Biotechnology Letters 23: 1057–1060, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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RNA assay as a method of viable biomass determination in the organic fraction of municipal solid waste suspension Ewa Liwarska-Bizukojc∗ & Stanislaw Ledakowicz Technical University of Lodz, Department of Bioprocess Engineering, ul. Wolczanska 213/215, 93-005 Lodz, Poland ∗ Author for correspondence (Fax: +48 42 631 37 38; E-mail:
[email protected]) Received 11 January 2001; Revisions requested 19 February 2001/28 March 2001; Revisions received 20 March 2001/3 May 2001; Accepted 5 May 2001
Key words: activated sludge, municipal solid waste, RNA assay, viable biomass determination
Abstract A method of RNA determination by means of alkaline hydrolysis and UV detection was applied to evaluate the amount of viable biomass during the aerobic biodegradation of the organic fraction of the municipal solid waste suspension. The relative standard deviation of this determination was from 0.7 to 4%. The viable biomass constituted only 9% of the volatile suspended solids in the activated sludge.
Introduction
Materials and methods
The simplest and most often used method for the biomass determination in the process of activated sludge wastewater treatment is volatile suspended solids (VSS) evaluation. In spite of its simplicity this method does not distinguish between living cells, dead biomass and non-viable organic particles. According to the literature, viable biomass in the typical activated sludge makes only 5 to 20% of VSS on average (Weddle & Jenkins 1971, Jørgensen et al. 1992). Therefore alternative methods to determine cellular ATP, RNA and DNA contents, Oxygen Uptake Rate (OUR) and various enzymes are being developed to achieve a more reliable determination of viable biomass in the activated sludge processes. Biomass determination is even more difficult when an aqueous suspension of organic solid waste is being analysed. In this case, the content and size of organic particles is so high that VSS determination is completely useless. Thus, a simple and reliable method was adapted from Benthin et al. (1991) for the determination of cellular RNA content to quantitatively assess the biomass concentration in the waste suspension for the purpose of modelling of the biodegradation process.
Substrate preparation Three types of substrate were investigated. The first substrate consisted of the fresh, organic fraction of a typical Polish municipal solid waste. The initial composition of waste was defined according to Steglinski (1999) and was identical in each process. The organic wastes were ground to achieve particles of size from 4 to 5 mm. The ground wastes were suspended in tap water to give total solids (TS): 15, 34, 55 and 66 g dm−3 and aerated in the bioreactors. The initial pH of each suspension was about 6.3. The second substrate was the activated sludge taken from Combined Wastewater Treatment Plant in Lodz (TS values are given in Table 1). The third substrate was a mixture of fresh organic solid waste suspension prepared as described above (TS = 55 g dm−3 ) and activated sludge in proportion 2:1 (v/v). The content of total solids in this mixture was 33.9 g dm−3 . The initial pH of the mixture was 6.6.
1058 Table 1. Volatile suspended solids (VSS) versus RNA biomass determination in the activated sludge. Sample number
1 2
Volatile suspended solids (VSS) (g dm−3 )
Relative standard deviation (RSD) (%)
Viable biomass (RNA)
Fraction of viable biomass in VSS
(g dm−3 )
Relative standard deviation (RSD) (%)
8.6 6.35
2.06 2.85
0.833 0.488
2.55 3.42
9.7 7.7
(%)
The bioreactors were equipped with standard control and measurement devices. The temperature, pH, pO2 (Ingold), CO2 in the exhaust gases (Servomex), the O2 flow rate and the rotation of stirrer were measured and controlled. The experiments were carried out at 25 ◦ C. To keep pO2 above 10% and to eliminate O2 limitation, pure O2 was applied to aerate the suspension. The aeration intensity was dependent on the microorganisms’ demands. Analytical procedures Fig. 1. RNA vs. time for various total solids values (pure O2 aeration, flow rate 30–300 dm3 h−1 to keep pO2 above 10%). , 15 g TS dm−3 ; •, 34 g TS dm−3 ; 55 g TS dm−3 ; , 66 g TS dm−3 .
Fig. 2. Linear dependence between RNA in the stationary phase and initial total solids; data obtained from four independent processes.
Bioreactors and process parameters The experiments were conducted in 6 litre working volume batch reactors. The aeration system consisted of a sparger and six bladed Rushton stirrers. The stirrers were mounted, in pairs on the shaft.
The RNA assays were performed according to Benthin et al. (1991). A sample, 5 ml, from the bioreactor was diluted, if necessary, washed three times with 3 ml cold 0.7 M HClO4 to destroy the cell walls of bacteria. The material was mixed with 3 ml 0.3 M KOH solution and held for 60 min at 37 ◦ C with occasional mixing to hydrolyse RNA. The post-hydrolysis supernatant was collected and the precipitate was washed twice with 3 ml cold 0.5 M HClO4 . Finally, all extracts were made up to 15 ml with 0.5 M HClO4 and centrifuged to remove any remaining solid particles. The obtained supernatant was subjected to the absorbance measurement. The absorbance of released purines and pirymidines was determined spectrophotometrically at 260 nm. The concentration of RNA was calculated from (Herbert et al. 1971): cRNA =
A260Mw R, εl
where Mw = 340 g mol−1 is the average nucleotide molar weight; R, dilution; ε = 10 800 mol cm−1 , molar absorption coefficient; l – length of measuring cell, cm. The linearity of Beer’s law was checked for the diluted samples in the absorbance range of 0.5–1.8. The new application of this method required statistical
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Fig. 3. Comparison of RNA concentration changes for two parallel biodegradation processes: with and without activated sludge inoculation (pure oxygen aeration, flow rate of 100 dm3 h−1 for the process without inoculation, and 80 dm3 h−1 for the process with activated sludge inoculation).
