Wat. Res. Vol. 35, No. 11, pp.

2702–2710, 2001
# 2001 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(00)00553-4 0043-1354/01/$ - see front matter

NUTRIENT ADDITION TO ENHANCE BIOLOGICAL

TREATMENT OF GREYWATER
BRUCE JEFFERSON, JOANNA E. BURGESS, AUDE PICHON,
JOANNE HARKNESS and SIMON J. JUDD*
School of Water Sciences, Cranfield University, Cranfield, Bedford MK43 0AL, UK

(First received 12 July 2000; accepted in revised form 1 November 2000)

Abstract}This study compares the chemical oxygen demand (COD) removal and respiration rates of a
microbial population treating real and synthetic greywaters dosed with nutrient supplements. The nutrient
composition of the real and synthetic greywaters was analysed and the dosing regime for nitrogen,
phosphorus and a range of trace metals planned accordingly. The doses consisted of eight single additives
(macronutrients and trace metals) to the control greywater and six trace metal additions to C : N : P
balanced greywater. The COD removal for the control real and synthetic greywater in lab-scale activated
sludge systems (0.038 and 0.286 kg COD kg MLSS
1 d
1, respectively) confirmed nutrient limitation and
the poor degree of greywater treatment. Nutrient dosing increased the COD removal rate and oxygen
uptake rate in many cases. The greatest stimulation of microbial activity was observed with zinc additions
to C : N : P balanced real greywater (1.291 kg COD kg MLSS
1 d
1 over 30 times the control). Inhibitory
effects to various extents were rare and limited mainly to the additions of metals to synthetic greywater.
The dominance of chemicals effects was observed on addition of some micronutrients; notably iron and
aluminium, metals on which many coagulants for use in biotreatment of other wastewaters are based. The
data indicate that the impact of understanding microbial processes and the nutrients required for
wastewater treatment can only serve to optimise process efficiency for the proposed treatment of
greywater. # 2001 Elsevier Science Ltd. All rights reserved

Key words}greywater, nutrient addition, microorganisms, micronutrients, trace metals

INTRODUCTION large-scale recycling at the Millennium Dome (Jef-

The water supply–demand balance in the UK is ferson and Judd, 1999). The current trend has been to
projected to reach critical levels in the future due to include biological treatment in the processing of
the anticipated growth in new housing developments greywater for all but the very small scale, to restrict
coupled with an increased per capita water usage and the propensity for regrowth of biomass in post-
decreasing supply in some parts of the country. treatment water. However, unlike sewage, little is
Water recycling forms one key component of the known about the fundamentals of greywater treat-
strategy to offset this imbalance. In the short to ment and hence its suitability for biological treat-
medium term, domestic recycling has been identified ment. Experience from pilot trials with biological
as one of the most promising options. Urban water systems treating greywaters has shown that such
use is higher in the UK than in many other European systems are generally limited to a low organic load,
countries, and is significantly more condensed into while analysis of the feed waters has shown a high
urban centres; 50% of the population live in towns of COD : BOD ratio coupled with nitrogen and phos-
100,000 or greater population (Laine et al., 1999). phorus deficiencies (Laine et al., 1999). It has been
The focus of domestic water recycling has been hypothesised that addition of macro- and trace
towards greywatery reuse for toilet flushing or nutrient addition may overcome both these pro-
recreational irrigation, offering a potential 30% blems, thus increasing the efficacy of biological
reduction in potable water use. This has been technologies for recycling applications.
implemented at a number of sites around the UK Biological cells require six macronutrients for
ranging from reuse within a single occupancy to metabolic processes: carbon, oxygen, hydrogen,
nitrogen, sulphur and phosphorus. The primary
nutrients are nitrogen, phosphorus and carbon
*Author to whom all correspondence should be addressed.
(Beardsley and Coffey, 1985). The trace element
Tel.: +44-1234-750111 ext. 2542; fax: +44-1234-
751671; e-mail: s.j.judd@cranfield.ac.uk requirements of bacterial cells can be determined
y
Wastewater from all domestic sources except toilet from the composition of the cells (Metcalf and Eddy,
flushing. 1991). However, as excess micronutrients can be
2702
 Nutrient addition in greywater 2703

Table 1. Microbial nutrient requirements and the concentrations present in greywater

Nutrient Reported Role of trace metala Concentration detected (mg l
1)

