Monitoring of microbial indicator groups in organically heavily loaded wastewater treatment systems by using RIDA@COUNT kits

Abstract

The versatility and robustness of RIDA®COUNT test plates have been tested in municipal wastewater, and from a decentralised source separation treatment plant, blackwater, grey water and compost derived from blackwater solids. The organic loads where high (BOD5 330-2400 mg/1) with counts of colony-forming units (CFU) exceeding 108 per ml. To observe the treatment efficiency, in the same sample the following microbial indicators were used: Escherichia coli, total coliform bacteria, Enterobacteriaceae, total heterotrophic bacteria, and yeasts and moulds. In the two wastewater treatment plants, the efficient removal of pathogens of enteric origin was demonstrated - more specifically, E. coli was drastically reduced, in some cases below the detection limit. Simultaneous use of different RIDA®COUNT culture media gives detailed data on the abundance and presence of different bacterial subgroups which can be correlated with environmental parameters. The bacteria of nonenteric origin in the blackwater treatment system frequently correlated with physical and chemical parameters (p< 0.05), but bacteria of enteric origin to a lesser extent. The RIDA®COUNT test plates can substitute agar plating, to a certain level, for microbial investigation of organically rich solid and liquid wastes. Relative standard deviations for total bacterial counts on RIDA®COUNT were for compost 17.2% and for blackwater 27.9%, respectively. Nevertheless, commercially available biological kits, such as RIDA®COUNT can be applied for routine monitoring of bacterial indicators in wastewater treatment plants as an alternative method to agar counting.

Full text

© by PSP Volume 21 – No 12a. 2012 Fresenius Environmental Bulletin
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MONITORING OF MICROBIAL INDICATOR GROUPS
IN ORGANICALLY HEAVILY LOADED WASTEWATER
TREATMENT SYSTEMS BY USING RIDA®COUNT KITS
Andreea Oarga1,*, Tjaša G. Bulc2, Petter D. Jenssen3 and Janez Mulec4
1University of Nova Gorica, Vipavska 13, 5000 Nova Gorica, Slovenia,
2University of Ljubljana, Faculty of Civil and Geodetic Engineering, Institute of Sanitary Engineering,
Faculty of Health Sciences, Department of Sanitary Engineering, Zdravstvena pot 5, 1000 Ljubljana, Slovenia
3Norwegian University of Life Sciences, Department for Plant and Environmental Sciences, P.O. Box 5003, 1432 Aas, Norway
4Karst Research Institute, Research Centre of the Slovenian Academy of Sciences and Arts, Titov Trg 2, 6230 Postojna, Slovenia
ABSTRACT
The versatility and robustness of RIDA®COUNT test
plates have been tested in municipal wastewater, and from
a decentralised source separation treatment plant, blackwa-
ter, greywater and compost derived from blackwater solids.
The organic loads where high (BOD5 330-2400 mg/l) with
counts of colony-forming units (CFU) exceeding 108 per
ml. To observe the treatment efficiency, in the same sample
the following microbial indicators were used: Escherichia
coli, total coliform bacteria, Enterobacteriaceae, total het-
erotrophic bacteria, and yeasts and moulds. In the two waste-
water treatment plants, the efficient removal of pathogens of
enteric origin was demonstrated - more specifically, E. coli
was drastically reduced, in some cases below the detec-
tion limit. Simultaneous use of different RIDA®COUNT
culture media gives detailed data on the abundance and
presence of different bacterial subgroups which can be corre-
lated with environmental parameters. The bacteria of non-
enteric origin in the blackwater treatment system frequently
correlated with physical and chemical parameters (p<
0.05), but bacteria of enteric origin to a lesser extent. The
RIDA®COUNT test plates can substitute agar plating, to
a certain level, for microbial investigation of organically
rich solid and liquid wastes. Relative standard deviations
for total bacterial counts on RIDA®COUNT were for
compost 17.2% and for blackwater 27.9%, respectively.
Nevertheless, commercially available biological kits, such
as RIDA®COUNT can be applied for routine monitoring
of bacterial indicators in wastewater treatment plants as
an alternative method to agar counting.