validation in these conditions. Therefore the relative error of the measurement was calculated and shown in Figures 1 and 3. Also relative standard deviation (RSD) was estimated (Table 1). Total solids (TS) and volatile suspended solids (VSS) were measured gravimetrically, according to Standard Methods for the Examination of Water and Wastewater (American Public Health Association, American Water Works Association, Water Pollution Control Facility 1989).
Results and discussion Benthin et al. (1991) used an RNA assay for pure culture of Lactococcus cremoris to obtain the data for the purpose of the growth and product formation modelling of the chemostat culture in the transient states. In the case described here the determination of RNA was performed for the mixed culture of autochthonic aerobic microflora existing in the suspension of the organic fraction of municipal solid waste. The adaptation of this method delivered the satisfactory results. The estimated relative standard deviation was low (0.7–4%) but at the same time higher than RSD values for this method given by Benthin et al. (0.4–1.1%). It is probably due to the non-homogeneity of the samples. On the other hand the RSD values for VSS determination were also low (2–3%). In spite of the accuracy of the method VSS concentration is not a good
indicator of viable biomass. The calculated relative error of the measurement of RNA concentration for each experimental point did not exceed 6% (Figures 1 and 3). Figure 1 shows RNA time courses in relation to various initial total solids. The obtained curves of RNA changes in time were similar to the typical biomass growth curve (Aiba et al. 1965). It proved that RNA increment was a good indicator of growing, viable biomass. The higher amount of waste was put into the bioreactor, the more autochthonic microflora was present in the system and the higher RNA concentration was observed. Hence, the linear dependence between initial TS and average RNA concentration in the stationary phase was achieved (Figure 2). In order to ultimately verify the correctness of this method, the RNA concentration was measured in a sample of activated sludge. This verification was essential because data on autochthonic biomass concentration in the suspension of the organic fraction of municipal solid waste have been not available in literature till now. On the basis of RNA content the biomass concentration was evaluated taking into account that RNA made 15–20% of dry weight of a typical bacterial cell (Ingraham et al. 1983). The obtained results of biomass determination were compared to VSS measurements. It occurred that viable biomass in this activated sludge was only about 9% of VSS (Table 1). This proved that VSS determination is a very approximate and inaccurate method of biomass evaluation in
1060 the activated sludge. Therefore, the application of an alternative method as RNA assay seems to be crucial. The investigated method was also applied to observe biomass growth during two parallel processes of the aerobic biodegradation of the waste suspension containing only autochthonic microflora and the suspension inoculated by activated sludge. The time courses of RNA for both cases are shown in Figure 3. The initial RNA concentration in the inoculated suspension was more than twice as high as in the non-inoculated one. What is even more interesting, after the exponential growth phase, RNA achieves no constant level in the inoculated suspension but seems to oscillate. This behaviour was never observed in the autochthonic microflora biodegradation processes (Figure 1). These oscillations are probably due to several causes: different conditions of the process than in activated sludge chamber, especially higher load of organic substances, low affinity of wastewater microflora to the substrate present in the waste suspension, competition between autochthonic and activated sludge microbes. To sum up, the applied method of RNA determination is reliable and repeatable and what is more important it enables a more accurate estimation of the amount of viable cells present in the mixed culture.
Furthermore, it delivers data for measurement, control and modelling of biodegradation processes, especially activated sludge processes.
References Aiba S, Humprey AE, Millis NF (1965) Biochemical Engineering. Tokyo: University of Tokyo Press. American Public Health Association, American Water Works Association, Water Pollution Control Facility (1989) Standard Methods for the Examination of Water and Wastewater, 17th edn. Washington DC. Benthin S, Nielsen J, Villadsen J (1991) A simple and reliable method for the determination of cellular RNA content. Biotechnol. Tech. 5: 39–42. Herbert D, Phipps PJ, Strange RE (1971) Chemical analysis of microbial cells. Meth. Microbiol. 5B: 209–344. Ingraham JL, Maaløe O, Neidhardt FC (1983) Growth of the Bacterial Cell. Sunderland: Sinnauer Associates Inc. Jørgensen PE, Eriksen T, Jensen BK (1992) Estimation of viable biomass in wastewater and activated sludge by determination of ATP, oxygen utilization rate and FDA hydrolysis. Water Res. 2: 1495–1501. Steglinski W (1999) The investigation of the composition and properties of the municipal solid waste from the area of Lodz. Lodz: OBREM (Research and Development Centre of the Urban Ecology). Weddle CL, Jenkins D (1971) The viability and activity of activated sludge. Water Res. 5: 621–640.