requirements
(mg l
1)a
Real greywater Synthetic
greywater

N, P, S 15, 3, 1 minb } 5.00, 1.37, 16.3 5.00, 0.047, 17.5

Ca 0.4–1.4 Cell transport systems and osmotic balance in all bacteria. 47.9 47.0
Bridging anionic ECP and aiding flocculation. Increase growth
rates. Requirements and effects vary. Interacts with other metals.
K 0.8–>3.0 Cell transport systems and osmotic balance in bacteria. 5.79 3.96
Fe 0.1–0.4 Growth factor in bacteria, fungi and algae. Adsorbed in 0.017 0.009
proportion to the concentration available. Electron transport in
cytochromes. Synthesis of catalase, peroxidase and aconitase. Ion
reduction for floc formation.
Mg 0.4–5.0 Enzyme activator for a number of kinases and phosphotransferase 5.29 5.02
in heterotrophic bacteria.
Mn 0.01–0.5 Activates bacterial enzymes. Often interchangeable with 0.04 0.02
magnesium in kinase reactions. Lower affinity for binding sites
than other metals but still can inhibit metabolism at 1 mg l
1.
Cu 0.01–0.5 Bacterial enzyme activator required in trace quantities. Can inhibit 0.006 0
metabolism. Chelates other substances, reducing their toxicity.
Al 0.01–0.5 Not known. Affects the species found in sludge (Annaka, 1977). 0.003 0
Zn 0.1–0.5 Bacterial metallic enzyme activator of carbonic anhydrase and 0.03 0
carboxypeptidase A. Dissociable on active site of enzymes.
Stimulates cell growth. Toxic at 1 mg l
1, especially to protozoa.
Can exacerbate toxic effects of other metals and inhibit
metabolism.
Mo 0.2–0.5 Molybdenum is a common limiting nutrient (Grau, 1991). 0 0
Co 0.1–5.0c Bacterial metallic enzyme activator. Dissociable on active site of 0 0
enzymes. Activates carboxypeptidase for synthesis of vitamin B12
(cyanocobalamin) but otherwise toxic. Can inhibit metabolism.
a
Burgess et al., (1999a).
b
From COD : N : P ratio, Beardsley and Coffey (1985).
c
Sathyanarayana and Srinath (1961).

adsorbed onto cell walls, the concentrations in effluent, reduced treatment efficiency and sludge-
biomass ash may exceed the actual required amount handling problems (Pala and Sponza, 1996; Soddell
(Wood and Tchobanoglous, 1975; Nicholas, 1963). and Seviour, 1996; Blackall et al., 1996). A clear
Actual requirements are influenced strongly by the example of this can be seen with activated sludge,
organic and hydraulic loading rates on the activated where pin point flocs, dispersed growth and poor
sludge microorganisms, the cell growth rate (Speitel flocculation have been attributed to deficiencies in
and Segar, 1995), the nature of the wastewater, and trace elements and have been shown to be amelio-
the mean cell residence time in the aeration basin rated by nutrient dosing (Gostick, 1991). Therefore
(Wood and Tchobanoglous, 1975). the application of biological processes to the treat-
As environmental conditions and nutrient supply ment of greywater, and its subsequent optimisation is
approach optimum the hydraulic retention time a logical progression. The aim of this study was to
necessary for wastewater treatment decreases (Speitel investigate the feasibility of optimising the biological
and Digiano, 1988; Benefield et al., 1979). The ratio treatment of greywater through nutrient supple-
at which nutrients should be supplied is contentious, mentation.
with COD : N : P ratios of 100 : 20 : 1 (Metcalf and
Eddy, 1991), 250 : 7 : 1 (Franta et al., 1994) and
MATERIALS AND METHODS
100 : 10 : 1 and trace sulphur (Beardsley and Coffey,
1985) quoted in the literature. Atomic emission spectrophotometry using an inductively
In the cases where treatment plants are dealing coupled plasma atomic emission spectrophotometer (Ther-
with a nutrient-limited wastewater, supplementation mo Jarrell Ash, Atomscan 16) was used to identify the
with macronutrients can result in an enhanced missing or deficient trace elements in synthetic and real
greywaters as compared to theoretical requirements of
degradation of organics (Valo et al., 1985; Singleton, micro-organisms. The concentrations of available nitrogen,
1994). Adequate trace nutrient concentrations, in- phosphorus and sulphur were also measured and compared
cluding metal ions and vitamins, are required to with the theoretical requirements of bacteria for these
support all the genera present in the activated sludge nutrients stated in the literature. This procedure produced a
treatment system in order for a diverse community to list of nutrients required as a supplementation dose
(Table 1).
survive. The lack of specific nutrients can result in an A series of tests was performed using a closed-cell aerobic
unbalanced mixed culture, leading to a poor-quality respirometer (CES Ltd. Aerobic Respirometer). This model
2704 Bruce Jefferson et al.

