A quacultural Engineering 6 ( 1987) 79-96

Circadian Periodicity of Biological Oxidation Under Three Different Operational Conditions G. Kriiner and H. Rosenthal
Biologische Anstalt Helgoland, Zentrale Hamburg, Notkestr. 31, 2000 Hamburg 52, FRG

ABSTRACT Daily cycles of biological oxidation efficiency were studied in three different biofihers: high-load trickling filter A, 64 kg fish m -s, B I O . N E T ~ material (Norddeutsche Seekabelwerke), 260 m 2 active surface area per m 3 volume; low-load trickling filter B, 1"2 kg fish m -s, lower section Hydropak*-foil (Friedrich Uhde GmbH), 200 m 2 m -3, upper section BIO-NET material, 260 m 2 m - S ; and low-load submerged rotating contactor (SRC), BIO-NET material, 380 m 2 m -3. The dissolved BOD 5 removal efficiency of trickling flter A was dependent on the p H and on the space-load of organic matter. The total ammonia-nitrogen oxidation efficiency decreased directly after feeding from 60% to just over 20% and returned 4 h later to the earlier oxidationrate of 60% fluctuating between 60% and 30% (initial total ammonia-nitrogen concentrations ranged between 0"78 and 2"89 mg N litre-1 8 h after feeding). This decreasing efficiency was caused by an increasing initial carbonaceous (BOD) level from 4 to 20 mg 02 litre- 1. In the low-load trickling filter B the total ammonia oxidation efficiency ranged between 35% early in the morning to 60%, 10 h after the first feeding. The removal efficiency in the SRC increased constantly from nearly 2% to more than 40% 7 h after the first feeding and decreased during night time to values of about 4%. The degradation efficiency of total nitrogen in both trickling filters fell drastically after feeding (from 75 to 23% and from 88 to 42%). The SRC showed a relatively constant increase from 28% directly after feeding to 58% 7 h later.

INTRODUCTION T h e removal of dissolved organic substances and simultaneous nitrification are important processes in the purification of recirculated fish 79 .Aquacultural Engineering 0144-8609/87/S03.50-- Elsevier Applied Science Publishers Ltd. England. 1987. Printed in Great Britain

80

G. Kriiner, H. Rosenthal

culture systems using biofilters as treatment units (Meade, 1974: Liao and Mayo, 1974; Kriiner and Rosenthal, 1983; Rosenthal et al., 1984; Klemetson and Cohen, 1984). In recent years at lest five types of biofilm reactor have been used for biological wastewater treatment (Rittmann, 1982). There are several advantages in using biofilters that are filled with plastic media in a fish culture recirculating system: (a) Clogging problems within the medium are minimal because of the shearing and hydraulic forces. (b) Aeration is easily and independently controlled by separate .aeration. (c) Most of the organisms are attached to the filter matrix as biomass; they are not easily washed out of the system during a hydraulic surge. Three potential metabolic sources of ammonia exist in biological treatment systems: (1) deamination of organic nitrogen compounds, (2) formation of ammonia from endogenous respiration and (3) release of ammonia by cell lysis (Painter, 1975). The adsorbed organic substances such as carbohydrates, fats and proteins are enzymatically sprit into small moieties. They are oxidized and accessible to the growth of newly formed bacterial cells. Intermediate products are converted to ammonia and acids through protein hydrolysis. The microbiological reactions result in additional microbial biomass, oxidation products containing smaller carbon chains, and carbon dioxide. Adams and Eckenfelder (1977) pointed out that the rate-limiting step in the nitrification reaction is the conversion of ammonia to nitrite by bacteria belonging to the Nitrosomonas group. It is well established that water quality parameters fluctuate considerably during day time in fish culture systems. These fluctuations may reach amplitudes as high as 200-400% above the baseline concentrations determined in morning samples. Maximum peaks may reach critical levels for ammonia and nitrite several times a day (Rosenthal et al., 1980, 1982, 1984; H6ner, 1984). Detailed knowledge of the shortterm variability of the most important water quality parameters is required in order to optimize operational procedures for fish culture recycling systems. Bio-technological procedures which are able to stabilize the performance of a biofilter can only be properly designed if: (a) the reactions of such filters to variations in water quality parameters are known, and (b) it is possible to predict the frequency and amplitude of these fluctuations under given culture conditions (i.e. defined biomass; system volume).

