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Biochemical Engineering Journal 17 (2004) 147–151 Software sensors based on titrimetric techniques for the monitoring

Biochemical Engineering Journal 17 (2004) 147–151

Biochemical Engineering Journal 17 (2004) 147–151 Software sensors based on titrimetric techniques for the monitoring

Software sensors based on titrimetric techniques for the monitoring and control of aerobic and anaerobic bioreactors

Heiko Feitkenhauer , Ulrich Meyer

Institut für Chemie- und Bioingenieurwissenschaften, ETH Zürich, Hönggerberg, CH-8093 Zürich, Switzerland

Received 9 January 2003; accepted after revision 28 May 2003

Abstract

Microbial activity influences the vicinity of a microbial cell in a way that changes the pH value of the surrounding medium. Key processes are the production or degradation of charged substances (e.g. volatile fatty acids) and the production of carbon dioxide (or hydrogen carbonate). In a technical system, the shift of the pH value may be prevented by a pH control system. It is proposed in this paper that the resulting base (or acid) consumption rates can be used as input for “software sensors” for substrate and biomass concentrations. This titrimetric technique was successfully applied to extreme thermophilic, aerobic bioprocesses (growth substrates phenol, aliphatic hydrocarbons) for the estimation of biomass and substrate concentrations (formation/degradation rates). In anaerobic experiments, the formation of volatile fatty acids (VFA) was monitored and the reduction of the VFA formation rate in the presence of an inhibitory compound investigated. The results show the versatility of software sensors based on titrimetric techniques and demonstrate the potential for process control in applications in which more sophisticated sensors are not available or affordable. © 2003 Elsevier B.V. All rights reserved.

Keywords: Aerobic processes; Anaerobic processes; Control; Software sensor; Thermophilic microorganisms

1. Introduction

Both software sensor and titration techniques have gained increasing popularity in recent years [1,2]. Reasons are their robustness and lower price compared to other, more sophis- ticated equipment. The latter is based on lower investment costs as well as on the relatively simple maintenance (and hence no need to employ additional, well trained techni- cians). Furthermore, the equipment necessary is installed in many plants (or laboratory bioreactors), but not used for automatic process control purposes.

1.1. Bioprocess fundamentals of the process control techniques

Nearly all activities of microorganisms affect their sur- roundings in a way that changes the pH value in the vicinity of the individual cell (or a microbial floc/pellet). Such a pH alteration can be, for example, expected during the aerobic degradation of uncharged substrates such as aliphatic hydro- carbons. Cells metabolize hydrocarbons and oxygen while they produce carbon dioxide and minor quantities of other

Corresponding author. Tel.: +41-1-632-3064; fax: +41-1-632-1074. E-mail address: feitkenhauer@tech.chem.ethz.ch (H. Feitkenhauer).

1369-703X/$ – see front matter © 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S1369-703X(03)00150-5

metabolites, e.g. fatty acids. Near the neutral pH most of the carbon dioxide will be converted to hydrogen carbon- ate and severely influence the pH value, while the rest will leave the reactor as gaseous carbon dioxide. The amount of carbon dioxide that leaves the reactor depends not only on the pH value, but also on the mass transfer characteristics within the treatment system. Therefore, not only microbial activities influence the pH value, but also process variables such as the aeration rate or stirrer speed. A further pH-shift is encountered when cells degrade compounds which in- fluence the pH value of the solution, e.g. the pH increases when an acidic compound is degraded or incorporated. One important example of that kind is also the incorporation of ammonium into growing cells. A similar pH change in the treatment system occurs in denitrification processes [3]. A very intense change is also found in two-phase anaerobic digestion systems. In this case no oxygen is involved in the reaction. In the first bioreactor (also called acidification reactor) the wastewater constituents are transformed into volatile fatty acids (and carbon diox- ide), while the same fatty acids are converted into biogas in the second reactor (methane reactor). Therefore, a strong change in pH accompanies the biological activities in both reactors. Concluding, in a characterized biological system it is not necessary to monitor the cells themselves (or their

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substrates) to detect the microbial activity, but it is sufficient to monitor the surroundings of the cells, e.g. by monitoring the pH. Unfortunately, a shifting pH influences the cells them- selves as well as their growth medium. An example for the latter is acetic acid, which can pass into microbial cells only in the neutral (protonated) form. In general, the bioavailabil- ity of many compounds changes with the pH. An example for the influence of the pH on the cells themselves is the distribution of the individual volatile fatty acids produced in an acidification reactor, which is dependent on the pH in the growth medium [4]. If a base (or acid) is supplied to the process to keep the pH value constant (e.g. by a pH controller), the amount of titrant (base/acid) added per unit time can be used as the relevant information instead of the pH value. This direct titrimetric technique will be used and discussed in this paper for the estimation of concentrations of biomass or degraded substrate.

