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Nutrient control for stationary phase cellulase production in Trichoderma reesei Rut C-30
Accepted Manuscript Title: Nutrient Control for Stationary Phase Cellulase Production in Trichoderma reesei Rut C-30 Author: Nicholas V. Callow Christopher S. Ray Matthew A. Kelbly Lu-Kwang Ju PII: DOI: Reference:
S0141-0229(15)30047-8 http://dx.doi.org/doi:10.1016/j.enzmictec.2015.08.012 EMT 8803
To appear in:
Enzyme and Microbial Technology
Received date: Revised date: Accepted date:
13-3-2015 16-8-2015 18-8-2015
Please cite this article as: Callow Nicholas V, Ray Christopher S, Kelbly Matthew A, Ju Lu-Kwang.Nutrient Control for Stationary Phase Cellulase Production in Trichoderma reesei Rut C-30.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2015.08.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nutrient Control for Stationary Phase Cellulase Production in Trichoderma reesei Rut C-30
Nicholas V. Callow, Christopher S. Ray, Matthew A. Kelbly, Lu-Kwang Ju* Department of Chemical and Biomolecular Engineering The University of Akron, Akron OH 44325, USA
*Corresponding author; e-mail address: [email protected] Highlights
Stationary phase Trichoderma reesei Rut C-30 for long-term cellulase production
Phosphorus limited cellulase production for extended fermentation
Airlift pellet fermentation of T. reesei Rut C-30 possible with nutrient limitation
Abstract This work describes the use of nutrient limitations with Trichoderma reesei Rut C-30 to obtain a prolonged stationary phase cellulase production. This period of non-growth may allow for dependable cellulase production, extended fermentation periods, and the possibility to use pellet morphology for easy product separation. Phosphorus limitation was successful in halting growth and had a corresponding specific cellulase production of 5 ± 2 FPU/g-h. Combined with the addition of Triton X-100 for fungal pellet formation and low shear conditions, a stationary phase cellulase production period in excess of 300 h was achieved, with a constant enzyme production
rate of 7 ± 1 FPU/g-h. While nitrogen limitation was also effective as a growth limiter, it, however, also prevented cellulase production.
Keywords Cellulase; Trichoderma reesei; Phosphorus; Nitrogen; Pellet
1. Introduction Cellulase enzymes have been used in textile and paper processing for finishing applications [1,2]. This enzyme group has potential applications in cellulosic biofuel production to hydrolyze biomass into fermentable sugars. However, expansion has been limited by relatively high production costs [1]. Members of the branched fungi genus Trichoderma are commonly employed to produce cellulase.
A variety of species, mutants, feeding, pH, temperature, mixing, nutrient supplements, and process control schemes have been identified to improve both volumetric and specific cellulase production [3–8]. A key advancement was removing the glucose repression of cellulase expression, which allowed for cellulase production on low cost sugars. The Rut C-30 mutant is lacking the cellulase expression dependency on glucose lean conditions [9]. Likewise, T. reesei Rut C-30, MCG-77, and NG-14 strains have all proven to be among the best cellulase producers [7]. The production of cellulase is a complex function of the growth environment. Cellulose, sophorose, and lactose have been shown to be effective cellulase inducing agents for T. reesei [10,11]. In some cases, improved cellulase production requires a complex feeding scheme to maintain low fungal growth and adequate cellulase production [12,13]. A simple control scheme
is desirable to arrest the growth of T. reesei for continuous cellulase production. Stationary culture conditions may allow for mycelial retention, long-term process stability, and potentially higher product yields with little waste of nutrients on fungal biomass production.
To achieve better process stability, recent process control schemes and chemical additives have been used to modify the morphology from filamentous to pellet for simplified industrial processing [14,15]. In submerged fermentation, specific shear ranges and surfactants have both been effective for morphology control. These morphological changes may help reduce the cost of cellulase production though improved separation. The use of pellets aids in extended periods of cellulase production with retained fungal biomass. Furthermore, the constant presence of fungal biomass, coupled with minimal nutrient availability may reduce the potential for contamination by competing organisms. Attempts have been made to create pellet T. reesei. However, these are limited by very specific operating conditions, pellet degradation, or overgrowth out of pellet form that leads to mixing failure [15–18].
It is hypothesized that phosphorus limitation will halt T. reesei Rut C-30 growth without limiting cellulase production. Reduced potassium phosphate levels have been previously shown to improve cellulase production [19]. Further, the application of nitrogen limitation may be effective at halting growth and providing insight into the nutrient allocation for synthesis of cellulose enzymes versus cell mass. Ideally, this non-carbon nutrient limitation can be combined with pellet morphology for continuous cellulase production under stationary phase conditions. Here, the fungal biomass concentration is stable and the cellulase activity is accumulating or continually harvested.
2. Methods 2.1. Strain and Culture Maintenance Trichoderma reesei Rut C-30 (NRRL 3469) was obtained from the United States Department of Agriculture’s Agricultural Research Service’s culture collection. It was maintained on potato dextrose agar plates (Sigma-Aldrich, St. Louis, MO). These plates were incubated at 30 °C for 4 days before storage at 4 °C. The culture was transferred every 4 weeks by streaking a fresh plate with a loop of cells taken from a mature plate. Fermentation inoculum was prepared by transferring 6 loops of cells, from a mature plate, into 100 mL of sterile potato dextrose broth (Sigma-Aldrich, St. Louis, MO), which was contained in a 250 mL glass Erlenmeyer flask covered by cheese-cloth cotton. The culture was incubated at room temperature for 3 days on a magnetic stir plate set to approximately 300 revolutions per minute (rpm) and then used for fermentation inoculation.
2.2. Experimental Setup This study is divided into two fermentation sections: high shear filamentous fermentation and low shear pellet fermentation. Within each section, the applicability of either nitrogen or phosphorus limitation was tested. The high-shear testing utilized filamentous T. reesei and determined how nutrient limitations influenced growth and enzyme production. The low-shear testing focused on Triton X-100 induced pellets for determining long-term process stability.
For all systems, inoculation was provided at 10% (v/v). The pH was allowed to naturally fall to 4.8, beginning at 5.6, and was then maintained by automatic addition of either 1.0 M hydrochloric acid or sodium hydroxide. Trans-278 (Trans-Chemco, Bristol, WI) anti-foam was added, as needed, to control foaming. All setups included periodic sampling to measure nutrient concentrations and T. reesei growth. During the fermentation, a proportion of limiting nutrient was added to observe the growth and cellulase production response. Unless otherwise specified, all medium components were purchased from Sigma-Aldrich (St. Louis, MO). The proteose peptone was from Remel (Thermo Fisher Scientific, Lenexa, KS) and the ammonium sulfate was from Fisher Scientific (Thermo Fisher Scientific).
In the high-shear fermentations, T. reesei Rut C-30 was cultured in a New Brunswick BioFlo 110 fermentor (Eppendorf North America, Enfield, NJ). The working volume was 1 L with an agitation provided by a 6-bladed Rushton turbine at 350 rpm. Air was supplied at 1 liter per minute (LPM) and with demand-controlled supplementation of pure oxygen to maintain a minimum 20% dissolved oxygen concentration, relative to air saturation.
In the pellet fermentations, inoculum was first added to 250 mL shake flasks and cultured for 5 days at 250 rpm in a Que Orbital shaker (Model 4703, Parkersburg, WV). These flasks contained the standard medium (Table 1) and 1.0 g/L of Triton X-100 to grow T. reesei pellets. These pellets were gently centrifuged at 1,046 g (Legend X1R, Thermo Scientific, Osterode, Germany) to isolate them from the growth medium and were then re-suspended in sterile water. The pellets were washed, re-centrifuged, and suspended in the final nutrient deficient medium for either phosphorus or nitrogen study. The pellets were used as the pre-culture for airlift fermentors. This
process reduced unused phosphates or ammonium from being transferred to the primary fermentor. A custom airlift fermentor provided gentle agitation with rising air bubbles. Aeration was supplied at 0.75 LPM, for 1.5 L working volume. In addition to the Trans-278 chemical antifoam, an external rotating cone foam breaker and pump to return the foamate back to the airlift fermentor accomplished foam control. Dissolved oxygen concentration was not monitored for this setup.
For this study, the growth medium contained lactose as the primary carbon source. Lactose has been shown to induce cellulase production and is an appropriate carbon source for growth [20– 22]. The included minerals were previously identified for successful growth and enzyme production [23]; these were used without modification. Suitable nitrogen sources for cellulase production have been described elsewhere [24,25]. T. reesei can consume a variety of nitrogen containing compounds with little change in cellulase production. In this work, urea and proteose peptone were replaced by ammonium sulfate.
T. reesei growth and cellulase production has not been reported for phosphorus limiting conditions. Potassium phosphate is generally included in excess of biological requirements for its pH buffering capacity. For this work, potassium phosphate was removed and potassium ions were added with potassium chloride. In phosphorus limited systems, the phosphates were withheld from the growth medium (Table 1). Urea and proteose peptone was also removed from the medium. Ammonium sulfate was increased to maintain the nitrogen content. The T. reesei was grown into an initial stationary phase. Then, a small amount of phosphate containing salt solution was added to observe the response.
The modified medium composition is indicated in Table 2 for the nitrogen limiting studies. The peptone, urea, and ammonium sulfate has been removed from the growth medium and combined in a new solution. This nitrogen rich solution was added occasionally during the fermentation to probe the response to nitrogen availability.
2.3. Analytical Techniques Samples were withdrawn from the fermentor by a syringe affixed sample tube and isolation valve. All samples were divided into 1.0 mL volumes and placed into polypropylene microcentrifuge tubes. These samples were centrifuged at 9,300 g in an Eppendorf 8150 bench top centrifuge for 10 min. The supernatant was carefully collected from the compacted fungal pellet. Both fractions were frozen at -20 °C until tested.
T. reesei concentrations were indirectly assessed through intracellular protein content. The fungal pellet was disrupted with 0.2 M aqueous sodium hydroxide, at equal volume to the original sample volume collected, and heating for 20 min at 100 °C. The protein release was determined by complexing with Coomassie Brilliant Blue G-250 dye (Bio-Rad Protein Assay Kit #500-002, Bio-Rad Laboratories, Hercules, CA). Bovine serum albumin was used for calibration. A previously established correlation to convert protein content to fungal biomass was used by dividing the intracellular protein concentration by 0.125 [26]. In this work, each sample drawn from the fermentor was analyzed with at least three analytical replicates. Values and error bars plotted in figures are the averages and standard deviations of the replicated tests.