Circadian periodicity of biological oxidation

81

Industrial, domestic and fish culture wastewaters are complex in their composition, containing particulate organic matter, dissolved organics and inorganic soluble components. In fish culture recycling systems, the organic waste components include mainly dissolved, colloidal and suspended matter, and their removal depends therefore not only on the efficiency of the biological treatment process but also on the effective mechanical separation of the suspended matter (Hilge and Rakelmann, 1984). Since dissolved substances are mainly transported by diffusion into the biologically active bacterial film coveting the biofilter substrate, the suspended matter will be partially adhered to the surface of the film, degraded there or being overgrown by bacteria. Commercially available plastic media, which are deployed as conventional substrates in biofilters, provide a suitable surface for bacterial attachment while the wastewater flows over it in a thin film. The surface area of slime varies with the type, distribution and packing characteristics of the filter medium and therefore the available slime surface is considered to be proportional to the specific surface of the medium (Gromiec et al., 1972). Removal rates measured on samples taken from the biofilter inlet and outlet must be carried out on a system where complete control of all parameters that influence the process is possible. In the present study, two trickling filters and a submerged rotating contactor (SRC), the latter being a prime example of a completely mixed biofilm reactor, were used to generate empirical data on the effect of fluctuating load on biofilter performance in two different recycling systems.

MATERIALS AND METHODS Two trickling filters equipped with different substrate combinations and a submerged rotating contactor (SRC) served as the technical units of the experiments (Table 1 ). Trickling filter A was equipped with BIO-NET ~ material (Norddeutsche Seekabelwerke AG, Nordenhamm). Trickling filter B was filled (lower section) with Hydropak*-foil (Friedrich Uhde GmbH, Dortmund) and with the BIO-NET material (upper section). Wastewater supplies to trickling filters A and B were provided by a rotary motordriven sprinkler system, operating at constant turning speed. The Hydropak material consists of a large number of closed, smooth channels; BIO-NET has a unique cylindrical net structure. Biofilter A was connected to a 'quasi-closed" recycling fish culture

82

G. Kriiner, H. Rosenthal

system of about 14 m 3 total water volume. The temperature was maintained at about 25C and the salinity was kept between 14 and 16%o. The water flow rate through trickling filter A was maintained at a constant 8 m 3 h - 1 (Table 1 ). The BODs-load in the culture system varied between 0.4 and 4.0 kg O2 day- ~corresponding to a level between 2 and 20 mg 0 2 litre- J in the in-flow of the biofilter. Total ammonia-.-nitrogen load ranged between 0.031 and 0.078 kg N-NH~- day -~ during the entire experimental period. The fish stocked were Tilapia (mainly Seratherodon mossambica, S. aurea and S. nilotica ) and eel (Anguilla anguilla ). During the experimental period the stocking density reached 64 kg fish m-3 corresponding to a maximum fish load of 896 kg. Fish were fed once per day (feeding time--08.00 h; feeding rate= 1-2% body weight, dry to wet weight basis).
TABLE l

Characteristics of Two TricklingFilters and the Submerged Rotating Contactor (SRC) with DifferentPackingMaterialsDuring Our Experiments
Operational data Trickling filter A BIO-NET Trickling filter B BIO-NET/ Hydropak SR C BIO-NET

Specific surface a r e a (m2m-3) Total volume (rna) Total surface (m2) Hydraulicload (m3day-l m-~) Flow rate (m3h-t) Flow rate (m3day- t m- z)

260 4.42 1 150 43.4 8.0 0.167

260/200 2.21/2.38 575/477 20.1 3.85 0.088

380 0.0076 29.0 20.8 0.066 0.055

Detailed information regarding the high-load culture system and the characteristics of trickling filter A has been given recently elsewhere (Rennhack, 1982; Kriiner and Rosenthal, 198 3). The rotating plastic package of the SRC exhibited a porous netstructure and was completely submerged while in operation (Sekoulov and Heinrich, 1981; Heinrich, 1984; Pliimke, 1985). The unit received its wastewater and its air supply through the central axis. Contact made between the wastewater, air and substrate could be largely controlled through adjustment of the rotation speed and wastewater flow rate, thereby providing a continuous flow towards the peripheral regions of the filter. The rotation speed affects oxygen transfer and maintains the

