RAZISKAVE IN RAZVOJ
RAZISKAVE IN RAZVOJ
Papir za notranjost revije PAPIR je prispevala Papirnica Vev~e, Biomatt 115 g/m
2
25
Papir za notranjost revije PAPIR je prispevala Papirnica Vev~e, Biomatt 115 g/m
2
24
EFFLUENT TREATMENT BY MIXED ALGAE-
BACTERIAL BIOMASS – PILOT EXPERIENCE
^IŠ^enJe OdP adne vOde Z meŠanO BIOmasO 
alG In BakterIJ – PIl OtnI POskus
Janja Zule
1
, Gabriele Weinberger
2
, Corinna Hentschke
2
, 
Ricardo Pereira
3
, Alp Erguensel
3
, Quentin Thiebaut
4
1. INTRODUCTION
Paper mills are obliged to reduce 
emissions and improve their effluent 
quality due to strict EU environmental 
legislation. Many companies have 
already implemented biological 
treatment as secondary stage waste 
water purification process in order to 
improve overall efficiency and thus 
comply with environmental standards. 
Conventional aerobic treatment provides 
good results, however on the other 
hand it is quite energy demanding. 
For this reason innovative techniques 
intended to improve performance and 
reduce costs are constantly observed 
and examined. One of possible ways 
of how to make biological cleaning 
more effective is introduction of 
microalgae into the conventional stage 
using activated bacterial biomass (1-
5). Heterotrophic bacterial organisms 
decompose organic impurities in water 
to CO
2 and H 2O for which they need 
oxygen which is conventionally supplied 
via aeration. An alternative source of 
oxygen supply may be autotrophic 
microalgae which consume available 
IZVLE^EK
Raziskali smo možnost uporabe alg za ~i{~enje odpadnih vod v papirni{tvu. Gre za nadgradnjo konvencionalnega aerobnega 
~i{~enja odpadne vode z uporabo me{ane algno-bakterijske biomase. Specifi~ne lastnosti mikroalg lahko v simbiozi z bakterijami 
pripomorejo k bolj{emu ekonomskemu in ekolo{kemu efektu ~i{~enja. Zaradi izmenjave kisika in ogljikovega dioksida med 
avtotrofnimi algami in heterotrofnimi bakterijami ni potrebno zunanje prezra~evanje, hkrati pa se izkori{~a sposobnost alg, da 
vežejo toksi~ne snovi, ki jih sicer bakterijski mikroorganizmi ne morejo razgraditi. Predstavljeni so rezultati pilotnega poskusa 
~i{~enja papirni{ke odpadne vode z uporabo nove tehnike. Raziskave so potekale v okviru mednarodnega projekta „Albaqua“.
Klju~ne besede: ~i{~enje odpadne vode, algno-bakterijska biomasa, prezra~evanje, flokulacija in posedanje, u~inek razgradnje.
ABSTRACT
Possible application of algae for effluent treatment in the papermaking process was examined. The conventional aerobic waste 
water treatment process may be upgraded by utilization of a mixed algae-bacterial biomass. Improved economic and ecologic 
efficiency is expected on account of specific properties of microalgae and symbiotic action of both microorganisms. Due to 
interchange of oxygen and carbon dioxide between autotrophic algae and heterotrophic bacteria no external aeration is needed, 
while on the other hand the algae present are able to consume some toxic compounds which can not be digested by bacteria. 
The novel technology of effluent treatment and the results of a pilot test which was performed in real papermaking conditions 
are presented. The research was conducted within the international project “Albaqua” (http://www.cornet-albaqua.eu).
Key words: effluent treatment, algae-bacterial biomass, aeration, flocculation and settling properties, degradation performance..
CO 2 (provided by bacterial action) and 
H 2O during photosynthesis for which 
they use energy in the form of sunlight. 
Oxygen is released in the process 
as waste product. The latter can be 
conveniently used by bacteria for their 
heterotrophic degradation of organic 
impurities. Symbiotic co-existence of 
two types of microorganisms within 
the same effluent can be exploited for 
improved cleaning performance. Due to 
efficient interchange of gaseous products 
between algae and bacteria no external 
aeration should be required, because all 
the necessary oxygen is to be provided by 
algae which at the same time efficiently 
consume CO
2 released by bacteria (figure 
1). In addition, improved cleaning 
efficiency is expected due to specific 
ability of microalgae to consume toxins, 
heavy metals as well as nitrogen and 
phosphorous compounds.