Cellulase activities were determined by the standard test method described by Ghose [27] and reported as Filter Paper Units per unit sample volume (FPU/mL). One FPU/mL corresponds to the release of 1 µmol/mL (or mM) glucose per minute, under standard testing conditions. The test was modified to improve temperature uniformity for larger numbers of test vials by including mixing in a heated water bath. The sample tubes were sealed and incubated partially submerged in water at 50 °C with mild shaking at 60 rpm in an orbital shaker (ORS 200, Bokel Scientific Inc., Fasterville, PA). The lactose concentrations were obtained from the blank enzyme sample values and were expressed as glucose equivalents. For each fermentation sample, the cellulase activity and lactose concentration was re-measured to obtain an average and standard deviation. The primary source of error in this test is in the tedious replicates, where liquid transfer errors are amplified, and in the non-homogenous test comprising a solid substrate and non-linear hydrolysis dependency on enzyme concentration [28].
Specific cellulase productivity is defined as the rate of cellulase production per unit amount of biomass, and carries the unit of FPU per gram-hour. These values were determined by fitting a linear regression equation to the measured cellulase activities (FPU/mL) during stationary phase periods, when there was no cell growth due to a nutrient limitation. The slope parameter, FPU/mL-h, was multiplied by 1000 (to convert the unit to FPU/L-h) and then divided by the average biomass concentration (g/L) for the given period. For systems with multiple stationary phase periods, each period was considered separately. To account for the differences in fermentation variation and errors during testing, replicate fermentations were included, where appropriate, to obtain average and standard deviation values.
3. Results A reference fermentation for T. reesei is shown in Figure 1. This fermentation was stopped when the lactose was depleted and the system reached carbon source limitation. The total duration was 62 h, during which time the cellulase activity increased. By averaging the incremental increase in both fungal biomass and cellulase, the specific rate of cellulase production during this period was determined to be 10 ± 2 FPU per gram of cell per hour. Given 8.3 g/L lactose consumed and 1 g/L proteose peptone, the system reached 3.5 ± 0.5 g/L of fungal mass.
3.1. High Shear with Phosphorus Limitation Phosphorus limited testing was replicated in three stir-tank fermentation sets. T. reesei was grown on the aforementioned potato dextrose broth, separated from this phosphorus containing nutrient solution and placed in a phosphorus free fermentation medium. Initially, the fungus continued growing until a phosphorus limitation was reached, assumed since all other nutrients were in excess throughout the fermentation. The amount of initial growth varied with the completeness of the phosphorus removal prior to inoculation. Initial growth reached cell concentrations that were between 3X and 13X of the inoculated concentrations. This initial growth can be seen in Figure 2 (A). The average fungal mass concentration during the first phosphorus limited period is indicated on the figure as a solid horizontal line. After the fungus reached a stationary phase, a stable biomass concentration for 48 h, a small amount of phosphorus containing solution was added to the fermentor. As indicated in the figure, growth resumed until a new stationary phase was reached. The average fungal mass concentrations for the second stationary period are indicated by the solid lines in Figure 2 (A). This stepwise
control of the fungal mass further indicates that phosphate was the limiting growth factor. The average of increased T. reesei concentration between the first and second stationary phases shown in Figure 2 (A) was used to determine the cell yield from phosphorus, which was 236 ± 11 (g/g). Figure 2 (B) indicates the cellulase profiles for each set. As shown, the cellulase production was not inhibited during periods of stationary phase created by the phosphorus limitation. By curve fitting the cellulase activity profile, indicated by the solid lines, a production rate was obtained. The average fungal concentrations during this period are indicated by the solids lines in subfigure A and were used to obtain the specific cellulase productivity. During the periods of non-growth, the average specific cellulase productivity, across all replicates, was 5 ± 2 FPU/g-h. Likewise, the FPU produced per lactose consumed was 227 ± 94 FPU/g, which was nearly twice that of the non-stationary systems at 124 ± 54 FPU/g (data provided in Supplementary Materials).
3.2. Low Shear with Phosphorus Limitation For low shear nutrient limited systems, pellet morphology was created in a shake flask and inoculated into a concentric airlift fermentor. The concentric airlift system contains a primary fermentor vessel, foam breaker, recirculation pump, pH controls, and sampling ports. Figure 3 shows the results from a fermentation set. The T. reesei reached stationary phase after 50 h with a cell concentration of 3.0 ± 0.3 g/L, indicating utilization of any free phosphorus that had been carried into the vessel with the pellet culture. Cellulase was produced at a constant rate of 27.3 FPU/L-h during the stationary phase until the carbon source was depleted. The specific cellulase production rate was 9 ± 3 FPU/g-h.
To test extended fermentation, periodic lactose addition was included to avoid carbon source limitations. The results are shown in Figure 4. Here lactose was periodically added, as indicated by the vertical arrows. The extended fungal biomass concentration was maintained at 1.9 ± 0.5 g/L. Here, the specific cellulase productivity was 7 ± 2 FPU/g-h, as was in a duplicate system (data not shown). The specific cellulase productivity, averaged across all replicated systems, was 7 ± 1 FPU/g-h. In these systems, the concentration of T. reesei was successfully halted for an extended duration of continuous cellulase production.
In the lower shear environment, the pellet diameter increased in size. The initial pellet size was set by the shake flask culturing conditions, at 0.8 ± 0.2 mm. During the fermentations, the size increased to 3.5 ± 0.8 mm. This increase occurred during the first 138 ± 8 h and then remained constant. One possibility is that the reduced shear forces allowed the pellet structure to loosen. Yet, it was also observed that the core of some pellets became hollow. Although the size increased, the pellets remained structurally together and easily mixed during the fermentation period. Furthermore, as indicated in Figure 4, there was no decrease in fungal concentration. This suggests that as the center of the pellet may have lacked adequate oxygen and died, the phosphorus was consumed by the outermost cells, maintaining the overall pellet concentration.
3.3. Nitrogen Limitation The nitrogen compounds were withheld from the growth medium during the growth of T. reesei. As shown in Figure 5, nitrogen limitation halted growth. However, as indicated in the profile, cellulase production was also stopped during nitrogen limitation. Following nitrogen addition,
the biomass concentration and cellulase activity increased. A delay in cellulase production was also observed.
In a replicate fermentation, 1 g/L Triton X-100 was added to assess the surfactant effects. Fermentation profiles are indicated in Figure 6. The stationary phase was reached after 24 h, indicating nitrogen depletion. The cellulase activity, FPU/mL, during 92 to 215 h remained unchanged during the nitrogen limited stationary phase, constant at 0.34 FPU/mL. Similar results were observed during the low shear airlift style pellet fermentation of T. reesei. The typical profile is indicated in Figure 7. Here nitrogen was added between 48 and 93 h at a rate of 0.99 mg-N/L-h. This yielded 1.2 g/L of T. reesei, for an N-based cell yield (YX/N) of 27.3. There was no statistically significant cellulase production (p-value = 0.75), though a slight increase of 17% could be observed during the nitrogen addition.
4. Discussion During T. reesei Rut C-30 fermentation under phosphorus limitation, it was observed that the specific cellulase productivity was 5 ± 2 FPU/g-h in stir-tank systems. The use of specific productivity has been periodically reported in previous works. However, in many typical batch fermentations the fungal biomass concentration changes with time. Since the cell concentration is not stable, the apparent cellulase production rate changes during the fermentation, as more biomass is produced, lost, or enters a nutrient limitation. This dynamic system makes determining and predicting enzyme production difficult. The use of nutrient limiting conditions allows for a stationary phase biomass concentration in batch fermentation. With a stable fungal culture, a predictable and measurable specific cellulase productivity can be determined. In this
work, the specific cellulase productivity is similar to T. reesei C5 grown in a stir-tank continuous fermentation process on 2% lactose, with productivities in the range of 4.6 FPU/g-h [29]. This comparison represents a critical improvement in cellulase production. Typical continuous cellulase production employs a growing fungal biomass, where a fraction of the cells are removed during continuous substrate addition. In this work, the phosphorus limitation eliminated the continuous biomass growth and maintained similar cellulase production rates. During phosphorus limitation, most of the substrate was utilized for cellulase production and not biomass formation. This was reflected in the increased cellulase activity per unit amount of lactose consumed during these periods of stationary phase.
Yet, in this work, the specific cellulase productivity during phosphorus limitation was half of that in the reference system under similar conditions, 10 ± 2 FPU/g-h. The reference productivity is in the range of other fed-batch fermentations grown on cellulose, 10 to 12 FPU/g-h [30,31]. T. reesei is expected to have a phosphorus reserve, including polyphosphates [32]. Yet, as shown in this work, the phosphorus reserve is unable to sustain prolonged growth during extended periods of deficiency. Phosphates are used in many basic cell functions. Yet, phosphorus is not consumed during the production of cellulase enzymes, as cellulase does not contain elemental phosphorus. There is not a simple material balance explanation as to why lower levels of phosphorus would cause lower cellulase production. The effects are, therefore, secondary. There are three aspects which are critical in this process. Phosphates are used to form the lipid membranes, in the DNA and RNA linkage, and energy intermediates. It is expected that phosphorus deficiency will prevent both membrane formation and DNA replication. These two aspects will prevent cell replication. Phosphates are also extensively used as an energy carrier,
adenosine tri-phosphate (ATP), and regulatory marker, via kinases, during carbohydrate catabolism. The reduction in cellulase productivity suggests the internal phosphate recycling is slowing the metabolic activity through limited phosphorus availability for RNA formation and ATP generation. This subsequently would slow cellulase production though decreased expression rates. Additional metabolite analysis is required to fully understand the shift in phosphorus containing molecules. Furthermore, it has been reported that the cellulase is actively excreted at the growing hyphal tips [33]. For the T. reesei in a phosphorus-limited non-growing state, the reduced hyphal tip growth would potentially decrease the points of enzyme release, possibly providing another cause for the slightly reduced productivity.