Circadian periodicity of biological oxidation

83

entire attached bacterial biomass in an aerobic condition. The rotation of the drum also provides a mechanical means to remove the accumulating solid particles adhered to the material. Tiffs is achieved mainly by the sheafing forces. It also maintains the sloughed solids in suspension. Both trickling filter B and the SRC were connected to a 'quasi-closed' recirculating culture system with a low biomass loading. System integration was reached via a by-pass connection ('multi-cycle' system: total volume including trickling filter, ozonation unit and anaerobic denitrification = 18 m3; salinity=20-23%o; temperature=23-25C; pH values---7.8-8.4; total ammonia-nitrogen = 0.008-0.078 mg N litre-1; BODs= 1-0-5.4 mg 02 litre-1; corresponding to 0.1 and 0.5 kg day -1 BOD5 load; all data based on daily observations during morning hours). Fish species stocked were Tilapia (mainly Seratherodon mossambica, S. aurea and S. galilaea), eel (Anguilla anguiUa) and mullet (Mugil cephalus). Stocking density during the experimental period reached 1.2 kg m- 3 corresponding to a maximum biomass fish load of approximately 28 kg. Fish were fed twice per day (feeding time = 09.00 h and 17.00 h; feeding rate = 1-2% body weight, dry to wet weight basis). All tanks of the systems were made of polyester-glassfibre. All plumbing was of PVC. Composite samples for water quality analysis were taken simultaneously at the inlets and outlets of the biofilters. Samples were taken on an hourly basis during night time and at half-hour intervals during day time. Water quality analysis included determinations of total ammonia (NH 3 +NH~-) and nitrite, pH-values and biological oxygen demand (BODs). Determined total ammonia- and nitrite-concentrations were calculated and presented as total ammonia-nitrogen (N-NH~-) and nitrite-nitrogen (N-NO ~-). Analytical methods used were slightly modified from Grasshoff et al. (1983). BOD 5 was determined as the oxygen consumption during 5 days of incubation at 20C in filtered samples. Inhibition of nitrifiers was achieved by adding 2 mg litre- 1 allylthiourea. The total removal of organic pollutants was measured on filtered samples in an attempt to describe the removal efficiency for dissolved BOD. Since colloids can be of a .size that is smaller than the pore size of the flter papers, colloids contribute to a certain degree to the measured dissolved BOD. No attempt was made to isolate or identify the mixed bacterial populations that developed on the biofilter substrates. It is important to note that the biofilters were operated at a constant hydraulic flow rate during each of the study periods (see Table 1 ).

84

G. Kriiner, tl. Rosenthal

RESULTS
Diurnal variations of water quality in various biofilters

High-load trickling filter A (64 kg fish m -3)

The diurnal variations of pH, total ammonia, nitrite and BOD concentrations at the inlet and outlet of trickling filter A are given in Fig. 1. The curves are based on half-hourly composite samples. The pH values varied between 6.0 and 6.3 at the filter inlet. The pH of the water was consistently lowered in the biofilter so that the outlet values were on average reduced by about 0.03 unit. The variations of pH and of total ammonia concentration at the inlet depended on those of the entire recycling system. Maximum pH values occurred at the same time as the maximum total ammonia values, usually within 4 h after feeding the fish. Total ammonia-nitrogen concentrations in the biofilter inlet varied considerably over a 12-h observation period, beginning directly after feeding. Values ranged from 0.78 to 2.89 mg N litre-~ 8 h after feeding. The outlet concentrations ranged between 0.19 and 1.33 mg N litreThe efficiency of total ammonia-nitrogen oxidation decreased directly after feeding from 60% to just over 20% and returned 4 h later to the earlier oxidation-rate of 60%, fluctuating between 60% and 30%. During night time the degradation efficiency was quite constant at levels around 60%. The nitrite-nitrogen concentration started at a relatively low value during morning hours (inlet 0"33 mg N litre-~, outlet 0.21 mg N litre -~ ) but increased constantly during the day, reaching a peak about 13 h after feeding (inlet 0.85 mg N litre-~, outlet 0.76 mg N litre-~). The decline began thereafter. After feeding, the reduction efficiency of nitrite decreased to almost zero, but increased steadily 4 h later, reaching the initial efficiency about 18 h after commencement of feeding. Carbonaceous (BOD) levels during the experimental period ranged from 20 mg O~ litre-~ directly after feeding to about 4 mg O, litreduring night time (biofilter inlet). The curve for BOD 5 degradation efficiency showed its minimum about 6 h after feeding and declined from 60% to 10%, indicating that the nitrification process became increasingly important about 1.5 h after feeding. Figure 2 shows at two different pH ranges (pH 6"1.-6.3 and 6.5-6.6) that the nitrification process is highly dependent on the load of organic matter, and that total ammonia-nitrogen conversion efficiency is almost negligible at high organic loadings.

Orcadian periodicity of biologicaloxidation

pH

85

631

= ~.,..&,,~

*-- , m e t ,-- outlet

N-NH~ I (rag L"t ) O3-i / ~ 02

I

o--

inlet

,;" :..:" ,/ /

i.~

ii
~.r

~,
*~.'K=

*--outle~

N-NO~ (rag L'II

,OOoo. t

...J"
""

_o,.,

100.

i
5 0-

t,.,,~ ~

,~

"~."',,"~'~

: W. ,-- outlet "~,..,,."-'%,....~ ........ . - ~--~ .......

t
~egradat,on efficiency

60

i6
20 i ~i .
,a.9 ''/

./'~"
=

80D 5 N-NH~

1
O (30! ~200

;'
16'00

p,

.-"
20'C0

.-NO:
2&'O0 4'00
tlme of d a y

Fig. 1. Water treatment in a high-load fish culture system using trickling filters: time curves for pH, total ammonia-nitrogen, nitrite-nitrogen, BODs and the related relative degradation efficiencies (based on initial concentration). Arrow = feeding time.