The main objective of our research was 
to introduce and optimize this innovative 
technique of biological effluent treatment 
first on laboratory level and than transfer 
the obtained experience and knowledge 
to pilot scale in real papermaking 
conditions in order to evaluate its 
potential and efficiency as alternative, 
energy saving effluent treatment 
technique.
The research was performed within EU 
sponsored Cornet “Albaqua” project 
(Combined algal and bacterial waste 
water treatment for high environmental 
quality effluents) which was coordinated 
by PTS (Papiertechnische Stiftung, 
Münich), and lasted 2 years. The other 
members of the project consortium 
were TUHH (Technische Universität 
Hamburg-Harburg), Celabor, Belgium, 
Pulp and Paper Institute, Slovenia and 
Paper Research Institute with Technical 
University, Hungary.
2. EXPERIMENTAL
In the first stage of the research it was 
necessary to examine which algae species 
were naturally present in papermaking 
waters, meaning that they were already 
adapted to technological conditions on 
the papermaking machines. Among 6 
different species found, the microalga 
Chlorella Vulgaris proved to be suitable 
for further experiments, because of its 
specific properties, such as relatively 
quick growth and good flocculation with 
bacterial biomass. Subsequently algae 
were cultivated on laboratory level in 
order to produce the relevant quantity 
of biomass for further experiments. The 
experimental conditions for optimal 
growth, such as light intensity, mixing 
velocity, growth medium, pH and 
temperature, were carefully observed and 
optimized.
Effluent treatment tests with mixed 
algae-bacterial biomass were performed 
in small laboratory bioreactors after 
optimization of experimental parameters, 
such as hydraulic retention time (HRT), 
biomass concentration or dissolved and 
suspended matter (DSM), algae-bacteria 
proportion, fixation of biomass on 
carriers, nutrition load (C:N:P), sludge 
retention time (SRT), sedimentation time, 
O
2 concentration, temperature, pH and 
light intensity. Real effluents from several 
different paper production processes 
were used in laboratory experiments 
in order to determine purification 
performance.
Pilot tests were performedin the paper 
mill Gori~ane using waste water from 
different production programs. The paper 
mill produces different printing paper 
grades from primary fibers. So far it has 
only chemo-mechanical waste water 
treatment plant which will have to be 
upgraded by a secondary stage water 
cleaning in order to improve effluent 
quality according to future legislation 
requirements. The schematic presentation 
of the pilot plant is shown in figure 2.
The pilot was equipped with two effluent 
collecting tanks (500 l), central bioreactor 
(500 l) equipped with mechanical stirrer 
and sensors for on-line measurement 
of temperature, pH, conductivity, redox 
potential and dissolved oxygen. The 
working volume of the bioreactor was 
340 l. The pilot was operated for four 
months from July till November. In the 
beginning it functioned in batch mode 
for one month, while afterwards its 
operation was continuous. The volume of 
mixed algae-bacterial biomass was 116 l 
while the volume of effluent to be treated 
was 224 l. Effluent retention time (HRT) 
was 2 days in batch mode. 
A sedimenter unit (40 l) was added when 
the pilot worked in continuous mode 
in which case the active volume of the 
bioreactor was 300 l, the sedimenter 
volume 40 l, the daily effluent inflow and 
outflow was between 80 l and 90 l and 
the sludge return flow was 250 l daily. 
After some time the daily inflow/outflow 
volume of effluent increased to 195 l 
daily. During the pilot experiments some 
nutrients in the form of N and P salts as 
well as some fresh algae had to be added 
Figure 1: Combined action of bacterial biomass and algae in effluent treatment plant
Figure 2: Effluent treatment pilot plant using mixed algae-bacterial biomass
to the reaction mixture in order to keep it 
in optimal condition. Experimental pilot 
parameters are collected in table 1.
The efficiency of the pilot performance 
was determined by measuring the 
following effluent parameters before and 
Parameter Value
DSM (biomass concentration) 0,5–2,5 g/l
oxygen concentration 2–8 mg/l O2
temperature 15–30 ˚C
pH 7,5–9,5
light natural daylight
HRT (hydraulic retention time) 1,8–3, 8 days
nutrients N, P compounds
weather sunny, cloudy, rainy
Table 1: Pilot parameters
after the treatment: COD, BOD 5, DOC 
(dissolved organic carbon), suspended 
solids, nitrogen and phosphorus 
compounds, such as NH
4, NO 2, NO
3
, PO 4 
as well as total phosphorus and nitrogen. 
ISO standard methods were used for the 
analyses.
Biomass concentration within the 
bioreactor was determined by DSM 
measurement. Algae content was 
characterized by spectrophotometric 
determination of chlorophyll 
concentration (6).
