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Chapter 45 : Physiological and Methodological Aspects of Cellulolytic Microbial Cultures

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Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, Page 1 of 2

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Abstract:

This chapter addresses three aspects of cultivating cellulose-utilizing microbes: growth media, fermentation equipment for small-scale laboratory cultivation, and methods to monitor cellulose consumption and fermentation dynamics with online and offline instrumental techniques. The choice of cellulose-containing substrate is a key aspect of medium formulation for cellulolytic microorganisms. Model substrates containing essentially pure cellulose, e.g., Avicel and Sigmacel, have significant merits for fundamental studies in that they have reproducible properties and are commonly accessible. The outcome of autoselection depends on the type of continuous cultivation. Multiply repetitive batch cultures can be used to monitor autoselection and evolution of microbial communities. The main operational difference between a pH auxostat and a chemostat is that fresh medium is delivered by the base pump rather than the peristaltic pump. To recycle microbial biomass, the fermentation system may include a tangential filter. The difference in gas exhaust versus background multiplied by N flow rate gives the formation rate of the respective gaseous product. The mass concentration of microbial biomass in cellulose-containing slurries can be estimated by analysis of some specific cellular constituent: DNA, membrane phospholipids, proteins, particulate N, muramic acid. Since the mass fraction of nitrogen in cells (e.g., 10%) is much higher than in most cell-free cellulosic substrates, nitrogen can be used as a proxy of microbial biomass in studies of cellulose-utilizing microorganisms.

Citation: Panikov N, Lynd L. 2010. Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, p 644-656. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch45

Key Concept Ranking

High-Performance Liquid Chromatography
0.46732005
Enzyme-Linked Immunosorbent Assay
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High-Performance Liquid Chromatography
0.46732005
Enzyme-Linked Immunosorbent Assay
0.46732005
Bacterial Growth
0.4458423
Vitamins and Growth Factors
0.40349975
0.46732005
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Figures

Image of FIGURE 1
FIGURE 1

Scheme of bioreactor designed for quantitative physiological studies of cellulose fermentation. 1, Medium carboy continuously stirred with magnetic stirrer to maintain microcrystal-line cellulose in suspended state; 2, N tank equipped with pressure regulator; 3, mass flow controller maintaining constant gas flow (40 to 200 ml/min) through reactor headspace (FMA5512; Omega); 4, manifold of three-way solenoid valves directing gas flow from several (6 to 12; 1 is shown) individual bioreactors to a QMS100 mass spectrometer (high-pressure gas analyzer; Stanford Research Systems); 5, manual sampling port, standard 60-cc syringe connected to neoprene tubing via female Luer connector; 6, automated sampling device consisting of solenoid valve and pump delivering 2- to 15-ml samples at regular intervals to the set of test tubes placed in refrigerated fraction collector (Bio-Rad); 7, liquid level sensor, based on conductivity principle, is used to maintain constant volume of the bioreactor during continuous cultivation; when the volume reaches the threshold level and the sensor’s tip touches liquid, the conductivity in the circuit jumps and activates the harvesting pump, pushing excessive cultural liquid to the waste bottle; 8, the condenser functions as a barrier for intensive medium evaporation; 9, pH probe and online optical NIR sensor (OD4, Dasgip); 10, Sartorius-Stedim BIOSTAT APlus controlling unit; 11, optional peristaltic pump; 12, tubing line used to discard cells which can be replaced by tangential filtration unit.

Citation: Panikov N, Lynd L. 2010. Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, p 644-656. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch45
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Image of FIGURE 2
FIGURE 2

Modification of the level control of a Sartorius-Stedim bioreactor (Dartmouth design). 1, Cultural liquid (electroconductive); 2, metal headplate; 3, stainless steel pipe used as level sensor and harvest tubing; 4, Teflon pipe (insulator); 5, flexible rubber tubing which allows changing the position of the pipe by moving it up and down without compromising the sterility of the fermentor; 6, temperature probe (electroconductive). When the liquid level is below the sensor, the electric circuit is open, the current is zero, and the pump is idle. At the instant of contact, the circuit is closed and the harvesting pump is activated.

