Low-cost Culture Research Articles

A new model of Algal Turf Scrubber for bioremediation and biomass production using seaweed aquaculture principles

An increase in the use of macroalgal biomass for different applications is required in development of efficient cultivation systems and low-cost culture media. With this in mind, the present work proposes modifications to the Algal Turf Scrubber (ATS) system inducing the establishment of a monospecific culture of Ulva ohnoi through a seeding process, speeding up the maturation time and increasing efficiency and productivity. Bench-scale units of Seeded Algal Turf Scrubber (sATS) were built to test different types of algal attachment substrates (nylon and jute fiber) and water flow conditions (stagnant and continuous) besides evaluating of coverage rate, daily growth rate, and nutrient removal capacity. The results were promising. The coverage rate by U. ohnoi seedlings was higher when nylon substrate was combined with continuous water flow (74.41 ± 6.79%). Changes in nutrient concentration demonstrated a dose-dependent pattern with complete removal of P-PO43− in 24 h at all concentrations tested when the system had 18 days of maturation and N-NH4+ was completely removed in 24 h at all concentrations tested in both maturation times (18 and 47 days). Daily growth rates presented satisfactory values, reaching maximum of 4.8 ± 0.6% day−1. In conclusion, it was demonstrated that the bench-scale sATS system was successful to enable cultivation of U. ohnoi by seeding, resulting in good quality biomass production and efficient nutrient removal, thus justifying upscale experiments.

Agro-industrial residues for the production of red biopigment by Monascus ruber: rice flour and sugarcane molasses.

Three culture media were studied for red pigment production by Monascus ruber in submerged cultivation: rice flour (20 g L-1), sugarcane molasses (30 g L-1), and, finally, molasses + rice flour (10 g L-1+10 g L-1); all culture media were added of 5 g L-1 glycine as nitrogen source. Rice flour showed pigment production of 7.05 UA510nm and molasses 5.08 UA510nm, and the mixture of rice flour and molasses showed the best result of 16.38 UA510nm. Molasses culture presented good results for cell biomass production of 11.09 g L-1. With these results, it was observed that one substrate presented good pigment production (rice flour) and another attained better results for cell biomass growth (molasses), and a third medium containing 10 g L-1 of rice flour + 10 g L-1 of molasses was formulated. The results for this mixture showed satisfactory results, with global pigment productivity of 0.097 UA510nm h-1 and maximum productivity rate of 0.17 UA510nm h-1. The high production and productivity obtained for the mixture of rice flour and molasses indicated that the production of red pigment by submerged fermentation, using the mixture of these low-cost culture media, may be promising in terms of commercial production.

Sustainable cultivation of Navicula incerta using cellulose-based scaffold incorporated with nanoparticles in air-liquid interface cultivation system

Microalgae cultivation using open cultivation systems requires large area and it is susceptible to contamination as well as weather changes. Meanwhile, the closed systems require large capital investment, and they are susceptible to the build-up of dissolved oxygen. Air-liquid interface culture systems with low water-footprint, but high packing density can be used for microalgae cultivation if low-cost culture scaffolds are available. In this study, cellulose-based scaffolds were synthesized using NaOH/urea aqueous solution as the solvent. Titanium dioxide (TiO2), silica gel and polyethylene glycol 1000 (PEG 1000) nanoparticles were added into the membrane scaffolds to increase the hydrophilicity of nutrient absorbing to support the growth of microalgae. The membrane scaffolds were characterized by FTIR, SEM, contact angle, porosity and porometry. All three nanoparticles additives showed their ability in reducing the contact angle of membrane scaffolds from 63.4 ± 2.3° to a range of 52.6 ± 1.2° to 38.8 ± 1.5° due to the hydrophilic properties of the nanoparticles. The decreasing in pore size when nanoparticles were added did not affect the porosity of membrane scaffolds. Cellulose membrane scaffold with TiO2 showed the highest percentage of microalgae Navicula incerta growth rate of 22.1% because of the antibacterial properties of TiO2 in lowering the risk of cell contamination and enhancing the growth of N. incerta. The results exhibited that cellulose-based scaffold with TiO2 added could be an effective support in plant cell culture field.

β-glucosidase from thermophilic fungus Thermoascus crustaceus: production and industrial potential.