The reuse of fungal biomass for continuous cellulase production can be achieved by retaining the cells during enzyme harvest. A simple method to contain the biomass is by auto-pellet formation, as achieved in the concentric airlift fermentor where T. reesei was presented with both a surfactant and lower shear environment. Yet, the use of pellet is not required for successful biomass retention; other schemes may be applied as well. In systems were the biomass is in a prolonged stationary phase, as in Figure 4, carbon source may be added without increasing the biomass concentration. With this improvement, fermentation time can be lengthened to improve cellulase activity without biomass accumulation. This reduces the amount of biomass waste and helps to eliminate mixing and air-transport limitations caused by high biomass concentrations. These effects on the overall production cost has been included in an economic analysis, described in the Supplementary Materials. By using a continuously-fed batch system, a 60% savings in production cost can be achieved. This savings also accounts for the lower specific productivity during the stationary phase. The estimated production cost reductions are by
eliminating the time for repeated biomass growth and decreased substrate utilization. Further savings may be obtained through the use of the pelleted examples presented. However, those assumptions require further study to fully understand the conditions necessary for long-term process stability.
The Triton X-100 promoted pellet formation and was previously shown to increase cellulase release by 70% [14]. This effect may account for the moderately improved cellulase production rate, 7 ± 1 FPU/g-h, when compared to the stir-tank systems, 5 ± 2 FPU/g-h. T. reesei was expected to produce slightly more enzyme under a lower shear environment [34]. However, the effect of surfactant cannot be separated from the pellet formation, as the surfactant was required to maintain the pellet structure for proper mixing. Furthermore, during the fermentation, the low shear environment allowed the pellet structure to expand in size. The expansion is likely due to a combined lack of oxygen in the center, causing death, and the lack of forces to re-pack the filaments [35]. When grown in the shake flasks, the orbital motion causes the pellet to periodically interact with flask wall and create a force along the pellet surface. These forces are absent in the airlift reactor. Nevertheless, the use of a phosphate limited condition allowed for the successful maintenance of the fungal pellet. The extended lifespan of the fungal pellet is a significant improvement, as the fungal mass can be retained in the vessel and reused for stable cellulase production.
While nitrogen limitation was successful in halting growth, the results indicated that T. reesei Rut C-30 would stop cellulase production when nitrogen sources are depleted, Figure 5. Nitrogen, typically in the form of amino acids, is utilized by the cells to produce proteins. Since
cellulase is a mixture of enzymes or functional proteins, it is anticipated that the fungal cells would normally cease enzyme production during periods of nitrogen deficiency. The use of nitrogen limitation is a convenient way to limit cell growth, because structural proteins cannot be formed and cell replication is inhibited. However, the Rut C-30 strain is a mutant that lacks full control over the cellulase expression. It was previously unknown if the cells could selectively stop cellulase production during a nitrogen limitation, while being provided a cellulase inducing substrate such as lactose. These results indicate that nitrogen limitation can induce a stationary phase. However, this stationary phase cannot be used for cellulase production. Cellulase production is stopped when nitrogen is depleted.
It was observed that, following nitrogen addition, the preference of T. reesei Rut C-30 is to form cell mass before the release of cellulase. In this study, the fungal mass was measured as a function of intracellular proteins. This method can over-exaggerate the biomass concentration during periods of intense internal protein formation, as the cell prepares for branching. As shown in Figure 5, a large spike in intracellular protein occurs following the nitrogen source addition at 124 h, yet no cellulase is produced during that cell growth period of 124 to 143 h. This effect is shown again in Figure 7, where the slow continuous addition of nitrogen source resulted in no significant cellulase production (p-value > 0.8). As shown in Figure 5, cellulase production briefly starts as the intracellular proteins are consumed from 150 to 190 h and then stops when the intracellular protein levels begin to reach a new stable value. This behavior indicates that cellulase regulation functions in extreme cases of nutrient limitation. It would otherwise be possible for the cell to consume structural proteins and lyse in an uncontrolled attempt to produce cellulase. These findings also confirm that T. reesei Rut C-30 does not produce
proteases to degrade the extracellular cellulase. Furthermore, since nitrogen addition after 100 h of nitrogen starvation was able to elicit an immediate growth response, it is evident the cells were not damaged during this period. Therefore, it is possible to use nitrogen limitation to successfully halt the growth of T. reesei for extended periods. However, this condition does not allow for simultaneous cellulase production.
In summary, nitrogen limitation successfully inhibited T. reesei Rut C-30 growth. The cell yield from nitrogen was 21 ± 4 g biomass per g nitrogen consumed. However, nitrogen limitation also inhibited cellulase production. On the other hand, stationary phase production of cellulase was achieved with phosphorus-limited conditions. T. reesei Rut C-30 under phosphorus limitation was found to require 1 g phosphorus per 236 ± 11 g biomass. In stir-tank fermentor without pellet formation, the specific cellulase productivity during the phosphorus-limited stationary phase was 5 ± 2 FPU/g-h. In low-shear airlift fermentor and with addition of Triton X-100 to induce formation of pellet morphology, the phosphorus-limited T. reesei Rut C-30 pellets had a cellulase productivity of 9 ± 3 FPU/g-h, similar to the productivity obtained in the reference fermentation made in this study (10 ± 2 FPU/g-h) and those reported in the literature for fedbatch fermentations with cellulose as substrate (10-12 FPU/g-h). With periodical lactose addition, this low-shear fermentation was successfully operated under phosphorus-limited stationary phase for more than 400 h. Cellulase production continued stably throughout the extended fermentation with an average specific productivity of 7 ± 2 FPU/g-h. This extended stationary phase cellulase production demonstrates that long-term process stability is possible with T. reesei Rut C-30. During the stationary phase production, the fungal biomass requires less oxygen for respiration. This feature may allow for improved volumetric productivity by using an
increased biomass concentration up to the oxygen and nutrient transport limitations of a given fermentor setup. Overall substrate utilization cost should also be lower because of the minimized substrate consumption for cell growth.
6. Acknowledgments This work was supported by the U.S. Department of Agriculture under the Biomass Research and Development Initiative (award # 2009-10001-05112). Data collection and sample analysis was greatly assisted by Rebecca Howdyshell and Alexander Dannemiller. Jacob C. Kohl assisted with figure formatting and proofreading.
7. Supplementary Materials Economic analysis details are provided in the supplementary materials.
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Table 1: The standard and phosphorus-limited media for T. reesei Rut C-30 are shown to indicate the removal of potassium phosphate, urea, and peptone. Potassium chloride was added to maintain the potassium content. Total nitrogen was maintained by increasing the ammonium sulfate.
Medium Component Monopotassium phosphate Ammonium sulfate Potassium chloride Urea Proteose peptone Magnesium sulfate heptahydrate Calcium chloride dihydrate Tween 80 Lactose Ferrous sulfate heptahydrate Manganese sulfate monohydrate Zinc sulfate heptahydrate Cobalt(II) chloride hexahydrate
Standard (g/L) 2.0 1.4 0.0 0.3 1.0 0.3 0.4 0.2 10.0 0.005 0.0016 0.0014 0.002
Modified (g/L) 0.0 2.65 1.09 0.0 0.0 0.3 0.4 0.2 10.0 0.005 0.0016 0.0014 0.002
Table 2: The standard medium for T. reesei Rut C-30 and the nitrogen-limited medium are shown to emphasize the removal of ammonium sulfate, urea, and peptones. These nitrogen-containing components were combined and used to spike the system to probe the cell response.
Medium Component Monopotassium phosphate Ammonium sulfate Urea Proteose peptone Magnesium sulfate heptahydrate Calcium chloride dihydrate Tween 80 Lactose Ferrous sulfate heptahydrate Manganese sulfate monohydrate Zinc sulfate heptahydrate Cobalt(II) chloride hexahydrate
Standard (g/L) 2.0 1.4 0.3 1.0 0.3 0.4 0.2 10.0 0.005 0.0016 0.0014 0.002
Modified (g/L) 2.0 0.0 0.0 0.0 0.3 0.4 0.2 10.0 0.005 0.0016 0.0014 0.002
Nitrogen Solution Component
Ammonium sulfate Urea Proteose peptone
(g/L) 1.4 0.3 1.0
Figure Captions: Figure 1: These profiles show the cellulase activity, lactose concentration, and T. reesei concentration in a stirred tank fermentor. Error bars indicate the standard deviations for duplicate cellulase and lactose analysis, and four sets of replicated measurements of biomass concentration. The fermentation ended when the lactose concentration reached zero. The effective cellulase production period was from 0 to 62 h. Figure 2: (A) The growth of T. reesei into phosphorus limitation and the subsequent growth following phosphate addition (vertical arrows) are indicated for 3 stir-tank fermentation sets. The solid horizontal lines represent the average fungal concentrations at stationary phases reached before and after the phosphate addition. (B) The cellulase profiles are shown with time-averaged production rates, indicated as the solid fitted lines. The specific productivity (5 ± 2 FPU/g-h) reported in the text is averaged from the values calculated for the periods that are indicated with lines. Error bars represent standard deviations about the mean in replicate testing (n = 2 in sets 1 & 2, and n = 3 for cellulase and n = 4 for biomass in set 3). Figure 3: Here T. reesei was grown into phosphorus limitation in an airlift fermentor. The average specific cellulase production rate was 9 ± 3 FPU/g-h during stationary phase, indicated by the dashed line. Error bars represent standard deviations of replicated testing (n = 6, 3, 3 respectively for biomass, cellulase, and lactose). Figure 4: These profiles are for an extended airlift fermentation with T. reesei pellets under phosphorus limitation. Cell growth occurred only during the first 48 h. The average stationary-phase fungal concentration was 1.9 ± 0.5 g/L. Vertical arrows indicate lactose addition. The average specific cellulase productivity was 7 ± 2 FPU/g-h during the extended stationary phase. Error bars represent standard deviations for replicated testing (n = 2). Figure 5: The growth of T. reesei into nitrogen limitation in a stir-tank fermentor is shown with the average stationary phase fungal concentrations ± standard deviations (n = 3 replicates) from 0 to 124 h. Nitrogen was added at 124 h, indicated by the vertical arrow. Cellulase production was inhibited during nitrogen limitation, shown by the enzyme activity profiles ± standard deviations (n = 3 replicates) before and after nitrogen addition (dashed lines). During 143-190 h, when the spiked biomass concentration (from intracellular protein measurements) triggered by nitrogen addition was decreasing to the new stationary-phase level, cellulase was produced at 3.1 ± 0.3 FPU/h. Figure 6: The growth of T. reesei into nitrogen limitation in a stir tank fermentor (with Triton X-100) (A) and the resulting cellulase production (B) are shown. Enzyme production occurs only during the minimal initial growth (0-100 h), while consuming carried over nitrogen. Cellulase production is inhibited during nitrogen limitation (114-215 h, p-value = 0.42). Error bars indicate standard deviations of replicates (n = 3). Figure 7: These profiles show the growth of T. reesei into nitrogen limitation for a pelleted airlift fermentation. Vertical arrows indicate the start (50 h) and end (93 h) of the period with nitrogen
addition provided at 0.99 mg-N/L-h. The stationary phase fungal concentration during 120-167 h was 1.5 ± 0.6 g/L. The cellulase production was statistically insignificant (p-value = 0.75) during the entire fermentation, even with minor additions of nitrogen. Error bars represent standard deviations for replicated tests (n = 2).