KEYWORDS:
wastewater, monitoring, RIDA®COUNT, microbial indicators.
* Corresponding author
1 INTRODUCTION
Microbial presence and organic load are among the key
parameters that must be taken into account before releas-
ing treated wastewater into the environment [1], especially
in sensitive areas such as mountains, karst, wetlands and
coast lines [2, 3]. On the other hand, domestic wastewater,
and faeces and urine especially, represent an important
source of nutrients that can be used in agriculture and
forestry [4]. That is why it is important to treat such efflu-
ents in a sustainable manner, e.g. use a minimum of energy
and water for transport and treatment, and recycle nutrients
[5]. Several systems for treatment of human excrement in
sensitive areas are available or under development [2, 6],
but efficient monitoring is often expensive, time consum-
ing and not easily executed, and demands training of labo-
ratory staff [1]. Currently there are a few commercially
available biological kits which require little equipment, are
easy to use, and the results of which are easy to read, there-
by simplifying the procedure. One of the most important
parameters to monitor is coliform bacteria and Escherichia
coli (Table 1).
In this paper we present and discuss an application of
RIDA®COUNT test plates for remote wastewater treat-
ment facilities. The plates were proven satisfactory for
insight on culturable microbial groups, and regular moni-
toring at wastewater treatment plants. RIDA®COUNT test
kit (R-Biopharm, Germany, http://www.r-biopharm.com)
is a ready-to-use and low cost test kit for quantitative
microbial detection which can be readily used by staff
with basic training in microbiology. Medium sheet
RIDA®COUNT plates have primarily been developed for
the alimentary industry [7] to check the critical points
during the industrial processes, and they give comparable
quantitative results with agar and Petri film media for food
or feed samples [8, 9]. RIDA®COUNT was also suc-
cessfully applied to monitor a microbially polluted river
in a cave [10].

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TABLE 1 - Some examples of easy-to-use kits for detection and enumeration of E.coli and coliforms based on simultaneous detection of ß-D-
glucuronidase and ß-D-galactosidase
Product Equipment
needed
Extra mate-
rial needed
Media status/Steps prior
sampling
Estimated time
for media
preparation (h)
Skills needed Time for
reading
results (h)
Web reference
3M™ Petrifilm™ E.
coli/Coliform Count
Incubator 37°C
Refrigerator 4°C
none Adjustment of pH 0 Advanced
basic
24 http://solutions.3m.com
.au
Bio-Rad
RAPID'E.coli2
Incubator 35°C
Petri plates
Sterile media/
Aseptical plate pouring
0.5 Advanced
basic
18-24 http://www.bio-rad.com
Colichrom-NPS Incubator 37°C
Petri plates
Sterile media/
Aseptical plate pouring
0.5 Advanced
basic
18 http://www.dr-moeller-
und-schmelz.de
RIDA®COUNT
E.coli/Coliform
Incubator 35°C none Immediate use for water
samples
0 Basic 24 http://www.r-
biopharm.com
SimPlate Incubator 35°C
UV lamp 365nm
none Rehidration of medium 0.1 Advanced
basic
48 http://www.idexx.com
TABLE 2 - Summary of treatment systems and samples characteristics
System/Population unit Sample Type Description
BW1 Liquid BW input from toilets in peat filters
BW2 Liquid BW output from peat filters
BW3 Liquid BW from biofilter after 6 h of recirculation
BW4 Liquid BW from evaporation module
GW1 Liquid GW input in HFTW from sinks
GW2 Liquid GW output from HFTW
GW3 Liquid GW output from VFTW
SW1 Solid Mixture of solid waste prior composting
Decentralized source separation/100
SW2 Solid Compost after 40 days
MW1 Liquid MW input from the city Centralized municipal/15,000
MW2 Liquid MW output after biological treatment processes
BW-blackwater; GW-greywater; HFTW- horizontal flow treatment wetland; MW- municipal wastewater; SW- solid waste;
VFTW- vertical flow treatment wetland
In addition to quantification of different microbial in-
dicator groups, correlations between microorganisms and
environmental parameters are frequently needed. These
simple analyses can provide data to the appropriate per-
sonnel at the plants and enable the optimization of down-
stream treatment processes and/or to manipulate the treat-
ment strategy, such as oxygen availability, pH, and tem-
perature.