operates in a similar way as the Warburg respirometer measurements of effluent COD were made at the end of each
(Heddle, 1980). The respiration rate is the rate at which run in the same way (Hach system, Camlab, adapted from
oxygen is used as the terminal electron acceptor, and APHA, 1992). The mixed liquor suspended solids (MLSS)
therefore can be used to indicate the condition of the sludge concentration of the sludge was determined using standard
in terms of metabolic activity. Six trace elements with and methods (APHA, 1992). Tests were duplicated to assess
without nitrogen and phosphorus balancing of the grey- repeatability.
water, as identified in literature (Table 1), were tested in
single supplements to both real and synthetic greywaters
(Table 2). The real greywater was collected from a purpose- RESULTS
built greywater collection facility fed from 16 residential
flats in the University’s halls of residence. The water was The inductively coupled plasma (ICP) atomic
collected from the flats’ baths, showers and handbasins emission spectrophotometer analysis showed that
only. The synthetic greywater recipe (Table 2) was devel-
oped from a recipe used in the UK water industry which the greywaters were deficient in nitrogen, phosphorus
represents a normalisation of the variation of real grey- and a number of micro-nutrients, namely iron,
waters found around the UK. Part of the remit of this study copper, aluminium, zinc, molybdenum and cobalt
was to evaluate the synthetic greywater recipe for differences (Table 1). The effects of nutrients on biomass range
in characteristics between the two types of greywater and from stimulatory to inhibitory:
assess the suitability of the recipe for use as a real greywater
substitute.
The sludge was taken from two membrane bioreactors, * Increased COD removal associated with increased
one treating real greywater (COD : N : P of 13 : 4 : 1) and or unchanged oxygen uptake indicates stimula-
the other treating synthetic greywater (COD : N : P of tion.
894 : 106 : 1). The sludge was maintained in porous pots
(Water Research Centre, 1978; USEPA, 1996) at a pH of
* Decreased COD removal and oxygen uptake
7.5  0.2. Aliquots of the activated sludge (50 ml) were also indicates metabolic inhibition.
taken from the same membrane bioreactors, at the same * Decreased COD removal and increased oxygen
time and placed in glass respirometer cells to which uptake indicates the uncoupling of metabolism, in
predetermined quantities of nutrient supplements had which anabolism is inhibited and catabolism is
already been added (Table 3). Control samples receiving
deionised water in place of supplements were also tested not.
when the test cells were dosed with micronutrients alone, * Increased COD removal and decreased oxygen
and controls receiving just nitrogen and phosphorus were uptake may indicate metabolic uncoupling and
used when the test cells received nitrogen, phosphorus and the inhibition of catabolism, but a more probable
micro-nutrient doses. The volumes in all cells were made up
to 55 cm3 with deionised water as required to standardise mechanism is increased adsorption of COD-
the partial pressures within the cells and make the tests imparting substrate components onto the bacter-
comparable. The respirometer cells were stabilised at 208C ial cell walls.
and stirred continually for 15 h, the maximum time possible
before oxygen in the headspace of the cells became
As the testing comprised several experiments, the
exhausted and the biomass placed under oxygen-limited
conditions. Oxygen uptake was recorded automatically at data were normalised so that the control results
5 min intervals. A sample of the original activated sludge equalled 100%, and the test results could be reported
was filtered for influent COD (Whatman GF/C filter) and as a percentage of the concurrent control, thus
making individual tests directly comparable (Tables
Table 2. Recipe for synthetic greywater 4–6, Figs 1 and 2).

The following are mixed and made up to 10.0 l with tap water
Nitrogen/phosphorus-limited greywater
0.64 g Synthetic soap Addition of nitrogen or phosphorus to real grey-
8.0 ml Hair shampoo
0.1 ml Sunflower oil water resulted in stimulation of the biomass (Fig. 1).
24.0 ml Tertiary effluent The addition of nitrogen to synthetic greywater
had the expected stimulatory effects on the sludge;
normalised oxygen uptake was 106% and COD
Table 3. Nutrient additions to the greywater samples removal 150% (Table 4, Fig. 2). Additions of cobalt,
Nutrient Dose added to real Dose added to synthetic
copper, iron and molybdenum to real greywater also
greywater (mg l
1) greywater (mg l
1) stimulated the biomass, resulting in normalised COD
removals of up to 200% with molybdenum (Table 4,
N 10.00 10.00 Fig. 1). Addition of micronutrients to synthetic grey-
P 1.63 2.95
S None None water led to stimulation of biomass by copper,
Ca None None molybdenum, zinc and aluminium. The COD re-
K None None movals achieved were 124, 117, 132 and 115%. Real
Fe 0.39 0.39
Mg None None greywater dosed with zinc and aluminium also
Mn None None showed chemical effects, as normalised COD removal
Cu 0.04 0.05
Al 0.05 0.05
increased to 154 and 225%, and oxygen uptake
Zn 0.97 1.00 decreased to 97 and 94%, respectively (Table 4,
Mo 0.70 0.70 Fig. 1). In synthetic greywater (Table 4, Fig. 2), the
Co 5.00 5.00
addition of cobalt, iron and phosphorus resulted in
 Nutrient addition in greywater 2705

decreases in oxygen uptake to 95, 81 and 88%, while

Repeatability (%)
the COD removal increased to 135, 103 and 125% of

0.3
3.2

2.2
3.1
2.1

1.1

1.1
1.7
1.6

5.7
3.8
4.3

23.3

26.3
the control, respectively.

