Optimization of Phytase Production from Escherichia coli by Altering Solid-State Fermentation Conditions

Abstract

Cultivation of Escherichia coli on wheat-bran substrate under various Solid-State Fermentation (SSF) conditions was evaluated for phytase yield along with the enzyme activity profile as a potential, low-cost alternative to submerged-liquid fermentation. The maximum phytase activity achieved by E. coli was 350 ± 50 SPU of phytase activity per gram of bran, incubated for 96 h with a substrate bed moisture content of 70% (w/v) at 37 °C with a relative air humidity of 90%, and supplemented with 10% (w/w bran) Luria-Bertani broth powder which translates into a 300% increase in phytase activity compared with an un-supplemented culture. The greatest improvements in phytase yield were associated with nutrient supplementation and the optimization of initial substrate moisture content. E. coli production of phytase utilizing solid-state fermentation technology was shown to be feasible utilizing the low-cost agro-residue wheat bran as substrate. Furthermore, the effect of pH and temperature on phytase activity was monitored from pH 2.5 to pH 7.5, and for temperatures ranging from 20 °C to 70 °C. Optimal phytase activity was at pH 5.5 and 50 °C when produced under the SSF optimized conditions.

Full text

Fermentation 2015, 1, 13-23; doi:10.3390/fermentation1010013
fermentation
ISSN 2311-5637
www.mdpi.com/journal/fermentation
Article
Optimization of Phytase Production from Escherichia coli by
Altering Solid-State Fermentation Conditions
Kyle McKinney 1,2, Justin Combs 2, Patrick Becker 2, Andrea Humphries 1, Keith Filer 2 and
Frank Vriesekoop 1,*
1 Department of Food Science, Harper Adams University, Newport, Shropshire TF10 8NB, UK;
E-Mails: kmckinney@alltech.com (K.M.); ahumphries@harper-adams.ac.uk (A.H.)
2 Alltech Biotechnology, Nicholasville, KY 40536, USA; E-Mails: jcombs@alltech.com (J.C.);
pbecker@alltech.com (P.B.); kfiler@alltech.com (K.F.)
* Author to whom correspondence should be addressed; E-Mail: fvriesekoop@harper-adams.ac.uk;
Tel.: +44-019-5281-0280.
Academic Editor: Hiroshi Kitagaki
Received: 22 May 2015 / Accepted: 24 July 2015 / Published: 30 July 2015
Abstract: Cultivation of Escherichia coli on wheat-bran substrate under various Solid-State
Fermentation (SSF) conditions was evaluated for phytase yield along with the enzyme
activity profile as a potential, low-cost alternative to submerged-liquid fermentation.
The maximum phytase activity achieved by E. coli was 350 ± 50 SPU of phytase activity
per gram of bran, incubated for 96 h with a substrate bed moisture content of 70% (w/v) at
37 °C with a relative air humidity of 90%, and supplemented with 10% (w/w bran)
Luria-Bertani broth powder which translates into a 300% increase in phytase activity
compared with an un-supplemented culture. The greatest improvements in phytase yield
were associated with nutrient supplementation and the optimization of initial substrate
moisture content. E. coli production of phytase utilizing solid-state fermentation technology
was shown to be feasible utilizing the low-cost agro-residue wheat bran as substrate.
Furthermore, the effect of pH and temperature on phytase activity was monitored from
pH 2.5 to pH 7.5, and for temperatures ranging from 20 °C to 70 °C. Optimal phytase activity
was at pH 5.5 and 50 °C when produced under the SSF optimized conditions.
Keywords: E. coli; enzyme; optimization; phytase; solid state fermentation
OPEN ACCESS