Citation: Panikov N, Lynd L. 2010. Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, p 644-656. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch45
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Image of FIGURE 3
FIGURE 3

Managing suspended solids in continuous culture. (A) Schematic illustration of forces affecting solid particles moving through narrow tubing: advective forces (vectors parallel to flow direction) are interacting with gravitational forces (vertical vectors), and as a result, solids sediment at the bottom of tube folding (enclosed area). (B) Sedimentation is maximal at junction point of wide and narrow tubing. (C) Wrong (crossed) and correct shape of the vertical pipe used to pump out substrate slurry. With a straight pipe inside a reservoir, the stirring of the carboy does not prevent sedimentation of particulate material back to the reservoir (arrows). The circular end of pipe completely stops sedimentation. (D) Wrong (crossed) and correct way of solids recovery (cells plus unused cellulose) from the fermentation vessel. Unintentional recycling of solids occurs because of their sedimentation in a vertical harvesting pipe (the tip of this pipe is positioned below the gas-liquid interface). Recycling is completely prevented if the harvesting pipe is positioned at the gas-liquid interface: sedimentation is blocked by gas bubbles, and the harvesting pipe serves as a level sensor (see also Fig. 2 ).

Citation: Panikov N, Lynd L. 2010. Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, p 644-656. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch45
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Image of FIGURE 4
FIGURE 4

Schematic representation of cultivation system listed in Table 2 . , medium flow rate; , feed substrate concentration; , residual substrate concentration; , cell mass concentration in fermentor; , cell concentration in outflow liquid; , metabolic products in fermentor; , products in outflow liquid.

Citation: Panikov N, Lynd L. 2010. Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, p 644-656. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch45
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Image of FIGURE 5
FIGURE 5

batch growth on Avicel, 10 g/liter. (Top) Example of online monitoring of gas production, titration rate, and turbidity; (middle) residual cellulose and cell biomass based on analyses of pellet C and N (example of offline growth monitoring in samples taken with fraction collector); (bottom) high-performance liquid chromatography data for the same experiment.

Citation: Panikov N, Lynd L. 2010. Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, p 644-656. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch45
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Image of FIGURE 6
FIGURE 6

Automated sampling which requires no preliminary tubing flushing. (Top) Configuration of the system between samplings (sampling frequency is typically every 1 to 2 h); the regulating solenoid valve is open and pump is idle. (Middle) The first sampling phase: the valve is closed and the pump is active. Cultural liquid is delivered to a tube kept in a refrigerated fraction collector. (Bottom) The second sampling phase: the valve is open and N gets in and interrupts the flow of cultural liquid; however, the pump keeps going for 5 to 10 s and cleans up the peripheral part of the sampling loop. Unidirectional flow of liquid and positive pressure gradient guarantee the sterility of the entire sampling line. This device can be used in a manual mode as an alternative to traditional sampling with disposable syringes or vials. In this case, the pump is activated manually and N flow is stopped with a clamp instead of a solenoid.

Citation: Panikov N, Lynd L. 2010. Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, p 644-656. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch45
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References

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Tables

Generic image for table
TABLE 1

Summary of nutrient media used to culture and related organisms

Citation: Panikov N, Lynd L. 2010. Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, p 644-656. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch45
Generic image for table
TABLE 2

Various cultivation techniques and their possible goals

Citation: Panikov N, Lynd L. 2010. Physiological and Methodological Aspects of Cellulolytic Microbial Cultures, p 644-656. In Baltz R, Demain A, Davies J, Bull A, Junker B, Katz L, Lynd L, Masurekar P, Reeves C, Zhao H (ed), Manual of Industrial Microbiology and Biotechnology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816827.ch45

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