COD removal
Nitrogen/phosphorus-balanced greywater
Dosing cobalt, copper, molybdenum and zinc into
Rate (kg kg MLSS d
1)

nitrogen/phosphorus-balanced real greywater stimu-

lated the biomass; COD removal increased to 130,
0.8
0.7
1.0
1.3
0.8
0.7
0.4
0.3
0.7
0.4
0.5
0.5
0.6
0.7
240, 150 and 290% of the control, respectively
(Table 4, Fig. 1). Dosing iron and molybdenum into
Synthetic greywater

synthetic greywater was also stimulatory, producing

normalised COD removals of 115 and 150%,
respectively. Adding aluminium to real greywater
Repeatability (%)

led to inhibition of the metabolism of the biomass;

COD removal dropped to 85% of the control, while

11.5

15.5
16.5

16.3
4.2

2.4

3.1
2.7

4.4
2.8
5.8
1.2
4.0

7.5
oxygen uptake rates remained the same (Table 4,
Fig. 1). Aluminium was also inhibitory in synthetic
greywater, as was zinc; both micronutrients caused
Oxygen uptake

the COD removal to decrease to approximately 80%

1
d )

of the control, and normalised oxygen uptake fell to

Table 4. Respiration and COD removal rates of activated sludge

1

70% for aluminium and 79% for zinc (Table 4,

Rate (kg kg MLSS

Fig. 2). Dosing iron into the real greywater resulted

1.7
1.4
2.2
2.5
1.6
1.5
0.8
0.6
1.0
1.0
1.0
1.0
1.2
1.4

in increased COD removal (185%) and a slight

decrease in respiration (97%).

Effects of greywater source and nitrogen/phosphorus

Repeatability (%)

balancing
Paired t-tests were applied to the respiration and
49.0
26.5
34.2
42.7
21.6
76.2

14.3
1.5

7.7

5.5
0.8
}

COD removal rates calculated per unit MLSS, and to

the normalised data. Paired t-tests perform a paired
two-sample Student’s t-test to determine whether a
COD removal

sample’s means are distinct. This t-test form does not

1
d )

assume that the variances of both populations are

1

equal. A paired test is appropriate when there is a

Rate (kg kg MLSS

natural pairing of observations in the samples, such

0.2
0.3
0.3
0.2
0.4
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.3
0.2

as when a sample group is tested twice}in this case,

testing micronutrients with and without nitrogen/
phosphorus balancing and with real and synthetic
Real greywater

greywater. Eight tests were carried out with con-

fidence limits of one standard deviation (Table 5);
Repeatability (%)

significant differences were found when comparing

respiration and COD removal rates of sludge with

4.0
0.9

0.9

5.7
0.8
26.7
27.8
32.1
25.6
22.9
31.3
}

and without nitrogen/phosphorus balancing, and

comparing normalised data obtained with real and
synthetic greywater. The results indicate that the
Oxygen uptake

origin of the greywater (i.e. real or synthetic) has an

1
d )

effect on the normalised data (i.e. the extent of

1

variation between a test sample and its concurrent

Rate (kg kg MLSS

control), although there was no significant effect on

1.1
1.1
0.9
1.2
1.3
0.9
0.5
0.4
0.6
0.6
0.7
0.6
1.0
0.9

the rates per unit MLSS per day. The t-test results
also show that the presence or absence of nitrogen/
phosphorus balancing in the respirometer does not
affect the normalised data, but has a significant effect
on the rates of uptake per unit MLSS.
Nutrient dose

The t-test results show that the origin of the

Mo
Co
Cu

greywater affects the behaviour of the sludge in

Zn
Fe

Al

response to nutrient dosing, in terms of the magni-

P,
P,
P,
P,
P,
P,

Mo
Co
Cu

Zn
Fe
N,
N,
N,
N,
N,
N,

Al

tude of the differences between test sludge samples

P
2706 Bruce Jefferson et al.

Table 5. Paired t-tests performed on respiration and COD removal data

Oxygen-uptake rate COD-removal rate Normalised oxygen Normalised COD

uptake removal

Real vs. synthetic greywater df=14 df=14 df=13 df=13

t stat=
5.47 t stat=
8.23 t stat=2.27 t stat=3.40
t crit=1.08 t crit=1.08 t crit=1.08 t crit=1.08
p=0.95 p=0.95 p=0.95 p=0.95
No significant No significant Significant difference Significant difference
difference difference
N/P-balanced vs. N/P-limited df=11 df=11 df=11 df=11
greywater
t stat=7.34 t stat=4.54 t stat=
0.02 t stat=
0.03
t crit=1.09 t crit=1.09 t crit=1.09 t crit=1.09
p=0.95 p=0.95 p=0.95 p=0.95
Significant difference Significant difference No significant difference No significant difference

Table 6. Summary of effects of nutrient addition

Nutrient Real greywater Synthetic greywater

With N/P balancing Without N/P balancing With N/P balancing Without N/P balancing

Co Stimulatory Stimulatory Chemical Chemical

Cu Stimulatory Stimulatory Inhibitory Stimulatory
Fe Chemical Stimulatory Stimulatory Chemical
Mo Stimulatory Stimulatory Stimulatory Chemical
Zn Stimulatory Chemical Inhibitory Stimulatory
Al Inhibitory Chemical Inhibitory Stimulatory
P } Stimulatory } Chemical
N } Stimulatory } Stimulatory

Fig. 1. Real greywater: normalised mean oxygen uptake and COD-removal rates.

and their concurrent controls. In this case, biomass per unit MLSS regardless of the source of the
receiving synthetic greywater showed a greater greywater.
response to nutrient dosing than sludge supplied
with real greywater. It can also be seen that the
addition of nitrogen/phosphorus dosing does not
REPRODUCIBILITY OF TRENDS
affect the increase in COD removal and respiration
rates in test cells compared to their controls, but that The measurements of MLSS, oxygen utilisation
nitrogen/phosphorus-balancing results in signifi- and influent and effluent COD made were used to
cantly different COD removal and respiration rates calculate respiration and COD-removal rates per unit
 Nutrient addition in greywater 2707

Fig. 2. Synthetic greywater: normalised mean oxygen uptake and COD-removal rates.