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1. Introduction
Monogastric livestock lack the enzyme phytase needed to digest phytate, the predominant form of
the essential nutrient phosphorus (P) in grains. To make more efficient use of the phosphorus in feed,
diets are routinely supplemented with exogenous phytase [1]. Phytase supplements used in animal
nutrition are typically of microbial origin, with commercial enzyme production by means of solid-state
fermentation (SSF) or submerged liquid fermentation (SLF). Solid-state fermentation is both
economically and environmentally advantageous in that SSF cultivation can be carried out in simpler
and therefore more cost-effective bioreactors; the enzymes produced typically can be used directly in
their crude form without need for purification or concentration steps [2], which negates the need for
capital and energy input; there is a significant reduction in effluent disposal and/or treatment cost,
because there is no need to remove vast amounts of water from the product steam [2], and low-cost,
nutrient-rich agro-residues can be recycled as substrates for enzyme cultivation [3]. Currently,
the majority of commercial SSF phytase is produced by growing the fungus Aspergillus niger on wheat
bran, which provides both a surface area for microbial attachment and carbon and nitrogen nutrients
from xylan and protein [4].
However, bacterial phytases offer some distinct advantages in terms of their stability and resistance
to proteolysis over phytases synthesized by fungi [5]. Traditionally, because of moisture requirements,
the commercial production of bacterial enzymes has been achieved by SLF, which utilizes free-flowing
substrates (e.g., molasses, broth). SSF technology offers many technical and economic advantages over
SLF, which is why the commercial potential of bacterial phytase production using SSF technology has
been of increased interest. Indeed, research has shown that SSF production of phytase by Bacillus spp.
is economically feasible when process conditions are optimized to enhance enzyme yields utilizing
low-cost substrates [6]. In contrast, whilst studies confirm that Escherichia coli can express phytase that
is stable under high temperatures and resistant to proteolysis [7], very little information has been
published that details E. coli phytase production under SSF conditions. To address this knowledge gap
we evaluated the effects of solid-state fermentation process conditions on phytase yield from E. coli
cultivated on a wheat bran substrate.
2. Experimental Section
2.1. E. coli Inoculum
Luria-Bertani (LB) broth containing 25 g dehydrated LB (Difco, Sparks, MD, USA) per liter was
autoclaved at 121 °C for 15 min, after which a 1-mL aliquot of E. coli (pAPPA1 plasmid in E. coli
(ATCC 87441)) stock culture (stored at −80 °C) was added. The prepared culture was transferred to a
shaking incubator (37 °C, 200 rpm) and typically grown for 8 h until it attained approximately
3.15 × 107 CFU mL−1.
2.2. Solid-State Fermentation
After completion of the liquid cycle, the culture was transferred to a wheat bran substrate to initiate
the solid-state fermentation. Five grams of soft, coarse wheat bran (Siemer Milling, Hopkinsville, KY, USA)

Fermentation 2015, 1 15
was sterilized in an autoclave at 121 °C, 15 PSI for 20 min in a 125 mL wide-necked Erlenmeyer flask
covered with a Bio-Shield wrap (Figure 1). The depth of the bran-bed was 1.3 cm inside the flask, which
corresponded to a surface area to volume ratio of 0.76, allowing for sufficient air exchange during
growth. The pre-grown liquid culture was mixed with sufficient sterile deionized water to achieve an
inoculum of approximately 1.13 × 107 CFU g−1 at 60% (w/v) SSF bed moisture content. The inoculated
flasks were placed into a Forma Scientific Incubator (Model 3033, Marietta, OH, USA) at 37 °C and
90% humidity. The cultures were incubated without agitation. Phytase activity was measured after SSF
completion at predetermined times in response to varied process conditions: nutrient additives, substrate
moisture level, inoculation rate, and incubation period. Enzyme activity of the phytase was evaluated by
creating a temperature and pH profile. All experiments were completed in triplicate.
Figure 1. Erlenmeyer flask containing 5 grams of sterilized inoculated wheat bran.
2.3. Effect of Nutrient Additives on Phytase Production
The following nitrogen-rich nutrient additives were individually tested: yeast extract, LB powder,
and tryptone. (Both yeast extract and tryptone are components of LB powder.) Nutrients were added at
concentrations of 10, 50, 100, and 250 mg·g−1 bran. Phytase production was measured for each nutrient
concentration utilizing 1.13 × 107 CFU E. coli g−1 of wheat for 96 h at 37 °C.
2.4. Effect of Substrate Moisture on Phytase Production
The influence of moisture on enzyme production was evaluated by varying the amount of water
applied to bran in addition to the standard inoculum. Moisture levels of 40, 50, 60, 70, and 80% (w/v)
were established, as determined by a Mettler Halogen Moisture Analyzer (Model HR83, Columbus, OH,
USA). Water activity was determined using a water activity meter (Model CX-2, AquaLab, Pullman,