Figure 1: These profiles show the cellulase activity, lactose concentration, and T. reesei concentration in a stirred tank fermentor. Error bars indicate the standard deviations for duplicate cellulase and lactose analysis, and four sets of replicated measurements of biomass concentration. The fermentation ended when the lactose concentration reached zero. The effective cellulase production period was from 0 to 62 h.
Figure 2: (A) The growth of T. reesei into phosphorus limitation and the subsequent growth following phosphate addition (vertical arrows) are indicated for 3 stir-tank fermentation sets. The solid horizontal lines represent the average fungal concentrations at stationary phases reached before and after the phosphate addition. (B) The cellulase profiles are shown with time-averaged production rates, indicated as the solid fitted lines. The specific productivity (5 ± 2 FPU/g-h) reported in the text is averaged from the values calculated for the periods that are indicated with lines. Error bars represent standard
deviations about the mean in replicate testing (n = 2 in sets 1 & 2, and n = 3 for cellulase and n = 4 for biomass in set 3).
Figure 3: Here T. reesei was grown into phosphorus limitation in an airlift fermentor. The average specific cellulase production rate was 9 ± 3 FPU/g-h during stationary phase, indicated by the dashed line. Error bars represent standard deviations of replicated testing (n = 6, 3, 3 respectively for biomass, cellulase, and lactose).
Figure 4: These profiles are for an extended airlift fermentation with T. reesei pellets under phosphorus limitation. Cell growth occurred only during the first 48 h. The average stationary-phase fungal concentration was 1.9 ± 0.5 g/L. Vertical arrows indicate lactose addition. The average specific cellulase productivity was 7 ± 2 FPU/g-h during the extended stationary phase. Error bars represent standard deviations for replicated testing (n = 2).
Figure 5: The growth of T. reesei into nitrogen limitation in a stir-tank fermentor is shown with the average stationary phase fungal concentrations ± standard deviations (n = 3 replicates) from 0 to 124 h. Nitrogen was added at 124 h, indicated by the vertical arrow. Cellulase production was inhibited during nitrogen limitation, shown by the enzyme activity profiles ± standard deviations (n = 3 replicates) before and after nitrogen addition (dashed lines). During 143-190 h, when the spiked biomass concentration (from intracellular protein measurements) triggered by nitrogen addition was decreasing to the new stationary-phase level, cellulase was produced at 3.1 ± 0.3 FPU/h.
Figure 6: The growth of T. reesei into nitrogen limitation in a stir tank fermentor (with Triton X-100) (A) and the resulting cellulase production (B) are shown. Enzyme production occurs only during the minimal initial growth (0-100 h), while consuming carried over nitrogen. Cellulase production is inhibited during nitrogen limitation (114-215 h, p-value = 0.42). Error bars indicate standard deviations of replicates (n = 3).
Figure 7: These profiles show the growth of T. reesei into nitrogen limitation for a pelleted airlift fermentation. Vertical arrows indicate the start (50 h) and end (93 h) of the period with nitrogen addition provided at 0.99 mg-N/L-h. The stationary phase fungal concentration during 120-167 h was 1.5 ± 0.6 g/L. The cellulase production was statistically insignificant (p-value = 0.75) during the entire fermentation, even with minor additions of nitrogen. Error bars represent standard deviations for replicated tests (n = 2).
Figure Captions: Figure 1: These profiles show the cellulase activity, lactose concentration, and T. reesei concentration in a stirred tank fermentor. Error bars indicate the standard deviations for duplicate cellulase and lactose analysis, and four sets of replicated measurements of biomass concentration. The fermentation ended when the lactose concentration reached zero. The effective cellulase production period was from 0 to 62 h. Figure 2: (A) The growth of T. reesei into phosphorus limitation and the subsequent growth following phosphate addition (vertical arrows) are indicated for 3 stir-tank fermentation sets. The solid horizontal lines represent the average fungal concentrations at stationary phases reached before and after the phosphate addition. (B) The cellulase profiles are shown with time-averaged production rates, indicated as the solid fitted lines. The specific productivity (5 ± 2 FPU/g-h) reported in the text is averaged from the values calculated for the periods that are indicated with lines. Error bars represent standard deviations about the mean in replicate testing (n = 2 in sets 1 & 2, and n = 3 for cellulase and n = 4 for biomass in set 3). Figure 3: Here T. reesei was grown into phosphorus limitation in an airlift fermentor. The average specific cellulase production rate was 9 ± 3 FPU/g-h during stationary phase, indicated by the dashed line. Error bars represent standard deviations of replicated testing (n = 6, 3, 3 respectively for biomass, cellulase, and lactose). Figure 4: These profiles are for an extended airlift fermentation with T. reesei pellets under phosphorus limitation. Cell growth occurred only during the first 48 h. The average stationary-phase fungal concentration was 1.9 ± 0.5 g/L. Vertical arrows indicate lactose addition. The average specific cellulase productivity was 7 ± 2 FPU/g-h during the extended stationary phase. Error bars represent standard deviations for replicated testing (n = 2). Figure 5: The growth of T. reesei into nitrogen limitation in a stir-tank fermentor is shown with the average stationary phase fungal concentrations ± standard deviations (n = 3 replicates) from 0 to 124 h. Nitrogen was added at 124 h, indicated by the vertical arrow. Cellulase production was inhibited during nitrogen limitation, shown by the enzyme activity profiles ± standard deviations (n = 3 replicates) before and after nitrogen addition (dashed lines). During 143-190 h, when the spiked biomass concentration (from intracellular protein measurements) triggered by nitrogen addition was decreasing to the new stationary-phase level, cellulase was produced at 3.1 ± 0.3 FPU/h. Figure 6: The growth of T. reesei into nitrogen limitation in a stir tank fermentor (with Triton X-100) (A) and the resulting cellulase production (B) are shown. Enzyme production occurs only during the minimal initial growth (0-100 h), while consuming carried over nitrogen. Cellulase production is inhibited during nitrogen limitation (114-215 h, p-value = 0.42). Error bars indicate standard deviations of replicates (n = 3). Figure 7: These profiles show the growth of T. reesei into nitrogen limitation for a pelleted airlift fermentation. Vertical arrows indicate the start (50 h) and end (93 h) of the period with nitrogen
addition provided at 0.99 mg-N/L-h. The stationary phase fungal concentration during 120-167 h was 1.5 ± 0.6 g/L. The cellulase production was statistically insignificant (p-value = 0.75) during the entire fermentation, even with minor additions of nitrogen. Error bars represent standard deviations for replicated tests (n = 2).
Figure 1: These profiles show the cellulase activity, lactose concentration, and T. reesei concentration in a stirred tank fermentor. Error bars indicate the standard deviations for duplicate cellulase and lactose analysis, and four sets of replicated measurements of biomass concentration. The fermentation ended when the lactose concentration reached zero. The effective cellulase production period was from 0 to 62 h.
Figure 2: (A) The growth of T. reesei into phosphorus limitation and the subsequent growth following phosphate addition (vertical arrows) are indicated for 3 stir-tank fermentation sets. The solid horizontal lines represent the average fungal concentrations at stationary phases reached before and after the phosphate addition. (B) The cellulase profiles are shown with time-averaged production rates, indicated as the solid fitted lines. The specific productivity (5 ± 2 FPU/g-h) reported in the text is averaged from the values calculated for the periods that are indicated with lines. Error bars represent standard
deviations about the mean in replicate testing (n = 2 in sets 1 & 2, and n = 3 for cellulase and n = 4 for biomass in set 3).
Figure 3: Here T. reesei was grown into phosphorus limitation in an airlift fermentor. The average specific cellulase production rate was 9 ± 3 FPU/g-h during stationary phase, indicated by the dashed line. Error bars represent standard deviations of replicated testing (n = 6, 3, 3 respectively for biomass, cellulase, and lactose).
Figure 4: These profiles are for an extended airlift fermentation with T. reesei pellets under phosphorus limitation. Cell growth occurred only during the first 48 h. The average stationary-phase fungal concentration was 1.9 ± 0.5 g/L. Vertical arrows indicate lactose addition. The average specific cellulase productivity was 7 ± 2 FPU/g-h during the extended stationary phase. Error bars represent standard deviations for replicated testing (n = 2).
Figure 5: The growth of T. reesei into nitrogen limitation in a stir-tank fermentor is shown with the average stationary phase fungal concentrations ± standard deviations (n = 3 replicates) from 0 to 124 h. Nitrogen was added at 124 h, indicated by the vertical arrow. Cellulase production was inhibited during nitrogen limitation, shown by the enzyme activity profiles ± standard deviations (n = 3 replicates) before and after nitrogen addition (dashed lines). During 143-190 h, when the spiked biomass concentration (from intracellular protein measurements) triggered by nitrogen addition was decreasing to the new stationary-phase level, cellulase was produced at 3.1 ± 0.3 FPU/h.
Figure 6: The growth of T. reesei into nitrogen limitation in a stir tank fermentor (with Triton X-100) (A) and the resulting cellulase production (B) are shown. Enzyme production occurs only during the minimal initial growth (0-100 h), while consuming carried over nitrogen. Cellulase production is inhibited during nitrogen limitation (114-215 h, p-value = 0.42). Error bars indicate standard deviations of replicates (n = 3).
Figure 7: These profiles show the growth of T. reesei into nitrogen limitation for a pelleted airlift fermentation. Vertical arrows indicate the start (50 h) and end (93 h) of the period with nitrogen addition provided at 0.99 mg-N/L-h. The stationary phase fungal concentration during 120-167 h was 1.5 ± 0.6 g/L. The cellulase production was statistically insignificant (p-value = 0.75) during the entire fermentation, even with minor additions of nitrogen. Error bars represent standard deviations for replicated tests (n = 2).