In this study we have studied total counts of hetero-
trophic aerobic bacteria, Escherichia coli, total coliforms,
Enterobacteriaceae, and counts of cultivable yeasts and
moulds in municipal wastewater, blackwater (toilet waste),
greywater (hand washing water), and compost derivated
from blackwater solids. RIDA®COUNT microbial indica-
tor groups were applied to observe microbial fluxes in
relation to environmental parameters, and to observe the
reduction of bacteria indicators (e.g. E.coli and Entero-
bacteriaceae) before release into the environment.
2 MATERIALS AND METHODS
2.1 Description of the wastewater samples and treatment
systems
The system used in this study is located in a sensitive
coastal Mediterranean area in Slovenia (Saline Natural
Park). The system design is based on separate treatment of
black- and greywater [3]. The blackwater is collected using
low flush vacuum toilets (sample BW1). The treatment
module is composed of a peat filter (sample BW2) where
separation of solid and liquid fraction takes place. The
liquid fraction passes over the biofilter filled with expanded
clay (sample BW3) and recirculates for six hours per day to
achieve a higher microbial mineralization of organic
matter. After the liquid circulation terminates, the liquid
fraction (sample BW4) flows to the evaporation module
where it is heated up (>60°C) to achieve disinfection, and is
finally evaporated in the environment. The peat filters which
contain blackwater solids were composted with bran and
bark in a mass ratio of 3:1. A sample designated as SW1
represented the initial mixture of solid waste before com-
posting and sample SW2 after 40 days of composting. In
the greywater hybrid treatment wetland [11, 12] the grey-
water (GW1) is treated in a horizontal flow treatment wet-
land (sample GW2) followed by a vertical flow treatment
wetland – (sample GW3). In a treatment wetland, biofilms
in association with plant roots stabilize and enhance the
treatment process [13]. Samples were taken in August and
September 2011 when the highest number of tourist vis-
ited the park, which resulted in a high load of black and
greywater. In this period a total of 2192 flushes from
vacuum toilets were recorded, with an average of 1.3 l per
flush giving the total input of 2849.6 l of blackwater.
Compost samples were taken at the beginning (September
2011) and after 40 days of composting process (Table 2).
At each sampling campaign approximately 0.5 l of mate-
rial was aseptically taken. Samples were transferred in a
cool box to the laboratory and subdivided to execute mi-
crobial and chemical analyses. Samples were processed in
triplicates (Figure 1).

© by PSP Volume 21 – No 12a. 2012 Fresenius Environmental Bulletin
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FIGURE 1 - Experimental design of wastewater treatment plants with indicated sampling points: BWI – blackwater input (sample BW1); PF
– peat filter (sample BW2); BF – biofilter (sample BW3); ET – evaporation module (BW4); C – compost (samples SW1 and SW2); GWI –
greywater input (sample GW1); HFTW – horizontal flow treatment wetland (sample GW2); VFTW – vertical flow treatment wetland (sam-
ple GW3); MWI – municipal wastewater input (sample MW1); TP – treatment pool; MWO – municipal wastewater output (sample MW2);
blank arrow – liquid flow; black arrow – solid flow.
As an example of another type of organically polluted
water, samples from the municipal wastewater treatment
plant from Postojna, Slovenia, were introduced into the study
(November 2011). The plant in Postojna treats municipal
waters (sample MW1) based on mechanical separation and
biological oxidation (sample MW2 after the treatment) for
15,000 population units (http://www.kovodpostojna.si/)
(Figure 1).