Fermentation 2015, 1 16
Washington, USA). Moisture levels were maintained by keeping the humidity inside the incubator at
90%. Phytase production was measured for each moisture level utilizing 1.13 × 107 CFU E. coli g−1 of
wheat bran for 96 h at 37 °C.
2.5. Effect of Inoculation Rate on Phytase Production
Culture flasks containing 5 g of sterile bran were inoculated with an 8 h bacterial culture. The overall
moisture level of the substrate was maintained at 60%, while five inoculum rates were established:
4.54 × 107, 2.27 × 107, 1.13 × 107, 5.4 × 106 and 9.07 × 105 CFU g−1 bran, representing culture to water
ratios of 1:1, 1:2, 1:4, 1:10 and 1:50, respectively. Phytase activity in response to each inoculum rate
was measured after 96 h of incubation at 37 °C.
2.6. Effect of Incubation Period on Phytase Production
Flasks were prepared containing E. coli at approximately 1.13 × 107 CFU g−1 of bran. Substrate
moisture was maintained at 60% (w/v) at 37 °C. Phytase production was measured after 24, 48, 72, 96,
120, 144 and 168 h of incubation.
2.7. Effect of Temperature and pH on Phytase Activity
Two-gram samples of dried SSF substrate were assayed for phytase activity at 20, 30, 40, 50, 60 and
70 °C at each of six pH levels: 2.5, 3.5, 4.5, 5.5, 6.5, and 7.5. The SSF substrate used had been grown
under the following conditions: 60% (w/v) moisture, 1.13 × 107 CFU E. coli g−1 of bran, 10% LB broth
powder, for 96 h at 37 °C.
2.8. Phytase Activity Assay
Incubation was stopped by adding an ammonium molybdate/acetone reagent, which produces a
colored complex. Phytase production was determined by assaying phytase activity based on the amount
of ortho-phosphate released by enzymatic hydrolysis of sodium phytate under controlled conditions
detailed in Engelen et al. (1994). The color absorbance of the ortho-phosphate was measured at 380 nm.
One solid-state fermentation phytase unit (SPU) is defined as the amount of enzyme required to liberate
1 mol of inorganic phosphate per minute at pH 5.5 and 50 °C. A control blank containing stop solution
was run simultaneously against test solutions. All other reagents were added and read at 380 nm against
a water blank. The blank absorbance was subtracted from the sample absorbance and the standard curve.
All measurements were performed in triplicate and the respective means reported.
2.9. Statistical Analyses
One-way analysis of variance (ANOVA) was performed to compare the differences between means;
regression analyses were performed to identify effects of independent variables on enzyme production.
Significance was declared at p < 0.05. All analyses were performed utilizing Minitab software
(State College, PA, USA).

Fermentation 2015, 1 17
3. Results and Discussion
3.1. Effect of Additives on Phytase Production
Because the effect of nitrogen supplementation varies between nitrogen sources and organism
species, testing to identify optimal rates is useful [8]. Wheat bran substrate typically offers an abundant
source of carbon to support microbial growth; however, supplementation of other growth-essential
nutrients such as nitrogen can further enhance growth [9]. In this work, wheat bran was supplemented
with a variety of nitrogen sources to determine whether phytase activity could be enhanced. A three-fold
increase up to ~300 SPU/g was observed when adding LB broth at 10% compared with the un-supplemented
control (Figure 2). Additive levels in excess of 10% were associated with decreased phytase activity,
with an addition level of 25% causing a reduction of phytase activity below that of un-supplemented
bran. Overabundance of nitrogen has been shown to reduce the production of hydrolytic enzymes due
to excess cell biomass [10]. When the components of LB broth (i.e., yeast extract, tryptone) were added
individually, phytase production increased compared with the control, but was numerically less
(p > 0.05) than that achieved with LB broth. When evaluating nutrient addition to any commercial scale
fermentation, cost is an important factor to consider in relation to the added benefit of enzyme
production. While LB is more expensive compared to any other nitrogen-based growth medium
ingredient; LB was included in this study because it: (a) represents a readily recognized, and
commercially available form of the two other nitrogen-rich media components used in this study; (b) LB
is one of the most commonly used media ingredient for culturing E. coli under experimental conditions;
and (c) on a large commercial scale, the ingredients that make up LB are readily available for a far more
sensible price that laboratory qualities of the branded products. Our estimates are that the increase
enzyme yield outstrip the increase in nutrient costs.
Figure 2. Effect of nitrogen-rich nutrient supplementation on phytase activity by E. coli
during solid-state fermentation (SSF). ● Yeast extract; ○ Tryptone; and ▼ LB Broth powder
were added at the concentration indicated. The dotted line represents phytase activity using
unsupplemented wheat bran (control). Data shown are the averages and standard deviation
(error bars) of three independent samples.
Nutrient Addition (% dw)
0 5 10 15 20 25 30
Phytase Activity (SPU/g)
0
50
100
150
200
250
300
350