S0141-0229(15)30047-8 http://dx.doi.org/doi:10.1016/j.enzmictec.2015.08.012 EMT 8803
To appear in:
Enzyme and Microbial Technology
Received date: Revised date: Accepted date:
13-3-2015 16-8-2015 18-8-2015
Please cite this article as: Callow Nicholas V, Ray Christopher S, Kelbly Matthew A, Ju Lu-Kwang.Nutrient Control for Stationary Phase Cellulase Production in Trichoderma reesei Rut C-30.Enzyme and Microbial Technology http://dx.doi.org/10.1016/j.enzmictec.2015.08.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Nutrient Control for Stationary Phase Cellulase Production in Trichoderma reesei Rut C-30
Nicholas V. Callow, Christopher S. Ray, Matthew A. Kelbly, Lu-Kwang Ju* Department of Chemical and Biomolecular Engineering The University of Akron, Akron OH 44325, USA
*Corresponding author; e-mail address: [email protected] Highlights
Stationary phase Trichoderma reesei Rut C-30 for long-term cellulase production
Phosphorus limited cellulase production for extended fermentation
Airlift pellet fermentation of T. reesei Rut C-30 possible with nutrient limitation
Abstract This work describes the use of nutrient limitations with Trichoderma reesei Rut C-30 to obtain a prolonged stationary phase cellulase production. This period of non-growth may allow for dependable cellulase production, extended fermentation periods, and the possibility to use pellet morphology for easy product separation. Phosphorus limitation was successful in halting growth and had a corresponding specific cellulase production of 5 ± 2 FPU/g-h. Combined with the addition of Triton X-100 for fungal pellet formation and low shear conditions, a stationary phase cellulase production period in excess of 300 h was achieved, with a constant enzyme production
rate of 7 ± 1 FPU/g-h. While nitrogen limitation was also effective as a growth limiter, it, however, also prevented cellulase production.
Keywords Cellulase; Trichoderma reesei; Phosphorus; Nitrogen; Pellet
1. Introduction Cellulase enzymes have been used in textile and paper processing for finishing applications [1,2]. This enzyme group has potential applications in cellulosic biofuel production to hydrolyze biomass into fermentable sugars. However, expansion has been limited by relatively high production costs [1]. Members of the branched fungi genus Trichoderma are commonly employed to produce cellulase.
A variety of species, mutants, feeding, pH, temperature, mixing, nutrient supplements, and process control schemes have been identified to improve both volumetric and specific cellulase production [3–8]. A key advancement was removing the glucose repression of cellulase expression, which allowed for cellulase production on low cost sugars. The Rut C-30 mutant is lacking the cellulase expression dependency on glucose lean conditions [9]. Likewise, T. reesei Rut C-30, MCG-77, and NG-14 strains have all proven to be among the best cellulase producers [7]. The production of cellulase is a complex function of the growth environment. Cellulose, sophorose, and lactose have been shown to be effective cellulase inducing agents for T. reesei [10,11]. In some cases, improved cellulase production requires a complex feeding scheme to maintain low fungal growth and adequate cellulase production [12,13]. A simple control scheme
is desirable to arrest the growth of T. reesei for continuous cellulase production. Stationary culture conditions may allow for mycelial retention, long-term process stability, and potentially higher product yields with little waste of nutrients on fungal biomass production.
To achieve better process stability, recent process control schemes and chemical additives have been used to modify the morphology from filamentous to pellet for simplified industrial processing [14,15]. In submerged fermentation, specific shear ranges and surfactants have both been effective for morphology control. These morphological changes may help reduce the cost of cellulase production though improved separation. The use of pellets aids in extended periods of cellulase production with retained fungal biomass. Furthermore, the constant presence of fungal biomass, coupled with minimal nutrient availability may reduce the potential for contamination by competing organisms. Attempts have been made to create pellet T. reesei. However, these are limited by very specific operating conditions, pellet degradation, or overgrowth out of pellet form that leads to mixing failure [15–18].
It is hypothesized that phosphorus limitation will halt T. reesei Rut C-30 growth without limiting cellulase production. Reduced potassium phosphate levels have been previously shown to improve cellulase production [19]. Further, the application of nitrogen limitation may be effective at halting growth and providing insight into the nutrient allocation for synthesis of cellulose enzymes versus cell mass. Ideally, this non-carbon nutrient limitation can be combined with pellet morphology for continuous cellulase production under stationary phase conditions. Here, the fungal biomass concentration is stable and the cellulase activity is accumulating or continually harvested.
2. Methods 2.1. Strain and Culture Maintenance Trichoderma reesei Rut C-30 (NRRL 3469) was obtained from the United States Department of Agriculture’s Agricultural Research Service’s culture collection. It was maintained on potato dextrose agar plates (Sigma-Aldrich, St. Louis, MO). These plates were incubated at 30 °C for 4 days before storage at 4 °C. The culture was transferred every 4 weeks by streaking a fresh plate with a loop of cells taken from a mature plate. Fermentation inoculum was prepared by transferring 6 loops of cells, from a mature plate, into 100 mL of sterile potato dextrose broth (Sigma-Aldrich, St. Louis, MO), which was contained in a 250 mL glass Erlenmeyer flask covered by cheese-cloth cotton. The culture was incubated at room temperature for 3 days on a magnetic stir plate set to approximately 300 revolutions per minute (rpm) and then used for fermentation inoculation.
2.2. Experimental Setup This study is divided into two fermentation sections: high shear filamentous fermentation and low shear pellet fermentation. Within each section, the applicability of either nitrogen or phosphorus limitation was tested. The high-shear testing utilized filamentous T. reesei and determined how nutrient limitations influenced growth and enzyme production. The low-shear testing focused on Triton X-100 induced pellets for determining long-term process stability.
For all systems, inoculation was provided at 10% (v/v). The pH was allowed to naturally fall to 4.8, beginning at 5.6, and was then maintained by automatic addition of either 1.0 M hydrochloric acid or sodium hydroxide. Trans-278 (Trans-Chemco, Bristol, WI) anti-foam was added, as needed, to control foaming. All setups included periodic sampling to measure nutrient concentrations and T. reesei growth. During the fermentation, a proportion of limiting nutrient was added to observe the growth and cellulase production response. Unless otherwise specified, all medium components were purchased from Sigma-Aldrich (St. Louis, MO). The proteose peptone was from Remel (Thermo Fisher Scientific, Lenexa, KS) and the ammonium sulfate was from Fisher Scientific (Thermo Fisher Scientific).
In the high-shear fermentations, T. reesei Rut C-30 was cultured in a New Brunswick BioFlo 110 fermentor (Eppendorf North America, Enfield, NJ). The working volume was 1 L with an agitation provided by a 6-bladed Rushton turbine at 350 rpm. Air was supplied at 1 liter per minute (LPM) and with demand-controlled supplementation of pure oxygen to maintain a minimum 20% dissolved oxygen concentration, relative to air saturation.
In the pellet fermentations, inoculum was first added to 250 mL shake flasks and cultured for 5 days at 250 rpm in a Que Orbital shaker (Model 4703, Parkersburg, WV). These flasks contained the standard medium (Table 1) and 1.0 g/L of Triton X-100 to grow T. reesei pellets. These pellets were gently centrifuged at 1,046 g (Legend X1R, Thermo Scientific, Osterode, Germany) to isolate them from the growth medium and were then re-suspended in sterile water. The pellets were washed, re-centrifuged, and suspended in the final nutrient deficient medium for either phosphorus or nitrogen study. The pellets were used as the pre-culture for airlift fermentors. This
process reduced unused phosphates or ammonium from being transferred to the primary fermentor. A custom airlift fermentor provided gentle agitation with rising air bubbles. Aeration was supplied at 0.75 LPM, for 1.5 L working volume. In addition to the Trans-278 chemical antifoam, an external rotating cone foam breaker and pump to return the foamate back to the airlift fermentor accomplished foam control. Dissolved oxygen concentration was not monitored for this setup.
For this study, the growth medium contained lactose as the primary carbon source. Lactose has been shown to induce cellulase production and is an appropriate carbon source for growth [20– 22]. The included minerals were previously identified for successful growth and enzyme production [23]; these were used without modification. Suitable nitrogen sources for cellulase production have been described elsewhere [24,25]. T. reesei can consume a variety of nitrogen containing compounds with little change in cellulase production. In this work, urea and proteose peptone were replaced by ammonium sulfate.
T. reesei growth and cellulase production has not been reported for phosphorus limiting conditions. Potassium phosphate is generally included in excess of biological requirements for its pH buffering capacity. For this work, potassium phosphate was removed and potassium ions were added with potassium chloride. In phosphorus limited systems, the phosphates were withheld from the growth medium (Table 1). Urea and proteose peptone was also removed from the medium. Ammonium sulfate was increased to maintain the nitrogen content. The T. reesei was grown into an initial stationary phase. Then, a small amount of phosphate containing salt solution was added to observe the response.
The modified medium composition is indicated in Table 2 for the nitrogen limiting studies. The peptone, urea, and ammonium sulfate has been removed from the growth medium and combined in a new solution. This nitrogen rich solution was added occasionally during the fermentation to probe the response to nitrogen availability.
2.3. Analytical Techniques Samples were withdrawn from the fermentor by a syringe affixed sample tube and isolation valve. All samples were divided into 1.0 mL volumes and placed into polypropylene microcentrifuge tubes. These samples were centrifuged at 9,300 g in an Eppendorf 8150 bench top centrifuge for 10 min. The supernatant was carefully collected from the compacted fungal pellet. Both fractions were frozen at -20 °C until tested.
T. reesei concentrations were indirectly assessed through intracellular protein content. The fungal pellet was disrupted with 0.2 M aqueous sodium hydroxide, at equal volume to the original sample volume collected, and heating for 20 min at 100 °C. The protein release was determined by complexing with Coomassie Brilliant Blue G-250 dye (Bio-Rad Protein Assay Kit #500-002, Bio-Rad Laboratories, Hercules, CA). Bovine serum albumin was used for calibration. A previously established correlation to convert protein content to fungal biomass was used by dividing the intracellular protein concentration by 0.125 [26]. In this work, each sample drawn from the fermentor was analyzed with at least three analytical replicates. Values and error bars plotted in figures are the averages and standard deviations of the replicated tests.