Biomass condition was also observed 
by microscopy in order that different 
microorganisms within the mixture were 
identified and their abundance evaluated. 
The condition of biomass in the reactor 
was monitored 5 times a week.
24 measurements of chemical parameters 
of input and output effluents were 
performed during pilot experiment. In 
batch operation incoming and outcoming 
water samples were collected at the start 
and in the end of each batch while the 
corresponding samples were collected 
twice a week during continuous work.

RAZISKAVE IN RAZVOJ
RAZISKAVE IN RAZVOJ
Papir za notranjost revije PAPIR je prispevala Papirnica Vev~e, Biomatt 115 g/m
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Papir za notranjost revije PAPIR je prispevala Papirnica Vev~e, Biomatt 115 g/m
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3. RESULTS AND DISCUSSION
It was established by laboratory 
experiments that algae could excellently 
integrate with activated sludge to form 
flocs. The latter showed good settling 
performance which was necessary for 
efficient separation of treated effluent 
from the remaining biomass. Fixation of 
mixed algae-bacterial mass on different 
plastic carriers was unsatisfactory. The 
microorganisms used did not need much 
time to adapt to experimental conditions 
including effluent characteristics. An 
exception were effluents from paper 
productions based on wood containing 
raw materials, most probably because 
of biocidal effects of some wood 
extractives. Other algae also appeared 
within flocs, meaning that a natural 
mix of microorganisms developed with 
time. In some cases Chlorella was the 
predominating species. Laboratory 
treatments of paper mill effluents 
with mixed biomass indicated good 
degradation results as the purification 
efficiency in most cases ranged between 
70 % and 80 %. The calculations 
were based on COD removal. The best 
performance was observed when HRT 
was adjusted between 1 and 3 days, 
depending on the waste water COD, 
however for highly polluted waters 
(COD > 800 mg/l) this technology can 
not be suggested. The most suitable 
biomass concentration was between 1,5 
g/l and 2,5 g/l and SRT value (sludge 
retention time) from 16 to 20 days. 
A sedimentation time of 2 to 3 hours 
could be easily achieved. Stirrers were 
needed in the system in order to keep the 
biomass suspended and ensure that all 
algae did come into contact with light. 
No external aeration was needed. Oxygen 
concentration was sufficient during night 
as well. Additional aeration with blowers 
favoured heterotrophic bacteria so it had 
to be avoided. The colour of the biomass 
(intensive green) was a good sign of 
system's health.
The laboratory results were confirmed by 
pilot experiment where overall treatment 
efficiency was similarly very high. COD 
and BOD values of inflow and outflow 
waters are presented in figures 3 and 4. 
The first 5 results were obtained in batch 
operation.
BOD values representing biodegradable 
organic compounds were also greatly 
reduced, in most cases the results did not 
exceed 3 mg/l which was quantification 
limit.
Effluents from the paper mill did not 
contain much nitrogen and phosphorous 
compouds, such as nitrites, nitrates, 
ammonia and phosphates. The initial 
values for N-NH 4, N-NO 3 and N-NO 2 
never exceeded 1,5 mg/l, however in 
most cases they were much lower. 
Inorganic phosphates P-PO 4 were never 
even detected.
The results of chlorophyll determination 
are expressed as mg per volume of 
reactor mixture and are presented in 
figure 5.
Chlorophyll concentrations moved 
between 2 mg/l and 12 mg/l which 
depended on various technological 
conditions. Concentrations were 
higher when there was enough N and 
P nutrients. The inflow waters were 
extremely poor in P and N compounds 
0
50
100
150
200
250
300
350
400
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Measurement
mg/l
input
output
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20
40
60
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100
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140
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Measurement
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input
output
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0,5
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Measurement
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d.m.
ash
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Measurement
mg/l
Figure 3: COD values of inflow and outflow effluents
Figure 4: BOD values of inflow and outflow effluents
Figure 6: Dry matter and ash content of the bioreactor mixture
Figure 5: Chlorophyll content of the reactor mixture
It is evident from the figure 3 that initial 
COD values differed significanly. The 
highest COD value of the incoming 
effluent was 337 mg/l and the lowest 
70 mg/l. In most cases COD values of 
the output effluents did not exceed 
30 mg/l which was the quantification 
limit. This meant that the efficiency of 
COD removal was very high despite 
different production programs and 
consequently differently polluted waters. 
The removal of soluble organic material 
expressed as DOC was also very high as 
in most cases final effluent values after 
treatment approached 5 mg/l which was 
quantification limit.
so occasional addition of nutrients was 
necessary. Decrease of chlorophyll could 
have been partly ascribed also to the 
presence of different insects, such as 
larvae which abundantly used algae for 
food. Larvae had to be regularly physically 
removed.