Fermentation 2015, 1 18
3.2. Effect of Moisture Level and Water Activity on Phytase Production
Moisture and water activity have been shown to be critical physiological parameters for enzyme
production with relatively small reductions to water values having a marked negative influence on
production [11]. Typical levels of substrate moisture levels for SSF enzyme production using fungi range
from 20% to 70% (w/v). In comparison, bacterial growth typically requires moisture levels of
approximately 70% [12]. The SSF bed moisture levels herein ranged from 40% to 80% (w/v). The
poorest phytase activity (i.e., 73 SPU/g phytase) was obtained at the 40% moisture level, whereas the
maximum phytase yield of 362 SPU/g phytase was achieved at the 70% moisture level, closely followed
by a yield of 309 SPU of phytase at the 60% moisture level (Figure 3). These moisture levels meet the
definition of solid-state fermentation: microbial growth on solid particles in the absence of free water.
At 60% to 70% moisture, the water present in SSF systems exists in a complexed form within the solid
matrix or as a thin layer either absorbed to the surface of the particles or less tightly bound within the
capillary regions of the solid. Free water becomes present only after the saturation capacity of the solid
SSF matrix is exceeded [13].
Figure 3. Effect of substrate moisture level on E. coli phytase activity during SSF on wheat
bran. Flasks were incubated for 96 h at 37 °C with a relative humidity of 90%. The data
reported are the average and standard deviation of three independent samples. Columns with
different superscript letters differ significantly (p < 0.05.).
The roles of water in biological systems are numerous and have a significant impact on growth rates
as discussed by Gervais and Molin [14]. The availability of water for biological reactions, especially
expressed as water activity (a), is directly correlated with growth rate. Water activity is defined as
the ratio of vapor pressure of a liquid solution to that of pure water at the same temperature. Substrate
water-binding properties can affect water availability. Many studies have addressed the importance of

Fermentation 2015, 1 19
maintaining water activity during fermentation and its effects on enzymatic stability, microbial growth,
and enzyme expression [15]. The highest phytase production occurred herein for a in the range of
0.96 to 0.97 (Figure 3).
3.3. Effect of Inoculum Rate on Phytase Production
Of the tested E. coli inoculum rates (ranging from 9.07 × 105 to 4.54 × 107 CFU g−1 bran), optimum
phytase activity was achived from 2.1 × 107 to 1.1 × 107 CFU g−1 bran (Figure 4). While a decrease in
inoculum rate from 4.5 × 107 to 2.27 × 107 was associated with an increase in phytase activity, further
decreases in inoculum rate were associated with a decline in phytase activity. Effects of the inoculum
rates on hydrolytic efficiency are known to vary between species and even strains of the same species [6].
Figure 4. Effect of E. coli inoculant rate on phytase activity. Effect of inoculant level with
sterile water and 8 h inoculum on phytase activity during SSF on wheat bran at 60% (w/v)
moisture. Flasks were incubated for 96 h at 37 °C with a relative humidity of 90%. The data
reported are the average and standard deviation of three independent samples. Columns with
different superscript letters differ significantly (p < 0.05).
3.4. Effect of Incubation Period on Phytase Production
The period required to achieve optimal enzyme yield is of great economic importance. Shorter
incubation periods translate into faster turnaround times between batches, shorter opportunity for
spoilage, and lower operating cost required to maintain culture conditions (e.g., temperature). Over
the 168 h period monitored herein, phytase activity was greatest (i.e., 380 ± 10 SPU/g) after 96 h and
remained relatively stable (Figure 5). In comparison, maximum enzyme production from fungal growth
generally requires up to 144 h [16].