Cellulase activities were determined by the standard test method described by Ghose [27] and reported as Filter Paper Units per unit sample volume (FPU/mL). One FPU/mL corresponds to the release of 1 µmol/mL (or mM) glucose per minute, under standard testing conditions. The test was modified to improve temperature uniformity for larger numbers of test vials by including mixing in a heated water bath. The sample tubes were sealed and incubated partially submerged in water at 50 °C with mild shaking at 60 rpm in an orbital shaker (ORS 200, Bokel Scientific Inc., Fasterville, PA). The lactose concentrations were obtained from the blank enzyme sample values and were expressed as glucose equivalents. For each fermentation sample, the cellulase activity and lactose concentration was re-measured to obtain an average and standard deviation. The primary source of error in this test is in the tedious replicates, where liquid transfer errors are amplified, and in the non-homogenous test comprising a solid substrate and non-linear hydrolysis dependency on enzyme concentration [28].
Specific cellulase productivity is defined as the rate of cellulase production per unit amount of biomass, and carries the unit of FPU per gram-hour. These values were determined by fitting a linear regression equation to the measured cellulase activities (FPU/mL) during stationary phase periods, when there was no cell growth due to a nutrient limitation. The slope parameter, FPU/mL-h, was multiplied by 1000 (to convert the unit to FPU/L-h) and then divided by the average biomass concentration (g/L) for the given period. For systems with multiple stationary phase periods, each period was considered separately. To account for the differences in fermentation variation and errors during testing, replicate fermentations were included, where appropriate, to obtain average and standard deviation values.
3. Results A reference fermentation for T. reesei is shown in Figure 1. This fermentation was stopped when the lactose was depleted and the system reached carbon source limitation. The total duration was 62 h, during which time the cellulase activity increased. By averaging the incremental increase in both fungal biomass and cellulase, the specific rate of cellulase production during this period was determined to be 10 ± 2 FPU per gram of cell per hour. Given 8.3 g/L lactose consumed and 1 g/L proteose peptone, the system reached 3.5 ± 0.5 g/L of fungal mass.
3.1. High Shear with Phosphorus Limitation Phosphorus limited testing was replicated in three stir-tank fermentation sets. T. reesei was grown on the aforementioned potato dextrose broth, separated from this phosphorus containing nutrient solution and placed in a phosphorus free fermentation medium. Initially, the fungus continued growing until a phosphorus limitation was reached, assumed since all other nutrients were in excess throughout the fermentation. The amount of initial growth varied with the completeness of the phosphorus removal prior to inoculation. Initial growth reached cell concentrations that were between 3X and 13X of the inoculated concentrations. This initial growth can be seen in Figure 2 (A). The average fungal mass concentration during the first phosphorus limited period is indicated on the figure as a solid horizontal line. After the fungus reached a stationary phase, a stable biomass concentration for 48 h, a small amount of phosphorus containing solution was added to the fermentor. As indicated in the figure, growth resumed until a new stationary phase was reached. The average fungal mass concentrations for the second stationary period are indicated by the solid lines in Figure 2 (A). This stepwise
control of the fungal mass further indicates that phosphate was the limiting growth factor. The average of increased T. reesei concentration between the first and second stationary phases shown in Figure 2 (A) was used to determine the cell yield from phosphorus, which was 236 ± 11 (g/g). Figure 2 (B) indicates the cellulase profiles for each set. As shown, the cellulase production was not inhibited during periods of stationary phase created by the phosphorus limitation. By curve fitting the cellulase activity profile, indicated by the solid lines, a production rate was obtained. The average fungal concentrations during this period are indicated by the solids lines in subfigure A and were used to obtain the specific cellulase productivity. During the periods of non-growth, the average specific cellulase productivity, across all replicates, was 5 ± 2 FPU/g-h. Likewise, the FPU produced per lactose consumed was 227 ± 94 FPU/g, which was nearly twice that of the non-stationary systems at 124 ± 54 FPU/g (data provided in Supplementary Materials).
3.2. Low Shear with Phosphorus Limitation For low shear nutrient limited systems, pellet morphology was created in a shake flask and inoculated into a concentric airlift fermentor. The concentric airlift system contains a primary fermentor vessel, foam breaker, recirculation pump, pH controls, and sampling ports. Figure 3 shows the results from a fermentation set. The T. reesei reached stationary phase after 50 h with a cell concentration of 3.0 ± 0.3 g/L, indicating utilization of any free phosphorus that had been carried into the vessel with the pellet culture. Cellulase was produced at a constant rate of 27.3 FPU/L-h during the stationary phase until the carbon source was depleted. The specific cellulase production rate was 9 ± 3 FPU/g-h.
To test extended fermentation, periodic lactose addition was included to avoid carbon source limitations. The results are shown in Figure 4. Here lactose was periodically added, as indicated by the vertical arrows. The extended fungal biomass concentration was maintained at 1.9 ± 0.5 g/L. Here, the specific cellulase productivity was 7 ± 2 FPU/g-h, as was in a duplicate system (data not shown). The specific cellulase productivity, averaged across all replicated systems, was 7 ± 1 FPU/g-h. In these systems, the concentration of T. reesei was successfully halted for an extended duration of continuous cellulase production.
In the lower shear environment, the pellet diameter increased in size. The initial pellet size was set by the shake flask culturing conditions, at 0.8 ± 0.2 mm. During the fermentations, the size increased to 3.5 ± 0.8 mm. This increase occurred during the first 138 ± 8 h and then remained constant. One possibility is that the reduced shear forces allowed the pellet structure to loosen. Yet, it was also observed that the core of some pellets became hollow. Although the size increased, the pellets remained structurally together and easily mixed during the fermentation period. Furthermore, as indicated in Figure 4, there was no decrease in fungal concentration. This suggests that as the center of the pellet may have lacked adequate oxygen and died, the phosphorus was consumed by the outermost cells, maintaining the overall pellet concentration.
3.3. Nitrogen Limitation The nitrogen compounds were withheld from the growth medium during the growth of T. reesei. As shown in Figure 5, nitrogen limitation halted growth. However, as indicated in the profile, cellulase production was also stopped during nitrogen limitation. Following nitrogen addition,
the biomass concentration and cellulase activity increased. A delay in cellulase production was also observed.
In a replicate fermentation, 1 g/L Triton X-100 was added to assess the surfactant effects. Fermentation profiles are indicated in Figure 6. The stationary phase was reached after 24 h, indicating nitrogen depletion. The cellulase activity, FPU/mL, during 92 to 215 h remained unchanged during the nitrogen limited stationary phase, constant at 0.34 FPU/mL. Similar results were observed during the low shear airlift style pellet fermentation of T. reesei. The typical profile is indicated in Figure 7. Here nitrogen was added between 48 and 93 h at a rate of 0.99 mg-N/L-h. This yielded 1.2 g/L of T. reesei, for an N-based cell yield (YX/N) of 27.3. There was no statistically significant cellulase production (p-value = 0.75), though a slight increase of 17% could be observed during the nitrogen addition.
4. Discussion During T. reesei Rut C-30 fermentation under phosphorus limitation, it was observed that the specific cellulase productivity was 5 ± 2 FPU/g-h in stir-tank systems. The use of specific productivity has been periodically reported in previous works. However, in many typical batch fermentations the fungal biomass concentration changes with time. Since the cell concentration is not stable, the apparent cellulase production rate changes during the fermentation, as more biomass is produced, lost, or enters a nutrient limitation. This dynamic system makes determining and predicting enzyme production difficult. The use of nutrient limiting conditions allows for a stationary phase biomass concentration in batch fermentation. With a stable fungal culture, a predictable and measurable specific cellulase productivity can be determined. In this
work, the specific cellulase productivity is similar to T. reesei C5 grown in a stir-tank continuous fermentation process on 2% lactose, with productivities in the range of 4.6 FPU/g-h [29]. This comparison represents a critical improvement in cellulase production. Typical continuous cellulase production employs a growing fungal biomass, where a fraction of the cells are removed during continuous substrate addition. In this work, the phosphorus limitation eliminated the continuous biomass growth and maintained similar cellulase production rates. During phosphorus limitation, most of the substrate was utilized for cellulase production and not biomass formation. This was reflected in the increased cellulase activity per unit amount of lactose consumed during these periods of stationary phase.
Yet, in this work, the specific cellulase productivity during phosphorus limitation was half of that in the reference system under similar conditions, 10 ± 2 FPU/g-h. The reference productivity is in the range of other fed-batch fermentations grown on cellulose, 10 to 12 FPU/g-h [30,31]. T. reesei is expected to have a phosphorus reserve, including polyphosphates [32]. Yet, as shown in this work, the phosphorus reserve is unable to sustain prolonged growth during extended periods of deficiency. Phosphates are used in many basic cell functions. Yet, phosphorus is not consumed during the production of cellulase enzymes, as cellulase does not contain elemental phosphorus. There is not a simple material balance explanation as to why lower levels of phosphorus would cause lower cellulase production. The effects are, therefore, secondary. There are three aspects which are critical in this process. Phosphates are used to form the lipid membranes, in the DNA and RNA linkage, and energy intermediates. It is expected that phosphorus deficiency will prevent both membrane formation and DNA replication. These two aspects will prevent cell replication. Phosphates are also extensively used as an energy carrier,
adenosine tri-phosphate (ATP), and regulatory marker, via kinases, during carbohydrate catabolism. The reduction in cellulase productivity suggests the internal phosphate recycling is slowing the metabolic activity through limited phosphorus availability for RNA formation and ATP generation. This subsequently would slow cellulase production though decreased expression rates. Additional metabolite analysis is required to fully understand the shift in phosphorus containing molecules. Furthermore, it has been reported that the cellulase is actively excreted at the growing hyphal tips [33]. For the T. reesei in a phosphorus-limited non-growing state, the reduced hyphal tip growth would potentially decrease the points of enzyme release, possibly providing another cause for the slightly reduced productivity.