A pH-measurement and an online correction system for

the pH value (pH-stat) is available in most laboratory scale wastewater treatment plants and is also used in many full scale (industrial) installations. However, online measure- ments of biomass concentrations are not as frequently used, as they are costly and require some maintenance. The same holds for online measurements of substrate concentrations or wastewater sum parameters. Offline measurements are time consuming and hence not very well suited for control pur-

poses. Furthermore, it is not very easy to determine the best sampling times, especially in batch-wise operated reactors. Also sampling may be required during the night (un-staffed operating hours). Therefore, it would be beneficial to use data that are easily available online such as the base con- sumption to maintain the pH constant.

In this paper, the “construction” of simple software sen-

sors based on titration techniques is described. Three ex- amples will be used to demonstrate the versatility of this technique in more detail:

(a)

Control of aerobic fermentation: estimation of biomass and substrate (phenol) concentrations.

(b)

Control of (anaerobic) acidification reactors: estimation of metabolite concentrations.

(c)

Control of (anaerobic) acidification reactors: estimation of inhibitory effects.

2. Materials and methods

2.1. Aerobic bioreactor and control system

The artificial wastewater either contained phenol or eicosane as the carbon source (concentrations indicated in the results). Furthermore, it contained the following mineral salts (in g/l): (NH 4 ) 2 SO 4 , 1.0; MgCl 2 ·6H 2 O, 0.1; CaCl 2 · 2H 2 O, 0.1; KCl, 0.04; FeSO 4 ·7H 2 O, 0.001; and 10 ml/l trace element solution according to DSMZ, Medium 141 [5].

It was sterilized in the treatment system (20 min at 121 C bioreactor). The phosphate buffer system (final concentra- tion: Na 2 HPO 4 , 0.42 g/l and NaH 2 PO 4 , 0.2 g/l) was adjusted to the desired pH and autoclaved separately and added be- fore the inoculation with the indicated Bacillus strain (phe- nol degradation: Bacillus thermoleovorans A2 [6]; eicosane degradation: B. thermoleovorans Hamburg I [7]). The 2 l bioreactor (liquid volume of 1.6 l, Typ VSF, Bioengineering, Wald, Switzerland) was equipped with three “disc turbine” stirrers on the axis and operated at 1500 rpm. The aeration rate (0.5 vvm) and temperature (65 C) remained unchanged during the experiment. The system was equipped with an oxygen probe and indicator (Bioengineering). The oxygen saturation was recorded and was above 5% air saturation at all times of the experiments. The system was also equipped with a pH-probe (Endress und Hauser, Weil am Rhein, Ger- many) and a pH controller. The pH was kept at the desired value by this controller connected to a peristaltic pump (us- ing 1 N NaOH solution). The base supply flask was placed on scales, which were read at the indicated times.

2.2. Anaerobic bioreactor and control system

The synthetic wastewater and system for the control of the anaerobic acidification reactor has been described before [8]. Briefly, it consists of a 7 l bioreactor with equipment for online sampling and a purpose build titration cell for the determination of volatile fatty acids and carbonate. The actual titration was performed using a commercial tirator (719, Metrohm, Herisau, Switzerland). All equipment was connected to a PC (Dell Optiplex, Austin, USA) using the serial interfaces of the computer (RS 232) or a multi-purpose I/O card (MIO 16E4, National Instruments, Austin, USA). The data acquisition and control software was written in Labview 5.1 (National Instruments). A pH controller kept the pH value in this reactor constant via a peristaltic pump (using 2 N NaOH solution). The time of operation of the base pump was recorded and converted into base amounts by the computer using a calibration curve. The same computer was also used in control of the first (acidification) reactor in a biogas system. This 2 l bioreac- tor was similar to the bioreactor described above, but in this case the titrator (Metrohm 719) was directly connected to the reactor for pH control. The pH value and amount of base (0.5 N NaOH) added were read every 90 s by the computer. The reactor was emptied and cleaned after every experiment to avoid cell growth on the reactor wall. The simplified wastewater contained the substrate glucose and the following mineral salts (g/l): NH 4 Cl, 0.15; MgCl 2 , 0.05; CaCl 2 , 0.05; FeSO 4 ·7H 2 O, 0.02; HNa 2 PO 4 ·12H 2 O, 0.35; KH 2 PO 4 , 0.35; yeast extract 0.02 NaCl as indicated in the experiment (adjusted to pH 6 with 0.5 N NaOH after inoc- ulation). It was inoculated with 160 mg/l (cell dry weight) of a mixed culture obtained from a continuously operated stirred bioreactor (substrate: starch). All cultures for inocu- lation were taken from a continuously operated bioreactor

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at the same time and stored at 4 C before the start of each experiment.