86

G. Kriiner, H. Rosenthal
d e g r a d a t i o n efficiency N-NH~, {*/,} 100 \~o o o \ o o oo y = 277-371n x r =-0.948 n=36 pH=6.S- 6.6

y :156-19 In x o~R. r=-0.820 n=36 k ;)H= 6.1 - 6.3 II

O

0 100

0 1000 flODs-space load {g.m ~3,d "I)

I 500

Fig. 2. Totalammonia-nitrogen removal in a tricklingfilter at two different pH ranges in relation to the BaD:space load. n = number of observations; r--coefficient of correlation; dots ---pH range 6.1-6.3; circles-pH range 6.5-6.6.

The dissolved B a D removal as a function of the B a D : s p a c e loading at two different pH ranges (pH 6.1-6.3 and 6-5-6.6) was demonstrated (Fig. 3) for one of the tested biofilters. From these data it is obvious that the removal of B a D is highly dependent on pH level and that the percentage removal increases as B a D : s P a c e load increases.

Low-load trickling filter B (1.2 kg fish m -~)

Figure 4 shows the daily fluctuations of pH, ammonia, nitrite and B a D concentrations at the inlet and outlet of trickling filter B (flow rate 4 m 3 h - ~ constantly). The pH varied between 8.0 and 8.1 at the inlet and was always higher (only one time lower) in the outlet (about 0.07 unit). Prior to feeding, the total ammonia-nitrogen concentration in the biofilter inlet varied between 0-078 and 0.039 mg N litre- ~ during 11 h after the first feeding. At the same time the outlet concentrations ranged between 0.016 and 0-031 mg N litre -~. The oxidation efficiency increased from 3 5 0 during the early morning to about 60% within 10 h after the first feeding.

Circadian periodicity of biological oxidation

d e g r a d a t i o n efficiency BODs P/=) Y

87

80-

=-105+25 l n x = 08Z.1 n = 2 7

pH = 6 5 - 6.6

o o

50

00

y =-6 * 9 In x r = 0 5 0 9 n = 36 pH = 6.1-6.3

/:Oo
o o 0 I i

100

500 1000 B O D s - s p a c e load (g m ' 3 . d -1)

Fig. 3.

Dissolved BOD~ removal efficiency in a trickling filter at two different pH ranges in relation to the BODs-space load.

The inlet nitrite-nitrogen level increased constantly and considerably over day time (0-03-1-07 mg N litre-'); maximum nitrite concentrations occurred simultaneously with the maximum total ammonia-nitrogen values. This trend is due to the drastically decreased degradation efficiency of the second step of nitrification ranging between 80 and 30% immediately after feeding but recovering to more stable values of about 60% 2 h later.

Low-load submerged rotating contactor (SRC) (1.2 kg fish m --~)

The diurnal variations of pH, total ammonia and nitrite at the inlet and outlet of the SRC and the associated efficiencies obtained for these factors are depicted in Fig. 5. The inlet values for the SRC are almost identical to those for trickling filter B (pH--8.0-8.1; N-NH~-=0-04-0.08 mg N litre-~; N-NO; -= 0.061-0-107 mg N litre- ~) since both units were operated in parallel bypasses of the same recycling system. The outlet values of pH were constant at 8.3, which means an increase of about 0.2 unit. The outlet concentrations of total ammonia-nitrogen in the SRC ranged from 0"039 to 0-031 mg N litre-'. The removal efficiency increased constantly from nearly 2% to more than 40% 7 h after the first feeding and decreased during night time to values of about 4%.

88
pH 831 e2 80 N'NH Z (rag L"I )
0 08-

G. Kriiner, H. Rosenthal

*-- i n l e t e-- o u t l e t

. . . . .

_:

""~' "~P'~t,-q~r,.,,,-~.~.~.*-,,o~..

..,,---~ , - , ~

0 0/.-

'~ ~"

~-- outlet

{mg.L -1 )
,.p,,"xe J " L ' ~ ' ~ ' ~ " ~ " ~ " ~ \ \

008-

.....,,""~' ..P 4)~

"" \ \ "",~-...,
*

0/,-

e-- o u t l e t

-o

,
efficiency

degrQdatlon

iO/o) /

' ;)

,d '~1

800 ~

12 O0

16 00~

2000

2/-00

~.00 h m e of day

Fig. 4. Water treatment in a low-load fish culture system using trickling filter B parallel to a submerged rotating contactor: time curves for pH, total ammonia-nitrogen, nitrite-nitrogen and the related relative degradation efficiencies (based on initial concentration). Arrow = feeding time.

Circadian periodicity of biological oxidation

pH
f

89

27
/
i

*- inlet
.-outlet

B t- I

L,

N-NH: (rag L "! }

008-

ft., ~P'Ko..~r '~

"~

00/.0- inlet
.-outlet -

~.