Fermentation 2015, 1 20
Figure 5. Effects of E.coli incubation time and pH on phytase activity. Effect of SSF
incubation period on phytase activity sampled every 24 h between 0–168 h. The flask
contained wheat bran moistened with a 24 h inoculum and sterile water at a ratio of 1:4.
○ pH; ● phytase activity (Solid State Fermentation Phytase unit (SPU) is defined as
the amount of enzyme that will liberate 1 mol of inorganic phosphate per minute at
pH 5.5 °C and 37 °C). The data reported are the average and standard deviation of three
independent samples.
3.5. Comparison between SmF and SSF on Phytase Productivity
Applying SSF to facilitate the production of phytase yields a maximum phytase activity of
350 ± 50 SPU per gram (Figures 2 and 3). This in itself represents a significant improvement compared
to the control, which achieved a phytase yield of approximately 110 SPU per gram (Figure 1). In a final
comparison for the application of a bacterial SSF application that employs E. coli as the fermentative
organism for the production of phytase, we undertook a SmF fermentation with E. coli in a shake flask
culture at 5% LB broth. We obtained our highest yield of phytase activity (64.5 SPU/g) within two days
of incubation (no further data shown). Hence, the least optimised SSF system yielded approximately
twice as much phytase activity compared to our best yield in SmF, while the optimised SSF conditions
yielded a more than five-fold increase in phytase activity.
3.6. Effect of Temperature and pH on Phytase Activity
The phytase produced by E. coli under optimal conditions (70% (w/v) moisture, 1.13 × 107 CFU
E. coli g−1 of bran, 10% LB broth powder, for 96 h at 37 °C) was assessed for stability and activity under
various pH and temperature profiles using 2 g of dried SSF product. It is of importance that the phytase
produced by this process will be able to withstand both the post-fermentation process and remain active
in the digestive system of monogastric animals. Most feed is pelletized, which occurs at elevated
temperatures; while the intestinal pH various between 3 and 6. The effect of pH and temperature on
phytase activity was monitored from pH 2.5 to pH 7.5, and for temperatures ranging from 20 °C to
70 °C. Optimal phytase activity occurred at pH 5.5 and 50 °C (Figure 6). This activity was the highest

Fermentation 2015, 1 21
at pH 5.5 throughout the temperature profile. A broad range of optimal pH and temperature values for
phytase activity has been reported in the literature across microbial species [17]. The optimal conditions
displayed in Figure 5 are consistent with other studies of E. coli [7].
Figure 6. E. coli phytase activity optimization. The temperature and pH profile of phytase
enzyme activity at various temperature and pH conditions. The temperature range was
between 20 and 70 °C. The pH range was between 2.5 to 7.5.
3.7. General Discussion
Our results show that the application of SSF for the production of phytase by E. coli provides a
marked improvement in yield in phytase activity over submerged cultivation (SmF). Under SSF
conditions, a maximum phytase activity of 350 ± 50 SPU per gram of bran was achieved by incubating
E. coli (2.27 × 107 CFU g−1) on a solid substrate of wheat bran supplemented with 10% LB powder at
70% (w/v) moisture at 37 °C for 96 h. The phytase activity achieved under these conditions was 3.5 fold
higher than the activity achieved under the least optimal conditions tested. Comparing our results to
previous studies, it is clear that the microbial source plays a major factor in the conditions for maximum
phytase activity. Typically, fungal SSF requires longer incubation periods up to 168 h, which presents
challenges for contamination and increased operating cost. Previous bacterial SSF studies evaluating
phytase have predominantly focused on Bacillus sp., which have shown similar results to the present
study. Our findings are similar to those described for Bacillus sp. Which indicate an incubation time of
72–96 h, with improved results after nutrient supplementation [6,18,19].
4. Conclusions
The results in the present study suggest that bacterial phytase production utilizing E. coli on SSF
technology is technically feasible, possibly offering a new, low-cost opportunity to produce a highly
stable phytase as an alternative to Bacillus sp for bacterial SSF. Additional studies are under way to

Fermentation 2015, 1 22
evaluate phytase production by Bacillus subtilis and also a mixed E. coli/B. subtilis culture utilizing a
wheat bran substrate.
Acknowledgments
We would like to express our gratitude to Alltech Inc. and T. P. Lyons for providing financial support
and laboratory facilities enable this to research.
Author Contributions
Authors Kyle McKinney, Keith Filer, Andrea Humphries and Frank Vriesekoop contributed to the
conception and design of the experiments; authors Kyle McKinney, Justin Combs, and Patrick Becker
performed the experiments; while all authors were involved in the analyses of the data and contributed
to the writing of the paper.
Conflicts of Interest
The authors declare no conflicts of interest.
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