MLSS. The repeatability of the duplicates was tions are well known. The addition of nitrogen to real
calculated by dividing the difference between dupli- and synthetic greywaters, and of phosphorus to real
cates by twice the mean, a method which indicates greywater resulted in stimulation of the sludge
the percentage repeatability of a measurement. biomass, as expected when biomass operates under
Percentage values in the range
10.0 to +10.0% nutrient-limited conditions. However, adding phos-
were taken as representing good data reproducibility. phorus to synthetic greywater led to effects indicative
The rates calculated for oxygen uptake and COD of adsorption/coagulation mechanisms. This suggests
removal per unit MLSS per unit time showed that that sludge receiving synthetic greywater was under
sludge supplied with synthetic greywater required nitrogen-limited conditions, and whilst also deficient
more oxygen and degraded more COD than the in phosphorus the overall imbalance remains nitro-
sludge receiving real greywater, under every dosing gen controlled. While the water quality determinands
regime (Table 4), although a t-test showed this of synthetic greywater are a good match for real
difference not to be significant (Table 5). It can also greywater, the two substrates are nutritionally
be seen that both COD removal and oxygen uptake different. Real greywater is more variable than
rates increase on the addition of nitrogen/phos- synthetic greywater, as the occasional use of a large
phorus to any dosing regime, indicating significantly number of different hygiene products affects real
increased metabolic activity of the sludge. The results greywater composition. The C : N : P ratio of real
obtained during tests using unbalanced greywater greywater will be affected to a certain extent by the
were more reproducible than tests using nitrogen/ use of detergents with and without phosphate, but as
phosphorus-balanced greywater, and synthetic re- such detergents contribute only a part of the
sults were more reproducible than real results (Table contaminant load, and flow balancing is an inevitable
4). Results from tests using cobalt, iron, molybdenum feature of a greywater treatment collection and
and aluminium varied most with both types of treatment system, they are not expected to be
greywater. In all cases except zinc addition without dominant factor in the characteristics of real grey-
nitrogen/phosphorus balancing, oxygen-uptake rates water.
were found to be less reproducible than COD- All of the trace metals added to greywater were
removal rates. The most striking trend was the lack found in concentrations lower than the theoretical
of reproducibility in data obtained from nitrogen/ requirements (Table 1), and so stimulatory effects
phosphorus-balanced tests compared to data from could be expected from their addition. Cobalt,
unbalanced greywater tests. copper and molybdenum stimulated the bacteria in
real greywater, whether nitrogen/phosphorus ba-
lanced or not. However, while molybdenum led to
DISCUSSION stimulation in nitrogen/phosphorus-balanced syn-
thetic greywater, and copper was stimulatory in
Effects of micronutrient additions unbalanced synthetic greywater, inhibitory effects
There is a paucity of published information were seen in other cases of molybdenum, copper and
regarding the mechanistic roles of micronutrients in cobalt addition. The reasons for these effects are not
aerobic bacterial cells, while chemical effects of metal certain; cobalt is a metallic enzyme activator required
ions on activated sludge in other wastewater applica- by some bacteria which synthesise cyanocobalamin
2708 Bruce Jefferson et al.