Dry matter and ash content of the 
bioreactor mixture is presented in figure 6.
The values ranged between 0,9 g/l and 
2,5 g/l with average value being 1,6 g/l. 
The lowest results were obtained when 
some degradation of biomass took place 
due to lack of nutrients. Inorganic portion 
of the dry reactor biomass averagely 
amounted to about 50 % of the total 
value. So only about half (44 – 63 %) of 
the reactor contents actually belonged 
to mixed algae-bacteria organic material. 
Most of inorganic matter was composed 
of carbonates, silicates, chlorides and 
sulphates (addition of Al2(SO4)3 in the 
first stage of water treatment) as well as 
the remaining nutrients.
A very important factor of the biomass 
condition and thus of the system 
performance was quantitative relation 
between algae and bacteria in the 
organic portion of the reactor mixture. 
The content of algae in the symbiotic 
mixture with bacteria moved between 
40 % and 70 % which indicated that the 
system was most of the time in good 
equilibrium (most values around 60 % in 
favour of algae).
Microscopic examination of the reactor 
biomass showed that there were 
other microorganisms present, such as 
protozoa, flagellates, vorticella, euplotes 
and others, however Chlorella Vulgaris 
distinctively predominated.
4. CONCLUSIONS
Effluent treatment with mixed algae-
bacteria biomass worked well for the 
paper mill effluents without wood 
extractives at low and medium loads. 
External aeration was not required as 
sufficient oxygenation was easily achieved 
by algae. Light intensity proved not to be 
a major factor as the pilot system worked 
also in dark and rainy days. Temperature 
and mixing had to be regularly monitored 
and optimized. Cooling (in summer) and 
heating (in autumn) within the bioreactor 
were conveniently achieved by available 
river water (18 ˚C) and effluent (37 ˚C). 
The system was in equilibrium when 
algae content was between 40 % and 
60 % in the mixed organic biomass. The 
major threat for algae represented insects 
(Chironomidae larvae) that consumed 
algae, so physical barrier (net) had to 
be applied for protection. In additon, 
it was established by some preliminary 
experiments that the generated mixed 
sludge could be used as substrate for 
biogas production, especially if it was 
mixed with primary sludge in order to 
improve C/N ratio.
Further research will be needed to 
optimize this novel energy saving effluent 
treatment technology and transfer it to 
the industrial scale.
Acknowledgement
Authors would like to express gratitude 
to their national funding agencies for 
financial support. 
Authors also greatly appreciate 
professional assistance of Borut Lazar 
and Robert Reinhardt from the company 
Algen Ljubljana by conducting pilot plant 
experiment. 
LITERATURE
1. SAFONOVA, E., KVITKO, K., IANKEVITCH, 
M., SURKO, L., REISSER, W. Biotreatment of 
industrial wastewater by selected algal-
bacterial consortia. Engineering in Life Science, 
2004, Band 4, Heft 4, str. 347–353.
2. PRECHTL, S., JUNG, R. Cost effective 
wastewater treatment with and algae reactor. 
Das Gas- und Wasserfach, 2002, Band 143, 
Heft 12, str. 872–877.
3. ASCHE, W. Utilisation of microalgae – 
natural substances from underwater. Journal 
Seifen, Öle, Fette, Wachse, 1996, Band 122, 
Heft 1, str. 28–29.
4. MEDINA-RODRIGUEZ, L. M. Floc formation 
in wastewater treatment systems using algal 
bacterial symbiosis. Hamburger Berichte zur 
Siedlungswasserwirtschaft, 2006, Band 59, 
str. 1–110.
5. NEIS, U., GUTZEIT, G. Biologische 
Abwasserreinigung mit symbiotischer Algen-
Bakterien-Biomasse. Hamburger Berichte zur 
Siedlungswasserwirtschaft, 2002, Band 40, str. 
159–168.
6. BARNES, B. A reappraisal of the use of 
DMSO for the extraction and determination 
of chlorophylls a and b in lichens and higher 
plants. Environmental and Experimental 
Botany. 1992, vol. 32, no. 2, str. 85–100.
1
Janja Zule, 
In{itut za celulozo in papir Ljubljana Slovenia
2
Gabriele Weinberger, Corinna Hentschke, PTS 
Papiertechnische Stiftung, München, Germany
3
Ricardo Pereira, Alp Erguensel, TUHH, Technische 
Universität, Hamburg-Harburg, Germany
4
Quentin Thiebaut, CELABOR SCRL, 
Herve, Belgium