The reuse of fungal biomass for continuous cellulase production can be achieved by retaining the cells during enzyme harvest. A simple method to contain the biomass is by auto-pellet formation, as achieved in the concentric airlift fermentor where T. reesei was presented with both a surfactant and lower shear environment. Yet, the use of pellet is not required for successful biomass retention; other schemes may be applied as well. In systems were the biomass is in a prolonged stationary phase, as in Figure 4, carbon source may be added without increasing the biomass concentration. With this improvement, fermentation time can be lengthened to improve cellulase activity without biomass accumulation. This reduces the amount of biomass waste and helps to eliminate mixing and air-transport limitations caused by high biomass concentrations. These effects on the overall production cost has been included in an economic analysis, described in the Supplementary Materials. By using a continuously-fed batch system, a 60% savings in production cost can be achieved. This savings also accounts for the lower specific productivity during the stationary phase. The estimated production cost reductions are by
eliminating the time for repeated biomass growth and decreased substrate utilization. Further savings may be obtained through the use of the pelleted examples presented. However, those assumptions require further study to fully understand the conditions necessary for long-term process stability.
The Triton X-100 promoted pellet formation and was previously shown to increase cellulase release by 70% [14]. This effect may account for the moderately improved cellulase production rate, 7 ± 1 FPU/g-h, when compared to the stir-tank systems, 5 ± 2 FPU/g-h. T. reesei was expected to produce slightly more enzyme under a lower shear environment [34]. However, the effect of surfactant cannot be separated from the pellet formation, as the surfactant was required to maintain the pellet structure for proper mixing. Furthermore, during the fermentation, the low shear environment allowed the pellet structure to expand in size. The expansion is likely due to a combined lack of oxygen in the center, causing death, and the lack of forces to re-pack the filaments [35]. When grown in the shake flasks, the orbital motion causes the pellet to periodically interact with flask wall and create a force along the pellet surface. These forces are absent in the airlift reactor. Nevertheless, the use of a phosphate limited condition allowed for the successful maintenance of the fungal pellet. The extended lifespan of the fungal pellet is a significant improvement, as the fungal mass can be retained in the vessel and reused for stable cellulase production.
While nitrogen limitation was successful in halting growth, the results indicated that T. reesei Rut C-30 would stop cellulase production when nitrogen sources are depleted, Figure 5. Nitrogen, typically in the form of amino acids, is utilized by the cells to produce proteins. Since
cellulase is a mixture of enzymes or functional proteins, it is anticipated that the fungal cells would normally cease enzyme production during periods of nitrogen deficiency. The use of nitrogen limitation is a convenient way to limit cell growth, because structural proteins cannot be formed and cell replication is inhibited. However, the Rut C-30 strain is a mutant that lacks full control over the cellulase expression. It was previously unknown if the cells could selectively stop cellulase production during a nitrogen limitation, while being provided a cellulase inducing substrate such as lactose. These results indicate that nitrogen limitation can induce a stationary phase. However, this stationary phase cannot be used for cellulase production. Cellulase production is stopped when nitrogen is depleted.
It was observed that, following nitrogen addition, the preference of T. reesei Rut C-30 is to form cell mass before the release of cellulase. In this study, the fungal mass was measured as a function of intracellular proteins. This method can over-exaggerate the biomass concentration during periods of intense internal protein formation, as the cell prepares for branching. As shown in Figure 5, a large spike in intracellular protein occurs following the nitrogen source addition at 124 h, yet no cellulase is produced during that cell growth period of 124 to 143 h. This effect is shown again in Figure 7, where the slow continuous addition of nitrogen source resulted in no significant cellulase production (p-value > 0.8). As shown in Figure 5, cellulase production briefly starts as the intracellular proteins are consumed from 150 to 190 h and then stops when the intracellular protein levels begin to reach a new stable value. This behavior indicates that cellulase regulation functions in extreme cases of nutrient limitation. It would otherwise be possible for the cell to consume structural proteins and lyse in an uncontrolled attempt to produce cellulase. These findings also confirm that T. reesei Rut C-30 does not produce
proteases to degrade the extracellular cellulase. Furthermore, since nitrogen addition after 100 h of nitrogen starvation was able to elicit an immediate growth response, it is evident the cells were not damaged during this period. Therefore, it is possible to use nitrogen limitation to successfully halt the growth of T. reesei for extended periods. However, this condition does not allow for simultaneous cellulase production.
In summary, nitrogen limitation successfully inhibited T. reesei Rut C-30 growth. The cell yield from nitrogen was 21 ± 4 g biomass per g nitrogen consumed. However, nitrogen limitation also inhibited cellulase production. On the other hand, stationary phase production of cellulase was achieved with phosphorus-limited conditions. T. reesei Rut C-30 under phosphorus limitation was found to require 1 g phosphorus per 236 ± 11 g biomass. In stir-tank fermentor without pellet formation, the specific cellulase productivity during the phosphorus-limited stationary phase was 5 ± 2 FPU/g-h. In low-shear airlift fermentor and with addition of Triton X-100 to induce formation of pellet morphology, the phosphorus-limited T. reesei Rut C-30 pellets had a cellulase productivity of 9 ± 3 FPU/g-h, similar to the productivity obtained in the reference fermentation made in this study (10 ± 2 FPU/g-h) and those reported in the literature for fedbatch fermentations with cellulose as substrate (10-12 FPU/g-h). With periodical lactose addition, this low-shear fermentation was successfully operated under phosphorus-limited stationary phase for more than 400 h. Cellulase production continued stably throughout the extended fermentation with an average specific productivity of 7 ± 2 FPU/g-h. This extended stationary phase cellulase production demonstrates that long-term process stability is possible with T. reesei Rut C-30. During the stationary phase production, the fungal biomass requires less oxygen for respiration. This feature may allow for improved volumetric productivity by using an
increased biomass concentration up to the oxygen and nutrient transport limitations of a given fermentor setup. Overall substrate utilization cost should also be lower because of the minimized substrate consumption for cell growth.
6. Acknowledgments This work was supported by the U.S. Department of Agriculture under the Biomass Research and Development Initiative (award # 2009-10001-05112). Data collection and sample analysis was greatly assisted by Rebecca Howdyshell and Alexander Dannemiller. Jacob C. Kohl assisted with figure formatting and proofreading.
7. Supplementary Materials Economic analysis details are provided in the supplementary materials.
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Table 1: The standard and phosphorus-limited media for T. reesei Rut C-30 are shown to indicate the removal of potassium phosphate, urea, and peptone. Potassium chloride was added to maintain the potassium content. Total nitrogen was maintained by increasing the ammonium sulfate.
Medium Component Monopotassium phosphate Ammonium sulfate Potassium chloride Urea Proteose peptone Magnesium sulfate heptahydrate Calcium chloride dihydrate Tween 80 Lactose Ferrous sulfate heptahydrate Manganese sulfate monohydrate Zinc sulfate heptahydrate Cobalt(II) chloride hexahydrate
Standard (g/L) 2.0 1.4 0.0 0.3 1.0 0.3 0.4 0.2 10.0 0.005 0.0016 0.0014 0.002
Modified (g/L) 0.0 2.65 1.09 0.0 0.0 0.3 0.4 0.2 10.0 0.005 0.0016 0.0014 0.002
Table 2: The standard medium for T. reesei Rut C-30 and the nitrogen-limited medium are shown to emphasize the removal of ammonium sulfate, urea, and peptones. These nitrogen-containing components were combined and used to spike the system to probe the cell response.
Medium Component Monopotassium phosphate Ammonium sulfate Urea Proteose peptone Magnesium sulfate heptahydrate Calcium chloride dihydrate Tween 80 Lactose Ferrous sulfate heptahydrate Manganese sulfate monohydrate Zinc sulfate heptahydrate Cobalt(II) chloride hexahydrate
Standard (g/L) 2.0 1.4 0.3 1.0 0.3 0.4 0.2 10.0 0.005 0.0016 0.0014 0.002
Modified (g/L) 2.0 0.0 0.0 0.0 0.3 0.4 0.2 10.0 0.005 0.0016 0.0014 0.002
Nitrogen Solution Component
Ammonium sulfate Urea Proteose peptone
(g/L) 1.4 0.3 1.0
Figure Captions: Figure 1: These profiles show the cellulase activity, lactose concentration, and T. reesei concentration in a stirred tank fermentor. Error bars indicate the standard deviations for duplicate cellulase and lactose analysis, and four sets of replicated measurements of biomass concentration. The fermentation ended when the lactose concentration reached zero. The effective cellulase production period was from 0 to 62 h. Figure 2: (A) The growth of T. reesei into phosphorus limitation and the subsequent growth following phosphate addition (vertical arrows) are indicated for 3 stir-tank fermentation sets. The solid horizontal lines represent the average fungal concentrations at stationary phases reached before and after the phosphate addition. (B) The cellulase profiles are shown with time-averaged production rates, indicated as the solid fitted lines. The specific productivity (5 ± 2 FPU/g-h) reported in the text is averaged from the values calculated for the periods that are indicated with lines. Error bars represent standard deviations about the mean in replicate testing (n = 2 in sets 1 & 2, and n = 3 for cellulase and n = 4 for biomass in set 3). Figure 3: Here T. reesei was grown into phosphorus limitation in an airlift fermentor. The average specific cellulase production rate was 9 ± 3 FPU/g-h during stationary phase, indicated by the dashed line. Error bars represent standard deviations of replicated testing (n = 6, 3, 3 respectively for biomass, cellulase, and lactose). Figure 4: These profiles are for an extended airlift fermentation with T. reesei pellets under phosphorus limitation. Cell growth occurred only during the first 48 h. The average stationary-phase fungal concentration was 1.9 ± 0.5 g/L. Vertical arrows indicate lactose addition. The average specific cellulase productivity was 7 ± 2 FPU/g-h during the extended stationary phase. Error bars represent standard deviations for replicated testing (n = 2). Figure 5: The growth of T. reesei into nitrogen limitation in a stir-tank fermentor is shown with the average stationary phase fungal concentrations ± standard deviations (n = 3 replicates) from 0 to 124 h. Nitrogen was added at 124 h, indicated by the vertical arrow. Cellulase production was inhibited during nitrogen limitation, shown by the enzyme activity profiles ± standard deviations (n = 3 replicates) before and after nitrogen addition (dashed lines). During 143-190 h, when the spiked biomass concentration (from intracellular protein measurements) triggered by nitrogen addition was decreasing to the new stationary-phase level, cellulase was produced at 3.1 ± 0.3 FPU/h. Figure 6: The growth of T. reesei into nitrogen limitation in a stir tank fermentor (with Triton X-100) (A) and the resulting cellulase production (B) are shown. Enzyme production occurs only during the minimal initial growth (0-100 h), while consuming carried over nitrogen. Cellulase production is inhibited during nitrogen limitation (114-215 h, p-value = 0.42). Error bars indicate standard deviations of replicates (n = 3). Figure 7: These profiles show the growth of T. reesei into nitrogen limitation for a pelleted airlift fermentation. Vertical arrows indicate the start (50 h) and end (93 h) of the period with nitrogen
addition provided at 0.99 mg-N/L-h. The stationary phase fungal concentration during 120-167 h was 1.5 ± 0.6 g/L. The cellulase production was statistically insignificant (p-value = 0.75) during the entire fermentation, even with minor additions of nitrogen. Error bars represent standard deviations for replicated tests (n = 2).