-Zg

t
N -N0} (rag L-i )

u= y'='e'~'o----o*...~.~x
0 08 = .,w p .~"*', "r ~ AP"qr4"~a~x x\

;=,=~=..

\\
x x

I a, fl,,,a..o, p,.u
L "~ P"~/

~ .... let
*-outlet

00/.,! "-,,,.,~""~

'~'~'

", " . ~" ~" . x "'8

0 J

0egrodot ion

efhoency

"!
20~

4 "v "--A' V' |

,,,? =
O I=!

11 / ",,~, ,,. ..~Y'----,.":~,,--... I -,1t" ,~ ~" :.:" =~.,~ '..P'~' ".-,.'..":~

/". ~:, ' ~ "i: -"
,,d = N - N I"I~_ N-NO~

T", " " . ~.

"~ "'---. "~ ""

8 00 t

12'00

16'00~

20'00

2L'00

& '00 t,me of doy

Fig. 5. Water treatment m a low-load fish culture system using a submerged rotating contactor parallel to trickling filter B: time curves for pH, total ammonia-nitrogen, nitrite-nitrogen and the related relative degradation efficiencies (based on initial concentration). Arrow = feeding time.

90

G. Kriiner, H. Rosenthal

The outlet concentrations of nitrite-nitrogen decreased from 0.043 to 0.007 mg N litre-~ 15 h after the first feeding. The degradation efficiency varied between 30% and 45% 6 h after the first feeding and decreased constantly from this level to 10% early in the morning.
Degradation efficiency of total nitrite-nitrogen

The nitrite values have to be considered in relation to the total nitrite derived from the inlet total ammonia. Since the total ammonia oxidized from the medium has to pass the nitrite step, nitrification of nitrite can be described by calculating the difference between the nitrite-nitrogen outlet values and the total nitrite-nitrogen produced in the unit (inlet minus outlet total ammonia-nitrogen calculated as nitrite-nitrogen plus inlet nitrite-nitrogen). Figure 6 shows the nitrification efficiency of the total nitrite-nitrogen of the three biofilters. The degradation efficiency in both trickling filters fell drastically after feeding the fish. The degradation efficiency in trickling filter A (N-NO2:0-02-0.2 mg N day -1 m -2) decreased from

degradation eff,ciency
(%)
o-qp

N-NO~

80

.
" i :

o
"'' 'e""

i
60

: "~" '

+"+~

+ , + ,0-0.o"

/.0 ./.

?+.+/
,+

~'+ ./

? r'V

+++
trickling trickltng submerged filter ftlter A B .

\.

x,...
"-..

~J ~, 20

rotating

contoctor

8/00

,2100

,6'0o

20'00

2~'00

~ bo

time

of day

Fig. 6. C o m p a r i s o n of s e c o n d - s t e p nitrification efficiency in biological filters during one daily cycle: triangle = t r i c k l i n g filter A c o n n e c t e d to high-density fish culture recirculating system; circles = trickling filter B; dots = SRC jointly c o n n e c t e d to a lowdensity culture system. A r r o w = feeding time.

Circadian periodicity of biological oxidation

91

75% to 23%, and trickling filter B (N-NO f: 0.02-0.01 nag N day- 1 m- 2) from 88% to 42%. A 70% degradation plateau was reached by trickling filter B after 2 h and after 4 h in trickling filter A. Filter A was, in contrast to filter B, very unstable and the efficiency varied greatly, with 45% of values falling between 45% and 80% removal. The SRC (N-NOf: 0.03-0-1 mg N day -1 m -z) had a relatively constant increase in nitrification efficiency from 28% directly after feeding the fish to 58% 7 h later, followed by a decreasing rate during night time to reach 15% early in the morning. DISCUSSION The results of our investigation indicate that high loading with suspended and colloidal particles significantly lowers the nitrification rate. A decreased nitrification rate, when colloidal particles are present, will necessarily affect the design of the pretreatment processes. A large part of the normal BOD will have to be removed immediately after the water leaves the fish tank (outlet) and prior to entry of wastewaters to the nitrification unit. Data from both studies describing BOD 5 removal and the combined removal of BOD 5 and nitrogen-species demonstrated that shock loads produce a dramatic and rapid deterioration in effluent quality. It was felt that a realistic appraisal of nitrification kinetics in the marine culture environment would be obtained if large mixed populations of nitrifying bacteria were maintained under a defined set of conditions for relatively long periods of time (in this study 8 months). Long-chained organic compounds containing a strongly polar group will form oriented layers at the water surface. It seems possible that these layers could act as a diffusion barrier for oxygen through surfaces. Such barriers would also alter surface dynamics and affect the transfer coefficient of oxygen and other gases. The performance of biological filters in removing organic material and nitrifying its nitrogen components is affected by many design factors such as the depth of the filter, hydraulic load and size and shape of the media. Furthermore, the physico-chemical characteristics of the wastewater, and the amplitude of daily fluctuations of various water quality parameters play a decisive role in treatment efficiency (Mehta et al., 1972; Stenquist et al., 1974; Porter and Smith, 1979; Kfiiner and Rosenthal, 1983; Hilge and Rakelmann, 1984). In an aerobic biological system, the BOD reduction results from biochemical activity of heterotrophic bacteria. Porter and Smith (1979) showed that the efficiency of

