Elsevier

Process Biochemistry

Volume 40, Issue 9, September 2005, Pages 3095-3102
Process Biochemistry

Production of cell-bound phytase by Pichia anomala in an economical cane molasses medium: Optimization using statistical tools

https://doi.org/10.1016/j.procbio.2005.03.059Get rights and content

Abstract

Statistical experimental designs were applied for the optimization of cell-bound phytase production by Pichia anomala in a cost-effective cane molasses medium. Using Plackett–Burman design, cane molasses, urea and inoculum density were identified as significant factors and these factors were subsequently optimized using a central composite design. The optimum variables that supported maximum enzyme titre were 8% cane molasses, 0.4% urea and 2% inoculum density. The validity of the optimized variables was verified in shake-flasks as well as in a laboratory fermenter. An overall 5- and 1.6-fold enhancement in phytase titres (324–1689 U/g DYB) and biomass production (6.4–10 g/l) were attained due to optimization, respectively.

Introduction

Phytic acid (myo-inositol hexakisphosphate) comprises 50–80% of the total phosphorus in most foods of plant origin [1]. It is the organic form of phosphorus that is largely unavailable to monogastrics such as poultry, pigs, fish and humans due to the lack or inadequate levels of phytate-hydrolyzing enzymes in their gastrointestinal tracts [2], [3]. Furthermore, phytates act as antinutritional factor in more than one ways [1] and their excess in the environment also leads to phosphorus pollution problems [4], [5]. Phytases (myo-inositol hexakisphosphate phosphohydrolase, EC 3.1.3.8 and 3.1.3.26) belong to a family of histidine acid phosphatases, which are capable of hydrolyzing phytic acid to myo-inositol and phosphoric acid in a stepwise manner forming myo-inositol phosphate intermediates [6], [7]. Supplementation of microbial phytase to animal diets tends to increase the bioavailability of proteins and essential minerals and consequently improves growth performance. It also reduces the amount of phosphorus in animal manure, thereby helping in combating phosphorus pollution [8], [9]. Phytase has been marketed as a feed additive in the US since 1996 and at the close of the twentieth century, annual sales of phytase, as an animal feed additive, were about US$ 500 million [10].

Any fermentation process is significantly influenced by various physical as well as chemical parameters. The first step in process optimization is screening of the important factors, followed by estimation of optimal levels of these factors. The conventional approach is to investigate one factor at a time, while keeping the others constant. This approach is tedious, time consuming and does not take into consideration the interactions among the factors [11]. A statistical approach provides an efficient alternative, which is economical and allows the study of interactions among the factors, and at the same time predicts the optimum values of the variables. Plackett–Burman design is a well-established and widely used statistical technique for screening of medium components in shake-flasks [12]. Following the initial screening, factorial design and response surface analysis is used to determine the optimum values of the factors studied. Several researchers in biotechnology have applied these techniques for optimization of culture conditions, determination of optimal values of process parameters and feeding rates [13], [14], [15].

The Ascomycetous yeast Pichia anomala produces a cell-bound phytase [16] with a proved beneficial effect in the poultry [17]. The enzyme also has the requisite characteristics, such as thermostability, acid stability and broad substrate specificity, for use as an animal feed supplement [18]. The high cost of phytases is the most cited limiting factor in its application in animal diets [9]. Cane molasses, the by-product of the sugar refinery process, containing 45–50% sugars, is the most economical source of carbohydrate for various industrial fermentations [19]. In this investigation, an attempt was made to optimize the cultural conditions for maximizing the production of phytase from P. anomala in an inexpensive cane molasses medium using statistical approaches. Further, a feasibility of large-scale production was attempted in a laboratory fermenter. This is the first report on use of statistical methods for improving yeast cell-bound phytase yield in cane-molasses medium.

Section snippets

Source of strain

The yeast strain was isolated from dried flower buds of Woodfordia fruticosa, and identified as P. anomala (Hansen) Kurtzman according to Barnett et al. [20]. The yeast strain was grown on malt extract-yeast extract-peptone-glucose (MYPG) agar slopes and maintained as described earlier [16]. The culture is deposited at the Microbial Type Culture Collection, Institute of Microbial Technology (Chandigarh, India) [MTCC-4133].

Cultivation medium and culture conditions

Erlenmeyer flasks (250 ml) containing 50 ml liquid medium (cane molasses

Optimization of phytase production from P. anomala

In addition to large amounts of sugars [approximately 50% (sucrose 33.5%, invert sugar 21.2%)], molasses contain small amounts of nitrogenous substances (0.4–1.5%), traces of vitamins such as thiamine, riboflavin, pyridoxine, folic acid, biotin, pantothenate, and various trace elements [24]. Due to the high C:N ratio, there is a requirement for supplementation with an additional nitrogen source and various nitrogen sources were added to the medium (0.02% nitrogen), and their affect on growth

Discussion

The phytase of P. anomala exhibited thermostability, acid stability, broad substrate specificity and lack of requirement for metal ions for activity, making it the enzyme of choice for poultry feed supplementation [18]. The supplementation of feed of broiler chicks with the yeast biomass resulted in better growth and phosphorus retention in the body, and reduced excretion of phosphorus in the droppings [17]. A major problem in the use of the yeast biomass as phytase source in the feed is its

Conclusions

The unoptimized cane molasses medium supported a low phytase titre (338 U/g DYB) and biomass (6.4 g/l). The optimization using statistical approaches and cultivation in a laboratory fermenter resulted in approximately 5- and 1.6-fold enhancement in phytase and biomass production, respectively. An increase in biomass yield (from 0.26 to 0.32) was also achieved. Thus, the production of cell-bound phytase from P. anomala with a potential application in animal feed industry was effectively optimized

Acknowledgement

We gratefully acknowledge financial assistance from the Department of Biotechnology, Government of India. One of us (Parvinder Kaur) is grateful to the Council of Scientific and Industrial Research (CSIR), New Delhi (India), for the award of fellowship during the course of this investigation.

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