2.2 Cultivation-based analysis and reading results
RIDA®COUNT plates are medium sheets, coated
with dry culture media which are activated when liquid
samples (1 ml) are applied. Different plates were used
to enumerate microorganisms from liquid and solid
samples: total counts of heterotrophic aerobic bacteria
(RIDA®COUNT Total Aerobic Count), Escherichia coli
and total coliforms (RIDA®COUNT E.coli/Coliform),
enterobacteria (RIDA®COUNT Enterobacteriaceae), and
cultivable yeasts and moulds (RIDA®COUNT Yeast&Mold
Rapid). On RIDA®COUNT test plates, microorganisms
form colonies that become coloured after the enzymatic
reaction with the substratum in the media. We combined
several different RIDA®COUNT test kits for the same
sample by simultaneous use of different selective growth
media allow us to extract more information on the pres-
ence of specific microbial groups. For example, the
RIDA®COUNT E.coli/coliforms kit gives the number of
E.coli (violet colonies) and non- E.coli coliforms (blue
colonies), while the RIDA®COUNT Enterobacteriaceae
kit gives the total number of enterobacteria: Cedecea,
Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella,
Kluyvera, Morganella, Proteus, Rahnella, Salmonella, Ser-
ratia, Shigella and Yersinia (http://www.r-biopharm.com).
All samples were aseptically applied on the
RIDA®COUNT plates, when possible in the field, and in-
cubated at 35°C for 24 h for bacteria and at 25°C for 48 h
for yeasts and moulds (http://www.r-biopharm.com). After
the incubation period, microbial colonies were enumer-
ated and expressed as colony-forming units (CFU) per
gram for solid material or CFU/ml for liquid samples. The
solid samples were resuspended in physiological solution
and appropriate dilution was applied on RIDA®COUNT
plates.
The comparative method for RIDA®COUNT Total
(% against Petrifilm AC plate) ranges between 94-110%
and is considered satisfactory (Quality Management Sys-
tem certified ISO 9001, ISO 13485, www.r-biopharm.com).
In this study, to check the repeatability and efficiency of
RIDA®COUNT plates, nutrient agar (Sigma, USA) was
tested in triplicates against RIDA®COUNT Total for liquid
and solid blackwater samples. Nutrient agar is widely used
to isolate nutrient non-demanding bacteria [14]. The com-
parison was based on sample streak and pour plating on
nutrient agar plates, and application of liquid sample on a
dry RIDA®COUNT medium sheet.
2.3 Physical and chemical analyses
Simultaneously with sampling for cultivable micro-
organisms, physical parameters of the samples were

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measured at the site (specific electrical conductivity-SEC,
pH, temperature-T, redox potential-ORP, dissolved oxy-
gen-DO) by Multimeter HACH HQ40d, USA. In the
laboratory the following parameters were measured: total
suspended solids-TSS (Analytical scale Mettler, USA),
NH4
+-N, NO2
--N, NO3
--N, orthophosphate (o-P), total
phosphorus (P-tot), COD (Spectrophotometer HACH DR
2800, USA), BOD5 (Manometer WTW OxiTop, USA),
after Standard Methods [15].
2.4 Statistical evaluation
The extracted data of defined cultivable microbial
groups (E.coli, non- E.coli enterobacteria, non- E.coli non
enteric bacteria, yeasts and moulds) and their abundance
were correlated with environmental variables. Relative
standard deviation (RSD) was calculated to evaluate the
repeatability of total bacterial counts on RIDA®COUNT
Total Aerobic Count compared to the number of cultiva-
ble bacteria on the nutrient agar plates.
3 RESULTS AND DISCUSSION
The results showed a notable reduction of bacteria
and fungi after the treatment process in the tested systems
(Table 3). As RIDA®COUNT E.coli/coliforms kit test
covers different subgroups of bacteria compared to
RIDA®COUNT Enterobacteriaceae, the numbers of coli-
forms and enterobacteria in some cases differed, for ex-
ample in samples BW2, BW4, GW1. This is one of the
parameters indicating changes in the abundance of a cer-
tain bacterial group. A reduction of bacteria was also ob-
served in the greywater module, although E.coli was pre-
sent in the last treatment wetland at the outlet. This unex-
pected result can be attributed to the high summer tem-
perature of stagnant water after accidental spilling from
the blackwater module. In the samples from municipality
wastewater treatment plant 91% of total bacteria, 91% of
Enterobacteriaceae, and 71% of E.coli were reduced at the
outflow from the system. 40 days of composting was
successful in complete removal of E.coli and reduction of
coliforms and enterobacteria, i.e. <0.03%.
