Reactor Theory

Reactor Theory and Practice

Students wishing to study biofilms in the field or laboratory have a wide selection of methods for collecting them, and if so desired, returning them to the lab.  The accompanying collection of exercises gives examples of how to collect biofilms from soil (Exercise 7 - Collecting Soil Biofilms by the Buried Slide Technique, and Exercise 16 - Isolation of Biofilm Populations from Soil Crumbs by Flotation), Water (Exercise 13 - Henrici's Microbial Fishing Technique) and cheek cells (Exercise 19 - Buccal Epithelial Cells with Adherent Bacterial Cells Revealed by Negative Staining) for example.  Although these methods are fine for examining naturally occurring Biofilms they were produced under conditions that are not under the control of the student.  In order to try to grow consistently uniform Biofilms for classroom or independent research one must employ some type of reactor.  Reactors are devices used to control at least some of the many variables, which contribute to the formation of a biofilm.  Even using a biofilm reactor, it is not usually possible to control all, relevant variables and no one type of reactor will “model” all conditions under which biofilms grow. Nevertheless engineers and microbiologists have devised a number of reactor types that reproduce many of the natural conditions under which biofilms grow. So, one reactor is not necessarily better than another, they are intended to model different habitats.

This introduction to reactor theory and practice is intended to help you to select the type of reactor which best suits the investigation you are contemplating and give you some pointers on how to construct, and maintain the reactor during biofilm development.  Some theory is included to help make sense of the properties of the various types of reactors

Why use a reactor?

Reactors permit the controlled development of a biofilm so that in replicate trials, rather consistent biofilm samples are produced to that the measurements of such parameters as biofilm mass, thickness, metabolic activity, cell number and resistance to chemical or physical challenge can be made.


Reactor systems can be established that permit investigators (that’s you) to control many experimental variables including1:

             
What sorts of reactors are there?


In the early days of biofilm research, commercial reactors were available from a limited number of suppliers and those that were available were very expensive.  Investigators like Bill Characklis, Barry Pyle and Gordon McFeters2 improvised using Mason jars ® and other readily available materials as reactor vessels.  Today, commercial reactor types are readily available but they are still very expensive. 

 

Figure 1. An early batch reactor, circa. 1992
constructed from a Mason Jar

Permissions
  1. Permissions Pending

In this collection we will revert to the fields infancy and provide instructions for building inexpensive but serviceable reactors from readily available materials, many of which you have in the lab already.
In their classic opus on biofilms, Characklis and Marshall describe three general types of biofilm reactors:

  1. Batch reactors
  2. Continuously Stirred Tank Reactors
  3. Plug Flow reactors1

Each of these has special properties, advantages and disadvantages and each can be configured in a variety of ways in order to mimic specific growth requirements or natural habitats the investigator wishes to model.

Batch Reactors

Batch reactors are examples of “closed reactors” that is ones that, once seeded or inoculated, receive no further inputs of mass or energy and permit no outputs of waste materials.  Batch reactors may be stirred or not stirred but in any case, conditions in the reactor are constantly changing.  Nutrients and other materials like oxygen are declining and metabolic waste products are increasing.  Perhaps the simplest example of a batch “reactor” is a nutrient broth culture tube, although microbial colonies growing on slants or plates are also examples of batch processes.

The earliest “Mason Jar Reactors” mentioned above, were also batch reactors in which glass microscope slides were suspended as coupons (Glossary) in the reactor medium.  In this reactor type cells will typically form biofilm on the coupons and on the walls of the reactor vessel but a large planktonic population will also co-exist in the reactor. Characklis and Marshall maintain that “a stable biofilm cannot be maintained in a batch or closed system”1.

The microbial growth pattern in a batch reactor is the well recognized growth curve memorized by every introductory microbiology student, consisting of a lag, an exponential, a stationary and, if the culture persists long enough, a death phase.