3. Results and discussion

3.1. Control of an aerobic fermentation: estimation of biomass and substrate concentrations

There are two main effects that can be exploited for measurements in aerobic systems: (a) Cells degrade (or incorporate) compounds which influence the pH value of the solution. One important example is the incorporation of ammonium into cell mass during the growth on a carbon source. (b) Cells release intermediates and products of the aerobic degradation in the wastewater, e.g. carbon diox- ide/hydrogen carbonate. These two processes are related to the metabolic activity of the cells and hence in most cases to the growth on a certain pollutant. Hence these data can be used to estimate the biomass or the degraded substrate concentrations. In both cases the initial concentration can- not be derived with a titrimetric technique. If required, they must be determined offline. In laboratory measurements they are often known from the set-up of the experiment (e.g. the start concentration of a substrate is known from the amount of substrate added to the reactor).

3.1.1. Estimation of substrate (phenol) concentrations Phenol was degraded as sole carbon and energy source by B. thermoleovorans A2 at 65 C in a batch-wise operated reactor (Fig. 1). In general, higher concentrations of phe- nol are very inhibitory to microorganisms (e.g. >200 mg/l), while lower concentrations are degraded fast and efficiently. Hence an online control of the degradation of phenolic com-

5 0 4.5 4 -2.5 3.5 3 -5 2.5 2 Phenol -7.5 1.5 OD 1
5
0
4.5
4
-2.5
3.5
3
-5
2.5
2
Phenol
-7.5
1.5
OD
1
Base
-10
0.5
0
-12.5
0
10
20
30
40
Phenol [mmol l -1 ], OD x 5 [-]
1 N NaOH [ml l -1 ]

Time [h]

Fig. 1. Phenol degradation and microbial growth of the thermophilic B. thermoleovorans A2 growing at 65 C and a pH of 6 in a 2 l bioreactor. The growth was monitored using the optical density OD at 570 nm as a parameter. On the second y-axis, the consumption of NaOH is plotted (negative values refer to the level of the NaOH reservoir and were not converted to make the good correlation with the phenol data more obvious).

pounds would be very beneficial, but traditional systems are often expensive and complicated. Offline measurements are time consuming and hence not very well suited for control purposes. In the experimental system, the pH was maintained con- stant by a pH controller, which pumped 1 N NaOH solution into the reactor. A variation of about 0.04 pH units was observed due to the “on/off” type controller used in the experiment. The amount of base used was measured on a balance (Mettler-Toledo, Switzerland, 0.1 g steps). A good correlation between the phenol degradation and the base consumption was found in this experiment (Eq. (1)). The following linear correlation was derived from these data to estimate the phenol consumption from the base consump- tion data (in ml 1 N NaOH /l bioreactor ):

Phenol degradation (mmol/l)

= 0.3884 × base consumption (ml/l)

(correlation coefficient R 2 = 0.99)

(1)

This correlation can be used to monitor online a degrada- tion experiment or to control continuous degradations (pH difference between feed and reactor have to be considered in this case). Deviations of measurement and correlation are caused by the relatively large steps of the balance (0.1 g) and the on/off type of control. The simple linear relationship also suggests that microbial ammonium uptake (proportional to phenol uptake) was equally important to carbon dioxide dissolution (where, e.g. saturation effects could yield a non-linear relation). Due to the use of NaOH insoluble sodium salts were probably produced from the dissolved carbon dioxide. Several types of biosensors for phenolic compounds are in use in different areas, including process technology and

medical applications [9,10]. Most sensors for process con- trol are based either on immobilized enzymes [11] or more sophisticated extraction and detections methods, e.g. em- ploying solid phase extractions and spectroscopic measure- ments [10]. All these methods have the advantage of being more specific for phenolic compounds. On the other hand, their drawback is the considerably high input in resources (investment, maintenance) to get reliable results.

3.1.2. Estimation of biomass concentrations Similar correlations can be derived for wastewater treat-

ment plants or for software sensors that estimate the cell density (optical density, OD) from the base consumption data. The following equation was derived for eicosane degra- dation by B. thermoleovorans at 65 C and pH 7 [12] and was confirmed also in experiments with hexadecane as the growth substrate:

OD = 0.24× base consumption (in ml 1 N NaOH /l bioreactor )

(correlation coefficient R 2 = 0.94)

(2)

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There are several types of sensors for the monitoring of cell/sludge densities in research bioreactors and industrial applications. Many of them are based on optical measure- ments. Problems are often encountered with air-bubbles and biofilms on the sensor, or, like in the above present appli- cation, with a second organic phase (aliphatic hydrocarbons like eicosane dissolve only in minor quantities in the aque- ous medium and hence form a second liquid phase even at low concentrations). An example for the development of an advanced sensor in this field is the multi-wavelength fluo- rescence sensor [13], which measures also substrate concen- trations. However, these methods are more complicated and expensive than the proposed use of titrimetric data.