92

G. Kriiner, H. Rosenthal

BOD removal in a biofilter of a given total volume is determined mainly by the surface area available for bacterial attachment per unit volume of plastic media. Oxidation of total ammonia to nitrate in aquatic solutions utilizes dissolved oxygen and will occur only if the oxygen is maintained at levels that prevent the development of anaerobic conditions. Oxygen concentrations in this study were stoichiometrically greater than that required for complete oxidation. The requirements for nitrification are the presence of ammonia, oxygen, trace nutrients, and a relatively low level of organic carbon in the biological filter. Little information exists on the optimum conditions for nitrification in a combined carbon oxidation-nitrification system involving plastic media. There are few data on the maximum specific growth rate for nitrifying organisms at 20C, indicating that such values are much lower than those for heterotrophic bacteria (Painter, 1975; Wickins, 1983), and that nitrification is generally dependent on the organic loading and on temperature (Stenquist et al., 1974). Under the physico-chemical conditions commonly found in wellaerated wastewaters, oxidation processes occur at a detectable rate only in the presence of certain chemo-autotrophic and heterotrophic bacteria (Focht and Verstraete, 1977). Table 2 provides the data obtained during our experiments. The rate of oxidation of carbonaceous matter is also dependent on the multiplication rate of bacteria. Most bacteria obtain their food and energy requirements from organic matter. The oxidation of carbonaceous material is carried out in the first stage BOD reaction. In general it was found that the upper half of the biofilter removes more
TABLE 2 Typical Ranges of Physico-chemical Variables in Well-aerated Recirculation Fish Culture Units
Variable System I 64 kg m - "~ System 11 1.2 kg m - "~

Temperature (C) Salinity (%o) pH NH 3 (mg litre- ~) N-NHj" (nag litre- ~) N-NOr (mg litre- ~) N-NO~ (mg litre-J) DO (mg litre-~) BOD5 (mg litre- ~)

25.8 12"9 6.05-6-27 0.000 4-0.003 0.47-2.81 0"21-0"88 36.1-39"7 5-9-7.7 3.4-20"0

21.5 20.0 7.97-8'07 0-001-0.004 0.03-0.08 0.04-0-10 22.6-23.2 7-1-8"5 1.0-5"4

Circadian periodicity of biological oxidation

93

organic matter than the lower half and that nitrification normally occurs in the deeper zones of the filter (Balakrishnan and Eckenfelder, 1969; Rennhack, 1982; Kriiner and Rosenthal, 1983). The nitrifying bacteria are not able to compete efficiently with the organo-heterotrophic bacteria unless they have plenty of substrate. The nitrifiers also-depend on the release of inorganic nitrogen compounds from the organic matter which is usually degraded by the heterotrophs. Normally, nitrification rates are strongly influenced by pH because nitrification produces two mole hydrogen ions for each mole of ammonia oxidized, which leads to pH lowering during the process. The decrease of pH value causes, in turn, the lowering of the nitrification rate. Apart from that, the change of pH may also influence the reaction rate indirectly. Srna and Baggaley (1975) showed that the optimum pH for complete nitrification is around pH 7.45 in a subgravel filter and effective nitrification is achieved between pH values of about 7"0-8.2 (temperature= 20-24C; salinity= 26 + 2%0). Wild et al. (1971) showed a pH optimum for nitrification in freshwater at pH 8.4. Haug and McCarty (1972) reported the adaptation of nitrifiers on a submerged filter to pH levels between 6.0 and 5.5 within about 10 days. Nitrification ceased completely below pH values of 5-5 (temperature= 25C; freshwater). McHamess and McCarty (1973) obtained evidence that nitrifying bacteria could be conditioned to operate at maximum efficiency even at relatively low pH values if they were held at low pH levels for a sufficient length of time (temperature = 20C; freshwater). The pH range investigated in our studies was between 6.05 and 6.27 in system I (64 kg fish m -3) and between 7.97 and 8.07 in system II (1.2 kg fish m-3). The reduction in pH values from 8"3 to 8"0 in the SRC (in trickling filter B the pH dropped only 0.1 unit) is a consequence of less efficient stripping of carbon dioxide from the mixed liquor by the relatively small volume of gas discharged to the atmosphere via aeration. Very little work has been carried out on the effect of high concentrations of oxygen and carbon dioxide on the growth and metabolism of the major bacterial groups involved in water treatment. Jones and Paskins (1982) reported that carbon dioxide concentrations in the aeration gas ranging between 0.03 and 2% of the total gas content do not affect the growth constants of Nitrosornonas (temperature=20C; pH=7.8), whereas high concentrations of oxygen are initially inhibitory to Nitros o m o n a s and may also be inhibitory to the growth of Nitrobacter. The series of varying peaks superimposed on the generally constant total ammonia-nitrogen degradation efficiency observed 5 h after feeding in trickling filter A probably represents a real phenomenon, because