(vitamin B12) and is also used in the production of orders of magnitude above those used in the current
carboxypeptidase (Wood and Tchobanoglous, 1975). study, where chemical rather than biological mechan-
Cobalt chloride and cobalt sulphate can stimulate the isms predominate. The inhibitory effect on respira-
synthesis of cyanocobalamin and some other B12-like tion and COD degradation was seen in all cases but
factors in aerobically fermenting sludge (Sathyanar- unbalanced real greywater.
ayana Rao and Srinath, 1961), suggesting a stimula- Small stimulatory effects were observed on the
tory action on metabolism. Doses of cobalt have addition of iron to nitrogen/phosphorus-balanced
shown potential in optimising the activated sludge synthetic greywater. In all other cases, adsorption
process with the addition of 51.0 mg l
1 cobalt. appeared to be the dominant process in the iron-
However, increases in metabolic activity may not be dosed sludge. Iron is an important element in
associated with any improvement in BOD or COD activated sludge in small quantities. Fe3+ reducing
removal, nor linked to any change in the nitrogen bacteria are found in most activated sludge mixed
and phosphorus content of the sludge (Sathyanar- cultures and Fe3+ and sulphate reduction are
ayana Rao and Srinath, 1961). These observations on important in floc formation. Iron-based co-precipi-
nitrogen and phosphorus interactions are corrobo- tants are commonly used in aerobic biological
rated in real greywater but contradicted in tests with wastewater treatment, as Fe3+ forms strong com-
synthetic greywater. Cobalt has also been reported to plexes with the condensed phosphates, pyropho-
be toxic to activated sludge (Geradi, 1986; Hunter sphate and tripolyphosphate, which may then be
et al., 1966); such inhibitory effects were not noted in removed by adsorption onto Fe3+ hydroxo-phos-
this study. phate surfaces (Jenkins et al., 1971). Work in which
Copper is found in wastewater streams from iron-based co-precipitants were added to an activated
industrial and domestic sources and exerts a toxic sludge system found adverse effects on COD and SS
effect on activated sludge (Gracia et al., 1994), an removal, although the most notable changes were to
effect noted when copper was added to nitrogen/ floc size and structure rather than effects on the
phosphorus-balanced synthetic greywater. However, microbial population (Clark et al., 1999b). The
copper addition in real and nitrogen/phosphorus- current study, in which the effects of iron indicate
limited synthetic greywater resulted in metabolic chemical reactions rather than biological effects,
stimulation, as expected in copper-limited substrates. corroborates previous literature and suggests that
Dosing low concentrations of copper into wastewater iron may not be a limiting nutrient in terms of
has been found to be beneficial in certain cases. Work bacterial metabolism.
in which 0.6–3.0 mg l
1 copper was added to indus- Zinc has been demonstrated to non-competitively
trial wastewater showed that concentrations above inhibit bacterial metabolism (Gökcay and Yetis,
the accepted toxicity threshold can chelate other 1996) and in these instances inhibited enzyme kinetics
toxicants in the effluent and improve process can be applied to the metabolism of the cells. Zinc
performance (Vandevivere et al., 1997). However, interacts with other metals (e.g. copper) to exacerbate
the effects of copper addition fade as the metal their effects and has been shown to reduce the rates of
becomes adsorbed onto other binding sites (Vande- reactions so that the kinetics of biodegradation in the
vivere et al., 1997), so it is possible that the presence of zinc and copper resembled those of
stimulatory effects noted here were temporary. biodegradation without the metals but with a very
Molybdenum can commonly be a limiting nutrient short sludge age (Beyenal et al., 1997). Clearly the
(Grau, 1991), and this appears to be the case in action of zinc on sludge biomass is strongly
greywater. In this study, addition of molybdenum influenced by the chemical conditions and as zinc
had beneficial effects on sludge supplied with all and copper were detected in real, but not in synthetic
greywaters except unbalanced synthetic greywater, greywater during ICP analysis it is reasonable to
where no conclusive effect was seen. Molybdenum expect the interactions occurring in the two types of
has been found to have stimulatory effects on greywater to differ.
activated sludge treating recalcitrant industrial waste-
water (Burgess et al., 1999b) although little is known
of its biochemical role. Reproducibility of data
The mechanism of the action of aluminium in The measurements taken during tests with real
aerobic bacteria is not known, and it appears from greywater showed more variability than those from
these results that if aluminium is required, the synthetic greywater. The difference in reproducibility
toxicity threshold is lower than previously thought. can be accounted for by the nature of the greywater.
Recently published work based on aluminium dosing The real greywater was collected daily, but its
has indicated beneficial effects on activated sludge composition was affected by trends in water use in
treatment of domestic wastewater; BOD and COD the source building, the wider range of possible
removal was improved (Clark et al., 1999a). Clark’s constituents and the water level in the collection
results contradict those in the current study, as COD sump at the time of sampling. The source used here
removal was decreased on the addition of aluminium, was naturally low in organics (mean BOD 48 mg l
1)
but they refer to coagulant concentrations of 2–3 which made the absolute rates of degradation and
 Nutrient addition in greywater 2709