Figure 1: These profiles show the cellulase activity, lactose concentration, and T. reesei concentration in a stirred tank fermentor. Error bars indicate the standard deviations for duplicate cellulase and lactose analysis, and four sets of replicated measurements of biomass concentration. The fermentation ended when the lactose concentration reached zero. The effective cellulase production period was from 0 to 62 h.
Figure 2: (A) The growth of T. reesei into phosphorus limitation and the subsequent growth following phosphate addition (vertical arrows) are indicated for 3 stir-tank fermentation sets. The solid horizontal lines represent the average fungal concentrations at stationary phases reached before and after the phosphate addition. (B) The cellulase profiles are shown with time-averaged production rates, indicated as the solid fitted lines. The specific productivity (5 ± 2 FPU/g-h) reported in the text is averaged from the values calculated for the periods that are indicated with lines. Error bars represent standard
deviations about the mean in replicate testing (n = 2 in sets 1 & 2, and n = 3 for cellulase and n = 4 for biomass in set 3).
Figure 3: Here T. reesei was grown into phosphorus limitation in an airlift fermentor. The average specific cellulase production rate was 9 ± 3 FPU/g-h during stationary phase, indicated by the dashed line. Error bars represent standard deviations of replicated testing (n = 6, 3, 3 respectively for biomass, cellulase, and lactose).
Figure 4: These profiles are for an extended airlift fermentation with T. reesei pellets under phosphorus limitation. Cell growth occurred only during the first 48 h. The average stationary-phase fungal concentration was 1.9 ± 0.5 g/L. Vertical arrows indicate lactose addition. The average specific cellulase productivity was 7 ± 2 FPU/g-h during the extended stationary phase. Error bars represent standard deviations for replicated testing (n = 2).
Figure 5: The growth of T. reesei into nitrogen limitation in a stir-tank fermentor is shown with the average stationary phase fungal concentrations ± standard deviations (n = 3 replicates) from 0 to 124 h. Nitrogen was added at 124 h, indicated by the vertical arrow. Cellulase production was inhibited during nitrogen limitation, shown by the enzyme activity profiles ± standard deviations (n = 3 replicates) before and after nitrogen addition (dashed lines). During 143-190 h, when the spiked biomass concentration (from intracellular protein measurements) triggered by nitrogen addition was decreasing to the new stationary-phase level, cellulase was produced at 3.1 ± 0.3 FPU/h.
Figure 6: The growth of T. reesei into nitrogen limitation in a stir tank fermentor (with Triton X-100) (A) and the resulting cellulase production (B) are shown. Enzyme production occurs only during the minimal initial growth (0-100 h), while consuming carried over nitrogen. Cellulase production is inhibited during nitrogen limitation (114-215 h, p-value = 0.42). Error bars indicate standard deviations of replicates (n = 3).
Figure 7: These profiles show the growth of T. reesei into nitrogen limitation for a pelleted airlift fermentation. Vertical arrows indicate the start (50 h) and end (93 h) of the period with nitrogen addition provided at 0.99 mg-N/L-h. The stationary phase fungal concentration during 120-167 h was 1.5 ± 0.6 g/L. The cellulase production was statistically insignificant (p-value = 0.75) during the entire fermentation, even with minor additions of nitrogen. Error bars represent standard deviations for replicated tests (n = 2).
Figure Captions: Figure 1: These profiles show the cellulase activity, lactose concentration, and T. reesei concentration in a stirred tank fermentor. Error bars indicate the standard deviations for duplicate cellulase and lactose analysis, and four sets of replicated measurements of biomass concentration. The fermentation ended when the lactose concentration reached zero. The effective cellulase production period was from 0 to 62 h. Figure 2: (A) The growth of T. reesei into phosphorus limitation and the subsequent growth following phosphate addition (vertical arrows) are indicated for 3 stir-tank fermentation sets. The solid horizontal lines represent the average fungal concentrations at stationary phases reached before and after the phosphate addition. (B) The cellulase profiles are shown with time-averaged production rates, indicated as the solid fitted lines. The specific productivity (5 ± 2 FPU/g-h) reported in the text is averaged from the values calculated for the periods that are indicated with lines. Error bars represent standard deviations about the mean in replicate testing (n = 2 in sets 1 & 2, and n = 3 for cellulase and n = 4 for biomass in set 3). Figure 3: Here T. reesei was grown into phosphorus limitation in an airlift fermentor. The average specific cellulase production rate was 9 ± 3 FPU/g-h during stationary phase, indicated by the dashed line. Error bars represent standard deviations of replicated testing (n = 6, 3, 3 respectively for biomass, cellulase, and lactose). Figure 4: These profiles are for an extended airlift fermentation with T. reesei pellets under phosphorus limitation. Cell growth occurred only during the first 48 h. The average stationary-phase fungal concentration was 1.9 ± 0.5 g/L. Vertical arrows indicate lactose addition. The average specific cellulase productivity was 7 ± 2 FPU/g-h during the extended stationary phase. Error bars represent standard deviations for replicated testing (n = 2). Figure 5: The growth of T. reesei into nitrogen limitation in a stir-tank fermentor is shown with the average stationary phase fungal concentrations ± standard deviations (n = 3 replicates) from 0 to 124 h. Nitrogen was added at 124 h, indicated by the vertical arrow. Cellulase production was inhibited during nitrogen limitation, shown by the enzyme activity profiles ± standard deviations (n = 3 replicates) before and after nitrogen addition (dashed lines). During 143-190 h, when the spiked biomass concentration (from intracellular protein measurements) triggered by nitrogen addition was decreasing to the new stationary-phase level, cellulase was produced at 3.1 ± 0.3 FPU/h. Figure 6: The growth of T. reesei into nitrogen limitation in a stir tank fermentor (with Triton X-100) (A) and the resulting cellulase production (B) are shown. Enzyme production occurs only during the minimal initial growth (0-100 h), while consuming carried over nitrogen. Cellulase production is inhibited during nitrogen limitation (114-215 h, p-value = 0.42). Error bars indicate standard deviations of replicates (n = 3). Figure 7: These profiles show the growth of T. reesei into nitrogen limitation for a pelleted airlift fermentation. Vertical arrows indicate the start (50 h) and end (93 h) of the period with nitrogen
addition provided at 0.99 mg-N/L-h. The stationary phase fungal concentration during 120-167 h was 1.5 ± 0.6 g/L. The cellulase production was statistically insignificant (p-value = 0.75) during the entire fermentation, even with minor additions of nitrogen. Error bars represent standard deviations for replicated tests (n = 2).
Figure 1: These profiles show the cellulase activity, lactose concentration, and T. reesei concentration in a stirred tank fermentor. Error bars indicate the standard deviations for duplicate cellulase and lactose analysis, and four sets of replicated measurements of biomass concentration. The fermentation ended when the lactose concentration reached zero. The effective cellulase production period was from 0 to 62 h.
Figure 2: (A) The growth of T. reesei into phosphorus limitation and the subsequent growth following phosphate addition (vertical arrows) are indicated for 3 stir-tank fermentation sets. The solid horizontal lines represent the average fungal concentrations at stationary phases reached before and after the phosphate addition. (B) The cellulase profiles are shown with time-averaged production rates, indicated as the solid fitted lines. The specific productivity (5 ± 2 FPU/g-h) reported in the text is averaged from the values calculated for the periods that are indicated with lines. Error bars represent standard
deviations about the mean in replicate testing (n = 2 in sets 1 & 2, and n = 3 for cellulase and n = 4 for biomass in set 3).
Figure 3: Here T. reesei was grown into phosphorus limitation in an airlift fermentor. The average specific cellulase production rate was 9 ± 3 FPU/g-h during stationary phase, indicated by the dashed line. Error bars represent standard deviations of replicated testing (n = 6, 3, 3 respectively for biomass, cellulase, and lactose).
Figure 4: These profiles are for an extended airlift fermentation with T. reesei pellets under phosphorus limitation. Cell growth occurred only during the first 48 h. The average stationary-phase fungal concentration was 1.9 ± 0.5 g/L. Vertical arrows indicate lactose addition. The average specific cellulase productivity was 7 ± 2 FPU/g-h during the extended stationary phase. Error bars represent standard deviations for replicated testing (n = 2).
Figure 5: The growth of T. reesei into nitrogen limitation in a stir-tank fermentor is shown with the average stationary phase fungal concentrations ± standard deviations (n = 3 replicates) from 0 to 124 h. Nitrogen was added at 124 h, indicated by the vertical arrow. Cellulase production was inhibited during nitrogen limitation, shown by the enzyme activity profiles ± standard deviations (n = 3 replicates) before and after nitrogen addition (dashed lines). During 143-190 h, when the spiked biomass concentration (from intracellular protein measurements) triggered by nitrogen addition was decreasing to the new stationary-phase level, cellulase was produced at 3.1 ± 0.3 FPU/h.
Figure 6: The growth of T. reesei into nitrogen limitation in a stir tank fermentor (with Triton X-100) (A) and the resulting cellulase production (B) are shown. Enzyme production occurs only during the minimal initial growth (0-100 h), while consuming carried over nitrogen. Cellulase production is inhibited during nitrogen limitation (114-215 h, p-value = 0.42). Error bars indicate standard deviations of replicates (n = 3).
Figure 7: These profiles show the growth of T. reesei into nitrogen limitation for a pelleted airlift fermentation. Vertical arrows indicate the start (50 h) and end (93 h) of the period with nitrogen addition provided at 0.99 mg-N/L-h. The stationary phase fungal concentration during 120-167 h was 1.5 ± 0.6 g/L. The cellulase production was statistically insignificant (p-value = 0.75) during the entire fermentation, even with minor additions of nitrogen. Error bars represent standard deviations for replicated tests (n = 2).