94

G. Kriiner, H. Rosenthal

the data points fall well outside the range of analytical error. Almost the same behaviour in total ammonia-nitrogen oxidation has been observed in the other two biofilters. Two hours after feeding, the total ammonia-nitrogen oxidation rate in both biofilters with a low biomass load (trickling filter B and SRC) was almost uniform during the entire experiment. This observation indicates that the nitrifiers worked near their maximum efficiency at all times, and were, therefore, largely independent of the residual of total ammonia-nitrogen concentration occurring during the early morning hours. Both Nitrosomonas sp. and Nitrobacter sp. are usually sensitive in their metabolic activity to changes in nitrogenous substrate concentrations, which are their essential substrates. Additionally, these nitrifying species are also influenced by the concentration of organic load which usually supports other bacterial groups such as the heterotrophs (Painter, 1970). Total ammonia-nitrogen and nitrite-nitrogen concentrations in wastewater from fish culture do not usually reach levels that inhibit the activity of nitrifiers. Recently Wild et al. (1971) found, among other factors, that total ammonia-nitrogen at a concentration of about 46.88 mg N litre- ~in the effluent did not have any inhibitory effect on the rate of nitrification at pH values of 8"5. This is another indication that the activity of nitrifying bacteria is not significantly affected by total ammonia-nitrogen concentrations normally encountered in fish culture systems (Table 2). During night time it seems that the total ammonia-nitrogen concentrations were below the critical levels needed to maintain an active nitrifying population. Knowles et al. (1965) showed that between 30 and 60% of a population of Nitrosomonas bacteria survived without nutrients for longer than a 5-day period. Between 80 and 90% of a Nitrobacter population survived for 0.8 day without nutrient supply. Nitrification efficiency has not been found to increase significantly with a decrease in carbon:nitrogen ratio (Balakrishnan and Eckenfelder, 1969). With other parameters (temperature, pH, DO, etc.) held con-" stant, the degree of nitrification decreases significantly with an increase of the organic load (Prakasam and Loehr, 1972). The removal of total ammonia-nitrogen in relation to the applied BODs-space load in trickling filter A is shown in Fig. 2. The removal was generally between 10 and 100% at a pH range of 6.5-6-6 (or 10-65% at a pH range of 6-1-6-3). There is usually some stratification of the heterotrophic and autotrophic micro-organisms in a biological filter, the heterotrophs tending to occupy the upper layers where the concentration of organic matter in the feed liquor is greatest, while the autotrophs flourish in the lower regions where their substrates are more abundant. In high-rate filtration

Circadian periodicity of biological oxidation

95

techniques, heterotrophic growth occurs throughout the depth of the filter, suppressing the autotrophic organisms. High B O D loadings in such filters would eventually cause nitrification to cease because of the washing-out effect of sludge, which would also affect nitrifiers.

ACKNOWLEDGEMENTS We are indebted to Ms Wiege for expert technical assistance during the experiments, water analyses and drawings. REFERENCES Adams, C. E. & Eckenfelder, W. W. (1977). Nitrification design approach for high strength ammonia wastewaters. J. Water Pollut. Control Fed., 49, 413-21. Balakrishnan, S. & Eckenfelder, W. W. (1969). Nitrogen relationships in biological treatment processes. I. Nitrification in the activated sludge process. WaterRes., 3, 73-81. Focht, D. D. & Verstraete, W. (1977). Biochemical ecology of nitrification and denitrification. Adv. Microb. Ecol., 1,135-214. Grasshoff, K., Ehrhardt, M. & Kremling, K. (1983). Methods of Seawater Analysis, Verlag Chemie, Weinheim, FRG. Gromiec, M. J., Malina, J. E & Eckenfelder, W. W. (1972). Performance of plastic medium in trickling filters. Water Res., 6, 1321-32. Hang, R. T. & McCarty, E L. (1972). Nitrification with submerged filters. J. Water Pollut. Control Fed., 44, 2086-102. Heinrich, D. (1984). Untersuchungen zur Nitrifikation von Abwiissern in iiberstauten Festbettreaktoren. Stuttg. Bet. Siedl.-wass.-wirtsch., 81, R. Oldenbourg, Miinchen. Hilge, V. & Rakelmann, U. V. (1984). Laboratory scale experiments on the nitrification of fish tank effluent in a fLxed film bed reactor. EMS special publication, No. 8, Research on Aquaculture, eds H. Rosenthal & S. Sarig, pp. 55-66. H6ner. G. (1984). Auswirkungen dichteabh~ingiger Umweltbedingungen in der Intensivhaltung auf Jungfische von Sarotherodon galilaeus. Dipl.-Arb. Universit~it Marburg, pp. 1-123. Jones, G. L. & Paskins, A. R. (1982). Influence of high partial pressure of carbon dioxide and oxygen on nitrification. J. Chem. Tech. Biotechnol., 32, 213-23. Klemetson, S. L. & Cohen, D. (1984). System analysis and design of biofilters for the three components of Macrobrachium production. Final Report BARD, No. US-60-80. pp. 1-117. Knowles, G., Downing, A. L. & Barrett, M. J. (1965). Determination of kinetic constants for nitrifying bacteria in mixed culture; with the aid of an electronic computer. J. Gen. Microbiol., 38,263-78. Kriiner. G. & Rosenthal, H. (1983). Eficiency of nitrification in trickling filters using different substrates. Aquacultural Engineering, 2, 49-67.