3.2. Control of (anaerobic) acidification reactors:

estimation of metabolite concentrations

The offline control of anaerobic digestion processes by buffer capacity measurements is widely used. In samples, the volatile fatty acid (VFA) content and carbonates can be distinguished and this method is available for online use [8]. A simpler way to monitor the acidification rate is presented here. It was shown that the base consumption correlates very well with the VFA concentration (as measured online with the above mentioned method and verified by a GC method with minor deviation). The base consumption was determined by measuring the operation time of the pump connected to the pH controller (and using a calibration curve for this pump). Not taking into account the initial concentration of VFA (which cannot be determined by direct titration), the VFA concentration in the system can be calculated from the base consumption by

VFA (mmol/l) = base consumption (mmol/l)/0.91

(3)

The factor in this equation is not one because a part of the organic acids is not dissociated at pH 6. On the other hand, some carbon dioxide produced by the cells was dissolved in the wastewater and changed the pH of the medium (at pH 6 the predominant species is carbon dioxide, but hydrogen carbonate has still a considerably share of the total carbonate alkalinity). Hence, when establishing such a simple correla- tion always similar conditions to the subsequent process con- trol must be used (especially pH, temperature and to some extent salinity). If these conditions change considerably, the whole process has to be modeled and the factors influencing the base consumption have to be incorporated in this model. There is an array of alternative methods to measure the VFA content in anaerobic bioreactors, most of them using more sophisticated techniques. One of the techniques with a lower investment necessary is the online titration of sam- ples [8]. At the other end of the spectrum, there are several techniques using simplified and sophisticated mass spec- trometers [14,15]. The advantage of these techniques is the

(correlation coefficient R 2 = 0.99)

80 0 g/l NaCl" 70 40 g/l NaCl" 60 50 80 g/l NaCl" 40 30
80
0 g/l NaCl"
70
40 g/l NaCl"
60
50
80 g/l NaCl"
40
30
0 g/l NaCl
10
g/l NaCl
20
40
g/l NaCl
10
80
g /l NaCl
0
0
1500
3000
4500
Base [ml 0.5 N NaOH]

Time [min]

Fig. 2. Determining the acidification rate of glucose (5 g l 1 ) in the presence of increasing NaCl concentrations in a 2 l bioreactor.

possibility to discriminate between different VFA species or at least between VFA and hydrogen carbonate (titration of samples). The main disadvantages are the much higher installation and maintenance costs, including the need for trained staff.

3.3. Control of (anaerobic) acidification reactors:

estimation of inhibitory effects

The examples described above relate to cases in which concentrations were determined directly from measured data. These data can also be used to gain information on in- hibitory substances [16]. One simple example is the follow- ing: the increase of the VFA concentration in a time interval is calculated from titration data. From this data, acidification rates in the presence of increasing concentrations of an in- hibitor (increasing NaCl concentrations) can be calculated. These decreasing rates (decreasing rates of base addition) can be used to calculate an inhibition constant (Fig. 2). A related technique has been used by Rozzi et al. [17] to determine inhibitory effects on methanogenic bacteria. In this case not the degradation, but the formation of an intermediate in the presence of an inhibitor substance was successfully studied.

4. Conclusions

Software sensors based on titrimetric techniques were successfully used to determine key concentrations and rates in wastewater treatment processes. In all cases useful correlations were established which are valid for specific systems and processes. The equations were obtained from experiments that can be performed relatively fast in other biosystems to establish the correlations in these biopro- cesses (specific correlations have to be established for each process to be controlled or monitored).

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In an aerobic process, the metabolism is mainly related to the degradation of a substrate and the formation of biomass. Both the substrate (shown for phenol) and biomass con- centration were described with a satisfactory accuracy. In anaerobic digestion, the metabolism of the involved groups of microorganisms is mainly a metabolism of volatile fatty acids, either production (acidification) or degradation (methanogenesis). In this case, the activity of the biomass can be determined using software sensors, e.g. for the acid- ification rate. It was also possible to relate the inhibitory effects to concentrations of inhibitory substances. For the further expansion of the utilization of this technique, we are suggesting a combination of titrimetric techniques with oxygen saturation and/or conductivity measurements and the use of more detailed models (to make this titration-based techniques also applicable in cases were, e.g. no linear relationships can be established).

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