respiration lower than with the synthetic greywater. independent of nitrogen and phosphorus balancing.
An additional factor was the length of time a sample Further, variations in results increased once nutrient
of real greywater might spend in the reservoir from balancing had occurred. This indicates that either
which the daily samples were taken, as previous work * after nitrogen and phosphorus balancing, other
has shown real greywater to degrade rapidly (Jeffer-
substances than those tested were imposing
son et al., 1999). Real greywater is highly site specific
process limitations,
in nature, with large variations in standard constitu-
ents such as BOD and COD. Greywater sampled or
from a range of sites around the UK has shown BOD * potentially toxic overdoses due to undetected
variations from 30 to 300 mg l
1 (Jefferson et al., variations in the existing background concentra-
1999). Synthetic greywater acts as a good analogue of tions of micro-nutrients that were being dosed.
this. However, the results presented here show
significant differences between the sources of Although the source of the greywater did not
greywater at the nutrient level. Thus, although significantly affect the rates of COD removal or
general trends appear consistent, complex nutrient oxygen uptake, it did affect the relative normalised
addition will undoubtedly be site specific. These difference between real and synthetic greywaters.
results imply that using nutrient addition in biologi- This highlights the site-specific nature of the nutri-
cal treatment of greywater may not produce results as tional characteristics of greywater, thus revealing a
consistent as those obtained in studies that employ limitation in the use of synthetic grey as a good
synthetic greywater to predict treatability in grey- medium for complex treatability studies.
water recycling systems. More variability was ob- Overall, significant improvements in greywater-
served in the measurements of oxygen-uptake rate treatment process efficacy are probable with simple
than in COD-removal rate with both real and nitrogen/phosphorus-balancing. Further, improve-
synthetic greywaters. ments are possible with more complex nutrient
Less repeatable results were obtained for cobalt, dosing but these benefits appear more spurious.
iron, molybdenum and aluminium than for other Although the independence of the effects of the
micronutrients. This may be owing to their action on complex nutrients to nitrogen/phosphorus balancing
biomass. Iron and aluminium are known to have indicate definite effects, the much greater variation
biochemical roles as well as varying coagulation in the results after nitrogen/phosphorus balancing
effects that are dependent on the prevailing chemical decrease the confidence with which these results
conditions. All four metals exhibited effects ranging could be exploited.
from stimulatory to inhibitory in the current study, The implications for greywater treatment are that
and have inconsistent effects on other biological nitrogen/phosphorus balancing alone allows the
wastewater treatment processes (e.g. cobalt–stimula- greatest potential for process enhancement. This is
tory effects, Wood and Tchobanoglous, 1975; likely to manifest itself through increasing the
Sathyanarayana Rao and Srinath, 1961}inhibitory maximum loading rates that can be applied without
effects, Geradi, 1986; Hunter et al., 1966). A decrease compromising effluent quality.
in reproducibility was observed when greywaters
were nitrogen/phosphorus balanced and then dosed
with micronutrients. This may be because the CONCLUSIONS
unbalanced greywaters were both nitrogen/phos-
Nutrient balancing has the potential to improve
phorus-limited, such that nitrogen/phosphorus-bal-
biological treatment of greywater, as nitrogen/
ancing caused another characteristic of the greywater
phosphorus-balancing eliminates the occurrence of
to become the limiting factor.
macronutrient-limiting conditions that often impede
substrate degradation. The data from the current
Impacts on process performance study demonstrate that biological treatment of the
real greywater employed can be maximised by
Nitrogen/phosphorus balancing made a significant
nitrogen/phosphorus balancing followed by supple-
difference to the rates of oxygen uptake and COD
ments of zinc or copper. Results from this study led
removal in both real and synthetic greywaters,
to the conclusions that
indicating that nitrogen and phosphorus play vital
roles in the biodegradation of greywater pollutants. * Nitrogen/phosphorus balancing can improve bio-
Increased COD removal rates were observed in 24 of logical treatment of greywater.
the 28 nutrient dosing regimes tested, suggesting that * Micronutrient addition can improve biological
macro- and micro-nutrient balancing will have a greywater treatment, but additions must be
potential role in greywater treatment and recycling in tailored to the greywater in each case.
the future. The effects of dosing with additional * Synthetic greywater does not react to nutrient
nutrients, beyond simple nitrogen and phosphorus addition in the same way as real greywater.
balancing, are unclear. The relative normalised * Complex interactions are evident during micro-
effects of each of the complex nutrients were nutrient dosing.
2710 Bruce Jefferson et al.