To get a better insight into changes in blackwater
communities, we used the following indicators: ratio of
total coliform bacteria vs. total bacteria, ratio of Entero-
bacteriaceae vs. total bacteria, and ratio of E.coli vs. total
bacteria. During the treatment, reduction of E.coli in the
system is clearly evident (Figure 2). Different sets of
RIDA®COUNT plates showed significant changes in the
bacterial community, for example in the samples BW2
and BW3 when percentage of bacterial subgroups of coli-
forms and enterobacteria notably differed (Figure 2).
The only cells that are able to form colonies on
RIDA®COUNT plates are those that can grow under the
conditions of the test (incubation media, temperature,
time and oxygen conditions). In bacteriology, nutrient
agar is frequently used for nutrient non-demanding spe-
cies to grow [14], which is why we compared the retrieval
of cultivable bacteria on RIDA®COUNT medium sheets
and on nutrient agar plates from blackwater liquid (BW1)
and compost at the beginning of the process (SW1).
The average values of bacterial counts on
RIDA®COUNT test plates after plating ten sheets with
the compost indexed were 100.0 (RSD 17.2%); the streak
plating on nutrient agar for compost was 146.0 (RSD
29.4%) and for pour plating on agar plates it was 122.9
(RSD 33.3%). This comparative agar method retrieved 22.9
to 46.0% more bacterial colonies than RIDA®COUNT
plates. RIDA®COUNT plate counts were in the range of
FIGURE 2 - Ranges of microbial indicators during blackwater treatment at input (1-BW1), separation from solid phase (2-BW2), treatment
of liquid (3-BW3), and ultimate phase of liquid treatment (4-BW4); CO/BA - ratio of coliform bacteria vs. total bacteria, EN/BA - ratio of
Enterobacteriaceae vs. total bacteria, EC/BA - ratio of E.coli vs. total bacteria

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TABLE 3 - Ranges of counts of cultivable microorganisms expressed as CFU/ml in liquid and as CFU/g in solid wastes; blackwater treat-
ment, BW1-BW4; greywater in hybrid constructed wetland, GW1-GW3; municipality wastewater treatment plant, MW1-MW2; blackwater
solid compost, SW1-SW2. Percentages of indicator group E.coli, total coliforms and Enterobacteriaceae compared to total bacteria are pre-
sented in brackets and referred to all samplings.
Sample Bacteria CFU/ml E.coli CFU/ml
(% of total)
Coliforms CFU/ml
(% of total)
Enterobacteriaceae CFU/ml
(% of total)
Yeast and moulds
CFU/ml
BW1 4.5×106-1.9×107 1.3×105-1.9×105
(1.02-3.43)
3.6×105-1.5×106
(4.60-13.00)
2.8×105-8.3×105
(3.51-13.47)
1.7×103-2.5×104
BW2 4.9×106-1.5×107 1.2×104-9.3×104
(0.24-0.97)
6.8×104-9.7×106
(0.94-10.14)
3.8×104-3.2×105
(0.78-3.30)
1.9×103-1.4×105
BW3 1.6×107-1.9×107 2.4×103-4.3×104
(0.01-0.26)
4.2×104-1.7×105
(0.21-1.00)
8.8×104-2.4×105
(0.52-1.26)
8.8×102-6.8×103
BW4 3.5×103-2.3×106 1.1×101-9.4×102
(0.00-0.32)
1.1×101-2.0×103
(0.01-0.32)
0-1.6×103
(0.00-0.07)
2.0-4.8×102
GW1 2.2×104-1.8×108 0-5.6×103
(0.00-0.00)
1.3×104-2.0×105
(0.11-57.17)
5.6×103-2.4×105
(0.13-25.00)
1.7×102-3.1×102
GW2 1.00×103-5.6×104 0-5.1×101
(0.00-0.09)
1.2×101-4.5×102
(0.39-6.84)
1.1×101-3.1×102
(0.55-3.83)
1.0-1.6×102
GW3 3.3×102-7.9×104 0-4.9×102
(0.00-0.62)
7.8-1.0×103
(1.00-2.33)
7.8-7.1×102
(0.90-2.33)
5.6-6.3×101
MW1 6.06×105 6.83×104
(11.28)
2.02×105
(33.30)
2.55×105
(42.11)
2.67×103
