Food and Industrial Microbiology

Module 5. Bioprocessing

Lesson 20
GROWTH CURVE, GROWTH MEASUREMENT, GROWTH CULTIVATION

20.1 Introduction

Growth is an orderly increase in the quantity of cellular constituents. It depends upon the ability of the cell to form new protoplasm from nutrients available in the environment. In most bacteria, growth involves the following steps.

1. Increase in cell mass and number of ribosomes

2. Duplication of the bacterial chromosome

3. Synthesis of new cell wall and plasma membrane

4. Partitioning of the two chromosomes

5. Septum formation

6. Cell division.

This asexual process of reproduction is called binary fission . For unicellular organisms such as the bacteria, growth can be measured in terms of two different parameters: changes in cell mass and changes in cell numbers .

20.2 Growth Curve

20.2.1 Batch experiment

It has been observed that if one of the essential requirements for growth is present in only limited amounts, the limiting factor affects on the rate of growth.

A batch culture system is one containing a limited amount of nutrient, which is inoculated with the microorganism. Cells grow until some component is exhausted or until the environment changes so as to inhibit growth. During batch fermentations the population of microorganisms goes through several distinct growth phases:

1. Lag phase
2. Accelerating growth phase
3. Log (exponential growth) phase
4. Declining growth phase
5. Stationary phase
6. Death or declining death phase
   

Log death phase

Fig. 20.1 growth phases of a simple batch culture ( Liu et al., 2003)

The salient points of the growth phases are as under

1. Lag Phase

a. Adaptation (acclimation) period

The lag phase is the initial phase which represents the period (time) required for bacteria to adapt to their new environment.

b. Constant number of cells

During this phase, the individual bacterial cells increase in size, but the number of cells remains unchanged.

c. Physiologically active

They are very active physiologically and are synthesizing new enzymes and activating factors.

2. Accelerating Growth Phase

Transition period from the lag phase to the log phase. .Cell is beginning to grow (increase in numbers) noticeably as enzyme systems are gearing up.

3. Log (Exponential Growth) Phase

a. Exponential growth during this phase, the bacterial cells divide regularly at a constant rate.

b. Straight line on semilog scale - The logarithms of the number of cells plotted against time results in a straight line.

c. Maximum Rate of Substrate utilization. A maximum growth rate occurs under optimal conditions, and substrate is removed from the medium at the maximum rate. The growth rate is limited only by the bacteria's ability to process the substrate. Food is in excess (not limiting) so that the rate of growth is only limited by the ability to process the food. Sometimes called "0-order growth" and growth rate is constant and maximum.

4. Declining Growth Phase

a. Transition period from the log phase to the stationary phase.

b. Decreasing growth rate

c. Exhaustion of essential nutrients

d. Accumulation of toxic metabolic products - the growth rate can be limited either by the exhaustion of essential nutrients or by the accumulation of toxic metabolic products. - food becomes limiting factor and therefore growth rate and mass of bacteria are dependent on the amount of food present.

5. Stationary Phase

a. The number of cells remains constant perhaps as a results of complete cessation of division or the balancing of reproduction rate by an equivalent death rate. Growth of new cells is balanced by the death of old cells. No increase in cell mass - population is "stable". net growth rate = 0

6. Death or declining Phase

a. The number of viable cells decreases slowly while the total mass may remain constant due to the fact that the death rate exceeds the production rate of new cells.

b. Depletion of essential nutrients

c. Accumulation of inhibitory products. - Death occurs primarily as a result of depletion of essential nutrients and/or the accumulation of inhibitory products.

7. Log Death Phase

a. Exponential death - "wholesale die-off" - system is dead - even if you add food, you will get no growth.

20.3 Microbial Growth Kinetics

The exponential growth phase is the most important phase of the growth cycle when the product you are trying to produce is, either the biomass itself or a growth associated product. Quantification of exponential growth rate (i.e. how fast cells grow) is the first fundamental step in the quantification of culture kinetics.

The establishment of exponential growth is dependent on a number of factors.

a. A viable inoculum

b. A suitable energy source

c. The presence of excess nutrients and growth factors

d. The absence of inhibitors

e. A suitable environment (i.e. temperature, dissolved oxygen)

Modelling and simulation of microbial cell growth is important both theoretically and practically. Although the Monod model has been the most widely used for the prediction of cell growth, it only fits the exponential growth phase of the growth, without any inhibition. The lag growth phase, decreased growth phase and the stationary growth phase of a typical microbial batch culture growth curve cannot be predicted using the Monod model.