* The effects of micronutrients are not confined to Heddle J. F. (1980) Respirometric oxygen demand tests for
the biology of the treatment system alone. wastewater. Proceedings of the Aquatic Oxygen Seminar,
Hamilton, November. Published by the Water and Soil
Division, Ministry of Works and Development, Well-
Acknowledgements}The authors would like to express their ington, New Zealand. Water and Soil Miscellaneous
gratitude to EPSRC, Thames Water Utilities and Yorkshire Publications, no. 29.
Water Services Ltd. for financial support of this research. Hunter J. V., Genetelli E. J. and Gilwood M. E. (1966)
Temperature and retention time relationships in the
activated sludge process. Proceedings of the 21st Industrial
REFERENCES Waste Conference, Purdue University, Indiana, vol. 121,
pp. 953–963.
Annaka T. (1977) Upgrading of existing sewage treatment Jefferson B. and Judd S. (1999). Water recycling: asking the
plants by chemical addition to aeration tank. Proceedings right questions? Water Environ. Int. 15B, 15–16.
of the fifth US-Japan Conference on Sewage Treatment Jefferson B., Laine A. and Judd S. (1999) Technological
Technology, Tokyo, April, pp. 1–17. requirements for water recycling. Environ. Prot. Bull. 60,
APHA (1992) Standard Methods for the Examination of 8–14.
Water and Wastewater, 18th ed. American Public Health Jenkins O., Fergusson J. F. and Menar A. B. (1971)
Association, Washington, DC, USA. Chemical processes for phosphate removal. Water Res. 5,
Beardsley M. L. and Coffey J. M. (1985). Bioaugmentation: 369–387.
optimizing biological wastewater treatment. Pollut. Laine A., Jefferson B., Judd S. and Stephenson T. (1999)
Engng. December, 30–33. Membrane bioreactors and their role in wastewater reuse.
Benefield L., Lawrence D. and Randall C. (1979) The effect Water Sci. Technol. 41(1), 197–204.
of sludge viability on biokinetic coefficient evaluation. Metcalf and Eddy, Inc. (1991) Wastewater Engineering}
J. Water Pollut. Control Fed. 51, 187–194. Treatment, Disposal and Reuse. In McGraw-Hill Series in
Beyenal N. Y., Özbelge T. A. and Özbelge H. O. (1997) Water Resources and Environmental Engineering, 3rd ed.
Combined effects of Cu2+ and Zn2+ on activated sludge G. Tchobanoglous and F. L. Burton. (Eds.), Macgraw
process. Water Res. 31, 699–704. Hill, New York.
Blackall L. L., Seviour E. M., Bradford D., Stratton H. M., Nicholas D. J. D. (1963). Inorganic nutrient nutrition of
Cunningham M. A., Hugenholts P. and Seviour R. J. microorganisms. In: Plant Physiology III, ed. F. C
(1996) Towards understanding the taxonomy of some of Stewart, Academic Press, New York, pp. 363–375.
the filamentous bacteria causing bulking and foaming in Pala A. I. and Sponza D. T. (1996) Biological treatment of
activated sludge plants. Water Sci. Technol. 34(5–6), petrochemical waste waters by Pseudomonas spp. added
137–144. activated sludge culture. Environ Technol. 17, 673–686.
Burgess J. E., Quarmby J. and Stephenson T. (1999a) The Sathyanarayana Rao S. and Srinath E. G. (1961) Influence
role of micronutrients in biological treatment of industrial of cobalt on the synthesis of vitamin B12 in sewage during
effluents using the activated sludge process. Biotechnol. aerobic and anaerobic treatment. J. Sci. Ind. Res. 20(c),
Adv. 17, 49–70. 261–265.
Burgess J. E., Quarmby J. and Stephenson T. (1999b) Singleton I. (1994) Microbial metabolism of xenobiotics:
Micronutrient supplements to enhance biological waste- fundamental and applied research. J. Chem. Technol.
water treatment of phosphorus-limited industrial efflu- Biotechnol. 59(1), 9–23.
ents. Trans. Inst. Chem. Eng. 77(B), 199–204. Soddell J. A. and Seviour R. J. (1996) Growth of an
Clark T., Stephenson T. and Arnold-Smith A. K. (1999a) activated sludge foam-forming bacterium, Nocardia
The impact of aluminium based co-precipitants on the pinensis on hydrophobic substrates. Water Sci. Technol.
activated sludge process. Trans. Inst. Chem. Eng. 77(B), 34(5–6), 113–118.
31–36. Speitel G. E. and Digiano F. A. (1988) Determination of
Clark T., Burgess J. E., Stephenson T., and Arnold-Smith microbial kinetic coefficients through measurement of
A. K. (1999b) The influence of iron-based co-precipitants initial rates by radiochemical techniques. Water Res. 22,
on activated sludge biomass. Trans. Inst. Chem. Eng. 829–835.
78 (B5), 405–410. Speitel G. E. and Segar R. L. (1995) Cometabolism in
Franta J., Wilderer P. A., Miksch K. and Sykora V. (1994) biofilm reactors. Water Sci. Technol. 31(1), 215–225.
Effects of operation conditions on advanced COD USEPA (1996) Prevention, Pesticides and Toxic Substances
removal in activated sludge systems. Water Sci. Techol. (7101). EPA 712-C-96-301. Fate, transport and trans-
29(7), 189–192. formation test guidelines. OPPTS 835.3220 (Porous pot
Geradi S. M. H. (1986) Effects of heavy metals upon the test). USEPA, Cincinnati.
biological wastewater treatment process. Public Works Valo R., Apajahalati J. and Salkinoja-Salonen M. (1985)
117(6), 77–80. Studies on the physiology of microbial degradation of
Gökcay C. F. and Yetis U. (1996) Effect of nickel(II) on the pentachlorophenol. Appl. Microbiol. Biotechnol. 21,
biomass yield of the activated sludge. Water Sci. Technol. 313–319.
34(5–6), 163–172. Vandevivere P., Ficara E., Julies E. and Verstraete W.
Gostick N. (1991) A study of the effect of substrate (1997) Adding copper to detoxify wastewater. Proceedings
composition on the settlement of activated sludge. of the Environmental Technology International Symposium
Ph.D. Thesis, Cranfield University, UK. April 21–23, Gent, Belgium, 405–411.
Gracia M. -P., Salvadó H., Ruis M. and Amigo J. M. (1994) Water Research Centre (1978) Porous Pot Method for
Effects of copper on ciliate communities from activated Assessing Biodegradability. Technical Report TR70,
sludge plants. Acta Protozool. 33(4), 219–226. Water Research Centre, Medmenham, UK.
Grau P. (1991) Criteria for nutrient - balanced operation of Wood D. K. and Tchobanoglous G. (1975) Trace elements
activated sludge process. Water Sci. Tech. 24(3–4), in biological waste treatment. J. Water Pollut. Control
251–258. Fed. 47, 1933–1945.