96

G. Kriiner, H. Rosenthal

Liao, P. B. & Mayo, R. D. (1974). Intensified fish culture combining water reconditioning with pollution abatement. Aquaculture, 3, 61-85. McHarness, D. D. & McCarty, E L. (1973). Field study of nitrification with the submerged filter. EPA Rept. No. R2-73-158, Washington DC. Meade, T. L. (1974). The technology of closed system culture of salmonids. Anim. Sci./NOAA Sea Grant, Technical Report 30, University of Rhode Island, Kingston. Mehta, D. S. et al. (1972). Oxygen theory in biological treatment plant design. J. Sanitary Engineering Division SA3. Painter, H. A. (1970). A review of literature on inorganic nitrogen metabolism in micro-organism. Water Res., 4, 393-450. Painter, H. A. (1975). Microbial transformations of inorganic nitrogen. IA WPR Conf. on Nitrogen as a Water Pollutant, Kopenhagen 1975. Pliimke, E. (1985). Wasseraufbereitung in einem Fischzuchtkreislauf: Abbauleistung eines Drehtrommelreaktors unter verschiedenen Betriebsbedingungen. Dipl.-Arb. Fachhochschule Hamburg, pp. 1-91. Porter, K. E. & Smith, E. (1979). Plastic-media biologicals. Water Pollut. Control, 78, 371-81. Prakasam, T. B. S. & Loehr, R. C. (1972). Microbial nitrification and denitrification in concentrated wastes. Water Res., 6, 859-69. Rennhack, H. (1982). Biologisch chemische Untersuchungen fiber die Eignung dreier verschiedener Materialien in einem Tropfk6rper zur Aufbereitung yon Fischabw~kssern in einem geschlossenem Warmwasserkreislauf. Dipl.-Arb. Universitiit Hamburg, pp. 1-70. Rittmann, B. E. (1982). Comparative performance of biofilm reactor types. Biotechnol. Bioeng., 24, 1341-70. Rosenthal, H., Andjus, R. & Kriiner, G. (1980). Daily variations of water quality parameters under intensive culture conditions in a recycling system. EIFAC/ 80/Symp.: E/59, pp. 1-10. Rosenthal, H., Hoffmann, R., J6rgensen, L., Kriiner, G., Peters, G., Schlotfeldt, H.-J. & Schomann, H. (1982). Water management in circular tanks of a commercial intensive culture unit and its effects on water quality and fish condition. ICES CM. 1982/F.'22, pp. 1-13. Rosenthal, H., Chiba, K. & Kriiner, G. (1984). Daily fluctuations of water quality in a recirculating system and its influence on the performance of a rotating bionet biological filter. ICES C.M. 1984/F.'20, pp. 1-11. Sekoulov, I. & Heinrich, D. (1981). Pilotversuche zur Nitrifikation in Festbettreaktoren. Wasserwirtschafi, 71, 331-3. Srna, R. E & Baggaley, A. (1975). Kinetic response of perturbed marine nitrification systems. J. Water Pollut. Control Fed., 47,472-86. Stenquist, R. J., Parker, D. S. & Dosh, T. J. (1974). Carbon oxidation-nitrification in synthetic media trickling filters. J. Water Pollut. Control Fed., 46, 2327-39. Wickins, J. E (1983). Studies on marine biological filters. Water Res., 17, 1769-80. Wild, H. E., Sawyer, C. N. & McMahon, T. C. ( 1971 ). Factors affecting nitrification kinetics. J. Water Pollut. Control Fed., 43, 1845-54.