When t = 0 (when exponential growth begins)

where X = X₀ (the biomass concentration at the start of the fermentation).

When t = t, X = X.

Equation (2) applies only to the duration of the exponential growth phase, beyond which either substrate limitation or toxin accumulation become rate determining.

Doubling Time is the time required to double the quantity of biomass, that is growing exponentially.

Specific growth rate ( µ ) can be defined as any point during the growth cycle. During the exponential growth period µ is constant and at a maximum for that process under the specified conditions.

It is often stated that µ , the specific growth rate is a function of growth limiting substrate concentration

µ = f (S)

S = concentration of growth rate limiting substrate

Relationship between growth rate and substrate concentration : This is generally defined in terms of the Monod Equation. Applies only where a single substrate (S) is limiting and the accumulation of growth associated toxins can be ignored (which is generally the case in dilute microbial suspensions). It is very similar to the shape of the Michaelis Menten equation for enzyme kinetics.

20.4 Monitoring Microbial Growth In Culture

During fermentation, methods are required for the routine determination of the microbial population, cell number and / or biomass, in order to monitor its progress. Numerous direct and indirect methods are available for this purpose. Direct procedures involve dry weight determination, cell counting by microscopy and plate counting methods. Indirect methods include turbidimetry, spectrophotometer, estimation of cell components (protein, DNA, RNA, or ATP), and online monitoring of carbon dioxide production or oxygen utilization.

20.4.1 Classical cultural methods

Conventional methods for the enumeration of bacteria in food are colony count methods. If low numbers of bacteria are suspected to be present in the food samples, numbers may be estimated by means of the most probable number method (MPN).

20.4.2 Automation as alternative method

Many improvements in this field have been made that permit laboratories to increase the efficiency and the number of samples processed such as agar preparation machines, automated dilutors, automated counting devices and spiral plate. As an example, the spiral plate is a semi-automated plating technique that greatly reduces manpower and material costs normally associated with the classical cultural method, in particular the colony count method.

20.4.3 Chromogenic and fluorogenic isolation media

The recognition of colonies of presumptive target organisms has been facilitated by the introduction of chromogenic and fluorogenic media. These are microbiological growth media that contain enzyme substrates linked to a chromogen (colour reaction), fluorogen

(fluorescent reaction) or a combination of both. The incorporation of such fluorogenic or chromogenic enzyme substrates into a selective medium can eliminate the need for subculture and further biochemical tests to establish the identity of certain micro-organisms.

20.4.4 Modified cultural methods

A variety of rapid methods have been elaborated, which predominantly aim to reduce the workload and facilitate the work flow by reducing the manipulations and/or the necessity for a full lab infrastructure and not necessarily shorten the time for detection. Some of these modified cultural methods are based upon the colony count method e.g. 3M Petrifilm and Compact Dry, whereas others make use of the principle of the MPN method e.g. TEMPO and SimPlate.

20.4.4.1 3M Petrifilm

Rather than a Petri dish, 3M Petrifilm makes use of thin plastic film as carrier of the culture medium. Generally the 3M Petrifilm plate comprises a cold-water-soluble gelling agent, nutrients and indicators for activity and enumeration. An important advantage of the 3M Petrifilm plate is the fact that it is very thin (a film), saving space in the incubator. After incubation, typical colonies can be counted either manually (facilitated by the grid on the background of the film and characteristic colored colonies) or automatically.

20.4.4.2 Compact dry (Nissui Pharmaceutical Co., LTD.)

The Compact Dry plates also have a dedicated user-friendly small plastic dish format that contains dehydrated nutrients and differentiating components. Similar to the 3M Petrifilm Compact Dry plates are thin, light and convenient to handle.

20.4.4.3 SimPlate (BioControl systems)

Detection and enumeration of micro-organisms by the SimPlate methods rely on a binary detection technology. It uses IDEXX’s Multiple Enzyme Technology (MET) to detect bacteria in food and in water. Visible colour changes occur as a result of bacterial enzyme interaction with substrates present in the liquid culture medium. The counting range is from <1 to 738 per plate (more than double of a standard pour plate).