Figure 2. Standard Growth Curve

Permissions
  1. Permissions Pending
These conditions created in a batch reactor are not commonly found in nature. Nutrient and waste exchange almost always occur in natural systems although not in many industrial (and pre-industrial) processes.  Sauerkraut, pickle, and kimchee production as well as the fermentation of beer, wine, and soy sauce are all carried out in batch culture.

Figure 3. (To appear)
A graph showing exhaustion of nutrients and accumulation of waste products in a batch reactor.

Permissions
  1. Permissions Pending

Exercise 1, Making and using a Batch Biofilm Reactor gives detailed instructions on making inexpensive biofilm reactors not so different from those original “Mason Jar Reactors” used by investigators at the Center for Biofilm Engineering early in their work2.

Exercise 5, the Static Coupon Reactor.   In this exercise, a piece of sterile filter paper is placed on the surface of a Tryptic Soy Agar plate.  The filter paper is saturated with dilute medium such as 1/10 Tryptic Soy Broth.  The filter paper is inoculated with the organisms of interest and a sterile microscope slide is placed on the filter paper.   A thin layer of cells forms on the glass slide with no further input of nutrient and, of course waste products accumulate within the system.  Although this is not a fluid system, it has some of the characteristics of a batch reactor as does any culture growing on a solid agar surface.  This protocol was devised by scientists at the S.C. Johnson Corporation in order to produce consistent biofilms for testing their line of cleansing and disinfectant products.

Continuous Flow Stirred Tank Reactor (CFSTR)

A Continuous Flow Stirred Tank Reactor (CFSTR) is one in which the contents are stirred so uniformly that it is assumed that no variation or concentration gradients exist within the vessel.  In theory, any sample taken from the overflow of the reactor will be identical with any sample taken from within the vessel.    In this reactor there is an in-flow of nutrient and an equal out-flow of nutrient, plus microbial waste products and microbial cells.

In order to describe the nature of a CFSTR one should understand three parameters affecting reactor dynamics, these are 1) flow rate, 2) residence time and 3) dilution time.


1)  Flow rate  Q=V/RT

That is Flow rate (Q) in ml/min is equal to the reactor volume (V) in ml, divided by the Residence Time (RT) in minutes.  It can be measured empirically in a reactor in steady state, as the volume of effluent from the reactor per unit of time.  In steady state the inflow and outflow of the reactor are equal and the culture volume of the reactor does not change.

2) Retention time   RT = V/Q

The residence time (RT) in minutes is the time it takes to entirely exchange the volume of the reactor and is expressed as shown above, where V = the volume of the reactor and Q is the flow rate of the effluent leaving the system.

1.1) Flow Rate   Q = V/RT

The flow rate (Q) (in ml/min) then is expressed as shown above, where V is the reactor volume and RT is the residence time. If for example the volume of the reactor is 400 ml and the flow rate is 40ml/min. then the Residence time R is 10 minutes.

3)  Dilution Rate   D = Q/V

The dilution rate equals the flow rate divided by the reactor volume.
In the example above, the dilution rate is:
                                                D = 40 ml/min  = 0.1 / min    
                                                         400 ml       
That is 0.1 or 10% of the volume of the vessel is changed every minute.

In microbiology classes a CFSTR is called a chemostat.  Adjusting the flow rate can alter the rate of growth of the culture therein.  Reducing the flow rate permits the exhaustion of some nutrients and the accumulation of wastes slowing growth as the culture approaches stationary phase.  Increasing the flow rate increases the nutrient concentration and reduces wastes bringing the culture closer to exponential phase.  In a chemostat at steady state u (the instantaneous growth rate of the culture is equal to D (the dilution rate).

If one increases the flow rate such that D > u then the planktonic culture is flushed from the system leaving only attached cells within the reactor.  At this point the reactor is operating as a biofilm reactor.  Of course planktonic cells are constantly being released from the biofilm by erosion or scheduled release (see chapter 2) but these cells are rapidly washed from the reactor.