MW2 6.53×104 2.33×103
(3.57)
2.98×104
(45.58)
2.99×104
(45.83)
5.67×102
SW1* 1.62×108 2.81x106
(1.74)
2.85x106
(1.76)
2.84×106
(1.75)
7.88×103
SW2* 2.33×107-5.57×108 0 2.22×101
-7.11×103
(<0.01)
0-7.43×103
(<0.03)
0
* microbial counts expressed as CFU/g
TABLE 4 - Relations between CFU counts on RIDA®COUNT Total and NA indexed on counts on RIDA®COUNT Total
Average counts of 10 replicates RIDA®COUNT Total index NA pour plating NA streak plating
BW 100.0 41.0 161.7
SW 100.0 122.9 146.0
BW-blackwater; SW-compost
7.27×107 and 1.11×108 CFU/g, streak plating 8.51×107 and
3.93×108 CFU/g, and pour plating in the range of 6.22×107
and 2.29×108 CFU/g. When blackwater liquid was ana-
lysed, streak plating on agar plates retrieved 61.7% more
bacterial isolates (RSD 16.4%) but pour plating with nutri-
ent agar gave 59.0% (RSD 22.2%) less bacterial colonies
(Table 4). RSD for RIDA®COUNT plates was 27.9%.
RIDA®COUNT plate counts were in the range of 1.08×107
and 1.43×108 CFU/ml, streak plating between 1.64×107
and 2.51×107 CFU/ml and pour plating in the range of
3.20×106 and 6.80×106 CFU/ml.
To demonstrate the eventual use of RIDA®COUNT
plates in microbial ecology research in complex systems,
we correlated the results of physical and chemical analy-
ses in the blackwater module (BW1, BW2, BW3) with
microbial groups. Physical and chemical parameters
were fluctuating between different sampling campaigns.
Temperature ranged from 20.9 to 28.8°C, pH from 8.00
to 8.71°C, DO from 0.05 to 6.8 mg/l, EC from 2.3 to
8.1 mS/cm, ORP from -107.7 to -71.4 mV, TSS from 295
to 2625 mg/l, NH4
+-N from 170 to 755 mg/l, NO2
--N 0.1 to
0.8 mg/l, NO3
--N from 47 to 282 mg/l, o-P from 24 to
263 mg/l, P-tot from 30 to 268 mg/l, COD mg/l from 910
to 2885 mg/l and BOD5 from 600 to 2400 mg/l. Varia-
tions in the environmental parameters were high, probably
due to occasional extremely high loads in the system. Pear-
son’s correlation analysis was used to correlate the data on
abundance of microbial groups, E.coli (EC), non-E.coli
enterobacteria (NECEN, calculated as number of E.coli
colonies subtracted from enterobacterial counts), non-E.coli
non enteric bacteria (NECNENBA, defined as bacterial
group which does not include EC and NECEN), and yeasts
and moulds (Y&M) to environmental variables, and micro-
bial subgroups. Our analysis showed that there were no
statistically significant correlations between microbial groups
and the following measured environmental parameters,
BOD5, DO, NO3
--N, o-P and P-tot. Analyses of microbial
groups detected by RIDA®COUNT and environmental
parameters in BW1 characterized by fresh blackwater in-
flow, showed that COD, ORP, pH and TSS had an impor-
tant impact (p<0.05). In BW2 (output from the peat fil-
ters) important parameters which influenced the commu-
nities were: NH4
+-N, NO2
--N, ORP, pH and SEC. Tem-
perature, pH and SEC had significant impact on some
microbial groups in BW3 (liquid fraction after recir-

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TABLE 5 - Summary of statistical significant Pearson’s correlation (bold, p<0.05) between microbial subgroups (BA, EC, CO, EN, NECCO,
NECEN, NECNENBA, Y&M) and physico-chemical characteristics and microbial parameters in three different blackwater treatment mod-
ules (BW1, BW2, BW3)
Group BW1 BW2 BW3
BA pH
0.998
0.037