20.4.4.4 TEMPO (Bio-Mérieux)

The TEMPO test is an automated MPN enumeration method and consists of a vial of culture medium and a card, which are specific to the test. Dedicated equipment and software support the inoculation and reading of the cards. Target micro-organism multiply in the culture medium resulting in a signal detected by the TEMPO Reader (based up on fluorescent pH indicator, β-glucuronidase activity, etc.).

20.4.4.5 Colilert_ (IDEXX Laboratories)

Colilert is used for the simultaneously detection and enumeration of total coliforms and E. coli in water and waste water based on the MPN principle. Colilert uses the patented Defined Substrate Technology (DST) and two chromogenic nutrient-indicators, ortho-nitrophenyl-β-D-galactopyranoside (ONPG) and 4-methylumbelliferyl-β-D-glucuronide (MUG).

20.4.4.6 Soleris (Neogen)

The Soleris technology monitors changes in the chemical characteristics of microbial liquid growth medium and detects micro-organisms with pH and other sensitive reagents. The reagents change their spectral patterns as the metabolic process takes place which can be detected photometrically by an optical instrument and monitored at predetermined time intervals. Sensitivity ranges from a single organism per vial to 10⁸ cfu/ml (upper limit), but the time at which growth is first detected is inversely proportional to the log number of bacteria in the sample.

20.4.5 Bio-chemically based enumeration methods

20.4.5.1 Impedance

This method is based on the principle that bacteria actively growing in a culture medium produce positively or negatively charged end-products (early stages of breakdown of nutrients) that cause an impedance variation of the medium. This variation, which is proportional to the change in the number of bacteria in the culture, makes it possible to measure bacterial growth. The time at which growth is first detected, referred to as detection time (DT), is inversely proportional to the log number of bacteria in the sample, which means that bacterial counts can be predicted from DT.

20.4.5.2 ATP bioluminescence

This technique measures light emission produced due to the presence of ATP, which is involved in an enzyme substrate reaction between luciferin and luciferase (bioluminescence). The quantity of light produced (measured as Relative Light Units RLUs) is proportional to the concentration of ATP and, thus, to the number of micro-organisms in the original sample. ATP bioluminescence can be used for enumeration of total count but it is only applicable if high numbers of bacteria are present (>10,000 cfu/g). As such this technique is mostly used to estimate the total surface cleanliness, including the presence of organic debris and microbial contamination, providing results within less than 5 min. The Milliflex Rapid system is an ATP based system provided by MilliPore.

20.4.6 Microscopic based enumeration methods

20.4.6.1 Flow cytometry

Flow cytometry quantitatively measures the optical characteristics of cells as they are presented separately in front of a focused light beam (from a high-pressure mercury vapour lamp or an assortment of lasers). As particles pass through the light beam three parameters are measured using photomultiplier tubes, the forward scatter, the side scatter and fluorescence. For routine analyses of milk quality, an automated instrument (Bactoscan 8000 method) was developed. This flow cytometry method uses ethidium bromide (intercalating with DNA) to stain bacteria in milk. The disturbing milk components are reduced and dispersed by treatment with detergent and enzyme at 50 ^ο C, and provides a result after 8 min.

20.4.6.2 Direct epifluorescent filter technique (DEFT)

The DEFT is a microscopic cell counting method. A pre-treated sample is filtered over a polycarbonate membrane. The microbial cells are concentrated and stained with fluorescent dyes. Incident light illumination (epifluorescence) is used to examine the filter surface. The actual staining and counting takes less than 0.5-1 h, but sample pre-treatment steps lengthen the total detection time. The detection limit is 10⁴ to 10⁵ cells/ml. COBRA instrument provided by Cobra Biomanufacturing Plc is an example of a DEFT based method.

20.4.7 Future trends for enumeration methods

20.4.7.1 Quantitative real-time polymerase chain reaction (Q-PCR)

PCR is a technique which actually quantifies genomic copies, the relation between genomic copies and the number of cells present in the sample needs to be determined as well.