Typically in setting up a reactor, the vessel is filled to capacity with nutrient medium, coupons are inserted and the reactor is seeded (inoculated) with the organism or combination of organisms desired.  A time interval with the nutrient inflow closed is permitted in order for cells to attach to the coupons and then the influent is adjusted to a level which will rapidly clear the planktonic population (i.e. growth rate > residence time).

Exercise 2 – Building and Using a Biofilm Continuous Flow Stirred Reactor, a part of the Hypertext biofilm laboratory exercise collection describes the construction of a CFSTR.

This reactor may be fed either by gravity or by a pump from a nutrient medium reservoir.  The waste medium is collected in a container and disinfected or autoclaved prior to disposal.  The waste medium container should be placed in a tray or basin with sufficient capacity to hold the entire contents of the system in the unlikely event of a catastrophic failure.  Here again the coupons used are typically glass microscope slides.  The preprinted slides distributed by various companies are ideal for this purpose since they have printed on them slightly raised circles of uniform dimension so that, if one wishes to harvest cells from a known surface area, this can be readily done using the expression (Area = πr2) .

Figure 4. Coupons.

Permissions
  1. Permissions Pending

Plug Flow Reactor (PFR)

The third general type of reactor is the Plug Flow Reactor (PFR).  In a plug flow reactor, nutrients (and sometimes organisms) are introduced to the reactor continuously and move through the reactor as a “plug”.  The system may be either contained (as in a water main, oil pipeline, or blood vessel) or open (as in a shower curtain, stream, or canyon seep). 

In an ideal plug flow reactor, it is assumed that there is no mixing of the medium along the long axis (X-axis) of the reactor although there may be lateral mixing in the medium at any point along the long axis (ie the Y-axis).

Due to the metabolic activity of the organisms in the biofilm, constituents of the reactor will change as medium flows along the long axis due to consumption of nutrients and elimination of waste products.  The nutritional conditions at any given point along this long axis should, however, remain constant in a stable reactor.  On the other hand, as an organism moves through the PFR it encounters constantly changing concentrations of nutrients, oxygen and waste products.

 

Figure 5. (This one need to be redrawn)

Permissions
  1. Permissions Pending

 

The Biofilm exercise collection contains several reactors that are of the Plug Flow variety. One very useful and instructive plug flow reactor is the Flow Cell.  Flow cells have been used to investigate the growth of biofilms in real time on the stage of a microscope.  Exercise 3 in the collection describes the construction of a flow cell from optical quality square capillary tubes (Exercise 3 - Building and Using a Flow Cell Biofilm Reactor). 

Figure 6. A student constructed flow cell
 attached to a peristaltic pump.

Permissions
  1. Permissions Pending

The flow cell has provision for introducing additional organisms, biocides, nutrients or dyes while the cell is in operation.  If one wishes to observe biofilms growing in as close to a natural environment as possible, this is the exercise for you.   One very instructive use of the flow cell is to pump water from an established aquarium continuously through the cell while observing the walls of the cell microscopically.  One can observe in real time attachment, micro-colony formation and the effects of fluid flow.  The observation of the viscoelastic properties of mature biofilms as long streamers wave back and forth in the current is unforgettable.

Sometimes liquid passes in a plug flow mode through a porous matrix such as soil, sand or porous rock.  Biofilms initiated in such a matrix can be used to slow the rate of flow of contaminants through the matrix in what is called a biobarrier.  Furthermore, if the appropriate organism is available, that is, one that can metabolize the contaminating substance, biobarriers can be used to remediate the contaminate reducing the concentration of contaminating material.  Exercise 18 (Biofilms as Biobarriers) in the collection describes the construction and operation of a biobarrier.  This exercise is appropriate for general microbiology but might be of particular interest to students in environmental science or engineering.

Figure 7. Left -Two biobarrier columns, packed with 3 mm glass beads and inoculated with Pseudomonas fluorescence
Right - Detail of the air filter and drain assembly

Permissions
  1. Permissions Pending

The plug flow reactors describe above may be described as “closed”, in that they are enclosed in a pipe-like geometry.  Another sort of plug flow reactor could be described as “open”.  A shower curtain, the walls of a bathtub, or a canyon wall seep all exhibit the properties of an open plug flow reactor.