NECNENBA
1.000
0.016
NH4+-N
1.000
0.019
NECNENBA
1.000
0.013
T
-0.999
0.025
NECNENBA
0.999
0.026
EC ORP
-0.999
0.033
COD
0.998
0.04
CO NECCO
1.000
0.008
NO2--N
0.998
0.045
NECCO
0.998
0.038
Y&M
1.000
0.009
SEC
-1.000
0.009
NECCO
0.998
0.038
Y&M
1.000
0.01
EN NECEN
1.000
0.019
pH
0.997
0.05
SEC
-0.998
0.031
NECCO CO
1.000
0.008
NO2--N
1.000
0.007
CO
0.998
0.038
Y&M
0.997
0.047
SEC
0.997
0.048
CO
0.998
0.038
Y&M
0.999
0.029
NECEN EN
1.000
0.019
pH
0.997
0.046
ORP
-1.000
0.008
pH
0.999
0.032
NECNENBA BA
1.000
0.016
NH4+-N
0.999
0.032
BA
1.000
0.013
T
-1.000
0.002
BA
0.999
0.026
Y&M TSS
-0.998
0.039
NECCO
0.997
0.047
CO
1.000
0.009
NECCO
0.999
0.029
CO
1.000
0.01
SEC
-1000
0.019
BA-total bacteria, EC-E.coli, CO-coliforms, EN-enterobacteria, NECCO-non-E.coli coliforms, NECEN-non-E.coli enterobacteria, NECNENBA-
non-E.coli non enteric bacteria, Y&M-yeast and moulds, shaded relations between microbial groups.
culation in expanded clay biofilter). Correlations between
abundances of different microbial groups were also ob-
served in modules - furthermore, in all three studied black-
water modules, we observed positive correlations between
total bacteria and non-E.coli non enteric bacteria, and
coliforms and non-E.coli coliforms (Table 5).
For this study we selected wastewater treatment sys-
tems with high (blackwater module) and moderate high
organic input (municipal wastewater). We demonstrated
the use of overlapping RIDA®COUNT culture media in
organically enriched wastewater treatment systems. The
plates used in the study revealed only a portion of the het-
erotrophic microorganisms. Use of culturable enrichment
techniques has a high value where selective media are re-
quired to demonstrate the presence and abundance of par-
ticular organisms in the system, for example E.coli, Salmo-
nella spp., coliforms, enterobacteria. E. coli is the most
common representative of the coliform group and is an
indicator for the presence of other pathogenic genera [16].
Total cultivable microbial counts decreased during the natu-
ral treatment process but, more specifically, the indicator
group for enterobacteria (E.coli and total enterobacteria
counts) demonstrated the efficient removal of pathogens of
enteric origin. In the treatment system for municipal waste-
waters, the reduction of E.coli in the treatment process was
71% (BOD5 in the input 330 and output 13 mg/l), in the case
of blackwater treatment the removal rate was 99.4%, and in
the compost it reached 100%. A similar trend was observed
when Enterobacteriaceae was used as an indicator group.
Yeasts and moulds were present in all samples with the
highest counts of 1.4×105 CFU/ml, except in the compost
sample SW2 after 40 days of composting. The absence of
fungal colonies in SW2 sample on RIDA®COUNT Yeast&
Mold Rapid plates is not necessarily linked to the absence of
this microbial group - more probably, slow growing fungi do
not successfully thrive on this medium even after pro-
longed cultivation of 72 hours.
The retrieval of cultivable bacteria on RIDA®COUNT
Total Aerobic Count medium sheets compared to nutrient
agar plates from blackwater liquid can be considered satis-
factory: the RIDA®COUNT test plates can substitute agar
cultivation for organically rich solid and liquid wastes for
monitoring and quick screening of the samples (Table 4).
