, the rate for methane production by adapted digesting sludge increases 2.43 times (Ea = 65.34 kJ mol -~) and the rate for substrate digestion by lake sediment and marine sediment increases 1.46 times (E~ = 28.23) and 1.37 times (Ea = 23.06), respectively. On the other hand, as shown in Table 7, the aerobic bacteria growth rate exhibits a lower temperature dependence than anaerobic digestion processes; thus, the rise in temperature from 20 to 30°C increases the reaction rate 1.21 times for Klebsiel la aerogenes

(E~ = 14.23 kJ mol-~). According to the present results, anaerobic

treatment o f a halophilic substrate can be carried out. Acceptable kinetic correlations may be obtained from the tested conditions. Although fitting of the experimental data by the Chen and Hashimoto model does not imply that the individual steps involved in the process could be accurately described by it, this fitting is useful for the reactor design and to compare the rate o f this process with other anaerobic treatments carried out with different inocula. As shown by the calculated free energy, the influence of the temperature on saline wastewater treatment is less important than in normal waters.

Acknowledgements--The authors are indebted to Mr Oscar Monroy (UAM, M6xico) for his valuable revision. This work was possible through grants FONDECYT (No. 1951004) and UNIDO (No. US CHI 93120).

Anaerobic treatment

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