This sort of reactor geometry is quite common in nature in any habitat where water flows over a surface either continuously or intermittently. One type of reactor designed to model these conditions is the Drip Flow Reactor (DFR).  Drip flow reactors typically produce biofilms grown under low-shear and laminar flow conditions.

Figure 8. Open Plug Flow Biofilms, formed on the rock walls
below seeps at White House ruins in
Canyon de Chelly National Monument, AZ.

Permissions
  1. Permissions Pending

Of course, drip flow reactors are commercially available (for example, http://www.imt.net/~mitbst/Drip_Flow_Reactor.html), but they are prohibitively expensive for most teaching laboratories.  A new exercise in the biofilm laboratory collection (Exercise 4 - Construction of a Drip Flow Reactor) describes the construction of a drip flow reactor from a readily available storage container and other inexpensive and generally available materials.  This reactor is not autoclavable but can be disinfected in a 1/10 Clorox® bath.

 

Figure 9. Drip flow reactor constructed from a food storage container.

Permissions
  1. Permissions Pending

 

Goeres et al. list the following characteristics of drip flow reactors3:

Other Considerations


In many teaching exercises it is only desired that students observe the properties of biofilms in a subjective manner with no intent to make quantitative measurements of what is being observed.  On the other hand, many studies students may wish to carry out will require quantatitive physical and numerical measurement of biofilm growth, increasing cell number, resistance to microbicides, biofilm thickness and mass.  Protocols enabling students to conduct each of these measurements are also found in the exercise collection.

Measuring the thickness of biofilms in a non-destructive manner is easily accomplished according to instructions given in Exercise 15 Measurement of Biofilm Thickness.  The measurement requires only a simple modification of a standard student microscope, which permits measurement of the vertical movement of the objective lens.

Harvesting and Dispersing of Cells from Biofilms 

Because biofilm cells are attached to a substratum and contained within a slimy matrix, their enumeration by plate counting requires techniques somewhat different than those used for planktonic populations.  The cells must first be harvesting from the surface and dispersed into suspensions of individual cells.  They can then be treated in a manner similar to planktonic cell populations by serial dilution and plate counting.  The procedures for harvesting, dispersing and enumerating biofilm cells are found in Exercises 11 and 12 (Harvesting and Dispersing of Cells from Biofilms (11 Standard Method) and (12 Alternate Method) and Exercise 9 - Drop Plate Method for Counting Biofilm Cells.

Figure 10. 24 Well Microtitre Plate.

Permissions
  1. Permissions Pending

Measurement of Biofilm Mass

 

Sometimes measurement of biofilm mass is a sufficient quantitative measurement.  Exercise 6 - Colorimetric Measurement of Biofilm Density  describes a method for measuring the relative mass of biofilm growing in a 24 well microtitre plate.  The biofilm is stained with crystal violet dye, then washed to remove unbound dye.  The bound CV is then eluted with alcohol.  The amount of crystal violet dye recovered, measured  colorimetrically, is proportional to the mass of biofilm in the well.

 

 


 

 

 

Nutritional considerations


Many biofilms grow in conditions that are oligotrophic, that is low in nutrient concentration.  In order to mimic these conditions, nutrient levels must be chosen that are appropriate for the environment in question.  Biofilms will grow, albeit slowly, in tap water with no added nutrient at all, an oligotrophic environment indeed.

One of the more commonly used media for culturing biofilms is R2A medium.  R2A medium is a low nutrient medium developed for the enumeration by plate count of bacteria from treated potable water.  This medium has been shown to favor the growth of stressed organisms such as those grown at low nutrient concentrations or exposed to chlorine and comes closer to modeling the natural conditions in which biofilms grow than richer media such as Plate Count or Brain Heart Infusion Agar.