Although commercially available kits, such as
RIDA®COUNT do not cover a wide range of different
bacterial groups, simultaneous use of different selective
plates, in combination with total bacterial counts for the
same sample, give comprehensive information on micro-
bial status. Data on abundance of an individual microbial
group obtained by cultivation can be correlated with envi-
ronmental parameters. The results of correlation analyses
of the blackwater modules in the Saline Natural Park in-
dicated very complex conditions in the source separation
treatment system. The bacteria of non-enteric origin fre-
quently correlated with physical and chemical parameters
in the blackwater modules, while bacteria of enteric origin
to a lesser extent (Table 5). This can be attributed to the
fact that conditions in the system are not favourable for
enteric indicators which are practically eliminated down-
stream in the system - for example, E.coli becomes less

© by PSP Volume 21 – No 12a. 2012 Fresenius Environmental Bulletin
3892
relevant in the process, but it is still a good indicator for
the treatment efficiency (Figure 2). It is worth stressing
that, in the mineralization processes, other bacteria in
the community which have not been retrieved on the
RIDA®COUNT test plates, have important roles in en-
ergy and matter fluxes. Human waste contains on average
1011.66±0.23 by direct microscopic count (wet weight) per
stool sample [17]. In general, a large proportion, (93%) of
the microscopic clump counts from human stool can be
cultivated in anaerobic conditions [18]. In blackwater liquid
on RIDA®COUNT plates, bacterial counts were up to
1.9×107 CFU/ml. In the treatment systems conditions are
different - for example, conditions in compost samples
resemble more closely the soil environment, where only 1
to 10% of microorganisms can be cultivated [19].
RIDA®COUNT test kits were not originally designed
to be used for wastewaters, but our results showed that
these plates can indeed be effective for routine use with
such complex environmental samples. Cultivation of bac-
teria on RIDA®COUNT test plates average the conditions
of streaked plates when microorganisms have normal avail-
able concentrations of oxygen, and poured plates when ex-
change of oxygen between atmosphere and the media is
slowed-down. Our study shows that RIDA®COUNT plates
can be used successfully as an alternative method to agar
counting, and can be used as a research tool. With only
basic training in microbiology, this method can also be
used for robust routine monitoring at municipal wastewa-
ter treatment plants, before treated water is released into
the environment. There are other more sensitive methods
available to identify and quantify microorganisms from
the environment, such as PCR and its derivatives [20].
4 CONCLUSIONS
Simultaneous use of different RIDA®COUNT test
plates in wastewater research and monitoring provides reli-
able results to observe: reduction of bacterial pathogens, and
fluctuation of cultivable microorganisms. RIDA®COUNT
plates can be used for routine monitoring in wastewater
treatment plants, as well as for checking efficiency of
sequential treatment modules for segregated effluents in
terms of reduction of biological indicator parameters. The
test kits also have a potential for use in microbial ecology
studies in environmental research. Future development of
new commercial cultivation kits, more specific and sensi-
tive, will provide even more comprehensive information of
these systems for research and monitoring.
ACKNOWLEDGEMENTS
The study was supported by the Sanbox: “Develop-
ment of an innovative sanitation and wastewater treatment
system for remote located tourist facilities”, Capacities,
Research for the benefit of SMEs of the EU 7th Frame-
work Programme, and by Research Programme “Karst
Research P6-0119” (Slovenia). The authors acknowledge
Nada Milharčič from Kovod Postojna, d.o.o. for field
assistance, Renato Babič and Aleksandra Krivograd
Klemenčič for laboratory support, Boris Kompare and Matej
Uršič for technical support, and Laura Marshall for termi-
nology assistance.
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Received: May 21, 2012
Accepted: August 02, 2012
CORRESPONDING AUTHOR
Andreea Oarga
University of Nova Gorica
Vipavska 13
5000 Nova Gorica
SLOVENIA
Phone: +386 5 700 1945
Fax: +386 5 700 1999
E-mail: dora_oarga@yahoo.com
FEB/ Vol 21/ No 12a/ 2012 – pages 3886 - 3893