20.4.8 Immunoassays

immunoassays are based on the highly specific binding reaction between antibodies and antigens. The selection of an appropriate antibody (monoclonal or polyclonal) is the determinant factor for the method’s performance. Usually, any positive result for pathogens obtained with immunoassays is considered as presumptive and requires further confirmation. Detection limit is approximately 10⁴ to 10⁵ cfu/ml, depending upon the type of antibody and its affinity for the corresponding epitope. Several types of immunoassays are available in food diagnostics of which lateral flow devices (LFD), Enzyme Linked Immunosorbent Assays (ELISAs) & Enzyme linked fluorescent assays (ELFAs) are widely used. Immunomagnetic separation (IMS) assays, although a sample preparation tool instead of a detection method, have been developed as an aid in reducing the time for the enrichment step prior to detection. and are therefore discussed in this paragraph.

20.4.8.1 Enzyme linked immunosorbent assay (ELISA) & enzyme linked fluorescent assays (ELFA)

ELISA is a biochemical technique that couples an immunoassay with an enzyme assay. In most of the alternative methods a sandwich ELISA is used. The sandwich ELISA comprised different steps. Specific antibodies are affixed to the surface of the wells of a 96 well microtiter plate. The sample, with an unknown amount of target antigen, is added and allowed to bind to the affixed antibodies. In the final step a substrate is added that the enzyme can convert to a detectable signal. Validated ELISA methods are available from BIO A.R.T., R-Biopharm AG, BioControl Systems, Rayal and 3M. The VIDA system developed by Bio-Mérieux

20.4.8.2 Immunomagnetic separation and concentration

Superparamagnetic particles can be coated with antibodies, allowing specific capture and isolation of intact cells directly from a complex sample suspension without the need for column immobilization or centrifugation. Monosized superparamagnetic polymer particles known as “Dynabeads” are available commercially from Invitrogen-Dynal. Pathatrix, an automated system, is a patented re-circulating immunomagnetic separation technology.

20.4.9 Bacteriophage-based detection methods

Phages are extremely host-specific. Bacteriophages or proteins of bacteriophages have been included in various ways in detection methods for pathogens. The specific bacteriophage tail-associated proteins can be attached to paramagnetic beads to capture bacteria in suspension. The bacteria-bead complex can be integrated in fast detection protocols.

20.4.10 Molecular based detection methods

20.4.10.1 Fluorescent in situ hybridization (FISH)

Fluorescence in situ hybridization (FISH) with ribosomal RNA (rRNA) targeted oligonucleotide probes is the most commonly applied technique among the ‘non-PCR-based’ molecular techniques. FISH is commercially exploited by e.g. Vermicon, which has detection kit for different pathogenic and non-pathogenic micro-organisms.

20.4.10.2 Conventional, real-time and multiplex polymerase chain reaction (PCR)

It is a three-step cyclic in vitro procedure based on the ability of the DNA polymerase to copy a strand of DNA. The presence of even 1 copy of the template within the reaction mixture can be detected within a couple of hours as about a million-fold of copies are created. In the early 1990s, the “second” generation of PCR technologies was introduced by the use of fluorescent double-stranded DNA dyes. Validated PCR methods are available from Bio-Rad, Roche, Qualicon/Oxoid, Genesystems, AES Chemunex, Applied BioSystems, Idaho Technology Inc., Lantmännen, IEH Laboratries and Consulting Group, ADNucleis and BioControl systems.

20.4.11 Future trends

20.4.11.1 Microarrays

Microarrays or gene chips provide a miniaturized system for the simultaneous analysis of hybridization of fluorescent-labelled single strand nucleotide chains to an array of oligonucleotide probes immobilized on a support such as glass or a synthetic membrane. PCR amplification is often used prior to hybridization to increase sensitivity of detection. DNA microarrays may be very useful for detecting multiple bacteria simultaneously on a single glass slide .

20.4.11.2 Biosensors

Biosensors are defined as analytical devices that combine biospecific recognition systems with physical or electrochemical signaling. Biosensors for the detection of pathogens in the food industry consist of immobilized biologically active material, like enzymes, antibodies, antigens or nucleic acids, in close proximity to a receiving transducer unit. Target recognition results in the generation of an electrical, optical or thermal signal that is proportional to the concentration of target molecules. Biosensors have the potential to shorten the time between sampling and results, but their future lies in reaching selectivity and sensitivities comparable to established methods at a fraction of the cost.