DifcoTM R2A Agar

                                           Approximate Formula* Per Liter
Yeast Extract .....................................................................0.5 g
Proteose Peptone No. 3 ..................................................0.5 g
Casamino Acids ................................................................0.5 g
Dextrose .............................................................................0.5 g
Soluble Starch ...................................................................0.5 g
Sodium Pyruvate ..............................................................0.3 g
Dipotassium Phosphate .................................................0.3 g
MagnesiumSulfate...........................................................0.05 g
Agar ....................................................................................15.0 g
        *Adjusted and/or supplemented as required to meet performance criteria.

 

Another medium that has been frequently used in biofim reactor studies is M9 Minimal Medium the recipe for which follows (from theLabRat.com):

M9 Minimal Media Recipe (1000 ml)

  1. Make M9 salts
  2. To make M9 Salts aliquot 800ml H2O and add
    • 64g Na2HPO4-7H2O
    • 15g KH2PO4
    • 2.5g NaCl
    • 5.0g NH4Cl
    • Stir until dissolved
    • Adjust to 1000ml with distilled H2O
    • Sterilize by autoclaving
  3. Measure ~700ml of distilled H2O (sterile)
  4. Add 200ml of M9 salts
  5. Add 2ml of 1M MgSO4 (sterile)
  6. Add 20 ml of 20% glucose (or other carbon source)
  7. Add 100ul of 1M CaCl2 (sterile)
  8. Adjust to 1000ml with distilled H2O

Students have also had good results growing biofilms in 1/10 or even 1/40 Tryptic Soy Broth or Tryptic Soy Agar.  Presumably other media at reduced concentrations would also serve and determining the optimum concentrations for any particular system would, in it self, be a worthy student project.

Shear forces or hydrodynamic stress

Shear is the force applied to a biofilm by the flow of water or medium, which tends to remove the biofilm by erosion or sloughing.  The amount of shear placed on a biofilm during growth has a profound effect on the architecture of the mature community.  For example, “a Pseudomonas aeruginosa biofilm formed in a reactor with high shear is denser and more tightly adhered to the surface as opposed to a P. fluorescens biofilm formed in a reactor under low shear, which is fluffy3.” Four typical environments, all experienced in nature by natural biofilms can be modeled in reactors of various sorts in the laboratory, high shear with turbulent flow, moderate shear, low shear and no shear3.  Both the Stirred Batch reactor (Exercise 1 and the Continuous Flow Stirred Reactor can be operated to produce High shear, turbulent flow or moderate flow depending on the rate of stirring.  The Static Glass Coupon Reactor (Exercise 13) is an example of a no flow reactor and the Drip Flow Reactor (Exercise 20) produces a low shear environment.
                  


Exercise

Reactor Type

Shear

1

Batch reactor

Static to high shear, no flow to turbulent flow

3

Continuous Flow Stirred Reactor

Low to high shear, laminar to turbulent flow

4

Flow cell Plug flow reactor

Low to high shear, laminar to turbulent flow

14

Biobarrier column, Plug flow reactor through a porous matrix

Low to moderate shear, turbulent flow

13

Static glass coupon reactor

No shear

20

Drip flow reactor, open plug flow reactor

Low shear, laminar flow

References

1 Characklis, William G. and Marshall, Kevin C. Biofilms, 1990. John Wiley and Son, Inc. New York.

2 Cargill, K.L., Pyle, B.H., Sauer, R.L., McFeters, G.A., 1992. Effects of Cultiure Conditions and Biofilm Formation on the Iodine Susceptability of Legionella pneumophila, Canadian Journal of Microbiology 38: 423-429.

3 Goeres, D.M., Hamilton, M.A., Beck, N.A., Buckingham-Meyers, K. Hilyard, J.D. Loetterle, L.R., Lorenz, L.A., Walker, D.K. and Stewart, P.S. 2009. A method for Growing a Biofiom Under Low Shear at the Air-liquid Interface Using the Drip Flow Biofilm Reactor.  Nature Protocols 4: 783-788.