Microbial β-glucosidases can be used in several industrial processes, including production of biofuels, functional foods, juices, and beverages. In the present work, production of β-glucosidase by solid state cultivation of the fungus Thermoascus crustaceus in a low-cost cultivation medium (comprising agroindustrial residues) was evaluated. The highest production of β-glucosidase, about 415.1 U/g substrate (or 41.51 U/mL), was obtained by cultivating the fungus in wheat bran with 70% humidity, during 96 h at 40°C. The enzymatic activity was optimum at pH 4.5 and 65°C. β-Glucosidase maintained its catalytic activity when incubated at a pH range of 4.0-8.0 and temperature of 30-55°C. The enzyme was strongly inhibited by glucose; even when the substrate and glucose concentrations were equal, the inhibition was not reversed, suggesting a non-competitive inhibition. In the presence of up to 10% ethanol, β-glucosidase maintained its catalytic activity. In addition to β-glucosidase, the enzymatic extract showed activity of 36 U/g for endoglucanase, 256.2 U/g for xylanase, and 18.2 U/g for β-xylosidase. The results allow to conclude that the fungus T. crustaceus has considerable potential for production of β-glucosidase and xylanase when cultivated in agroindustrial residues, thereby reducing the cost of these biocatalysts.

Ecophysiology of two endemic Amazon quillworts

Abstract Isoetes L. is a genus of lycophytes widely distributed around the world, with many endemic species. In our work we document the first successful large scale ex situ cultivation of the threatened species from Brazilian Amazon Isoetes cangae and Isoetes serracarajensis, which are endemic of a lake and close ponds nearby, respectively. This study aimed to develop the ex situ cultivation methods of the species I. cangae and I. serracarajensis. The capacity of regrowth and to tolerate high soluble iron in the water was monitored. We also studied plant leaf nutrition, growth, photosynthesis, and plasma membrane H+-ATPase activity. Both species were cultivated in a low-cost system at a greenhouse during three seasons through a year. The survival rate of plants was higher as 98 %, and both species developed effectively on the culture system with different patterns during linear growth: I. cangae showed faster leaf growth but was slower on new leaves production compared to I. serracarajensis. The mechanism of leaf expansion seems to be related to H+-ATPase activation, near to 2-fold higher in I. cangae. Contrarily, the effective quantum yield of photosystem II was higher in I. serracarajensis than in I. cangae. Both species accumulate very high concentration of minerals in the leaf tissue, and I. serracarajensis presented more iron than I. cangae, and remarkably showed more new leaves on higher iron concentration exposure. I. cangae showed the ability to reproduce during ex situ cultivation. The results of this work have broad applicability, assisting other low-cost cultivation studies, which are critical to ecosystem recovery of mining areas and conservation strategies.

Low-cost culture medium for biomass production of lactic acid bacteria with probiotic potential destined to broilers

The aim of this study was to evaluate different low-cost culture media for biomass production of 3 potential probiotic L. salivarius strains, which could be destined to broilers at farms. Different formulated media based on whey permeate (WP) supplemented with nitrogenous sources were evaluated: yeast extract (YE), whey hydrolysate (WH) and MnSO4.H2O (Mn), MgSO4.7H2O (Mg). The growth of each strain in the formulated media and the cost was compared with their growth and cost in commercial medium (MRS). L. salivarius DSPV008P did not grow adequately in any of the formulated media. On the other hand, addition of YE and Mn in the formulated media increased L. salivarius DSPV002P and L. salivarius DPSV011P growth. In contrast, WH and Mg addition increased the L. salivarius DSPV002P biomass only. L. salivarius DSPV011P was the only strain that had similar growth performance in MRS as in the selected medium: WP + YE 8g/l + Mn. In this sense, L. salivarius DSPV011P reached a biomass of 9.22 Log (CFU/ml) in the selected formulated medium, with a low-cost growth medium 12 times less than in MRS. Although the effect of supplements added to the culture medium on kinetic parameters are strain dependent, L. salivarius DSPV011P is the strain with the best technological characteristics, capable of growing in a medium based on a by-product of the dairy industry supplemented with YE and Mn and at a much less cost than in MRS medium.

Parámetros cinéticos del cultivo de Lactobacillus plantarum y Saccharomyces boulardii en un medio de cultivo a base de melaza de betabel

Regular consumption of probiotic microorganisms as part of the diet can contribute to improving health and mitigating the effects of metabolic syndrome diseases. Then, it is essential to design production processes for this type of microorganisms, which are optimal from the point of view of production time and cost. This paper presents a study of the effect of using a low-cost culture medium, formulated with beet molasses, which manages to enhance the growth of two types of probiotic microorganisms, Saccharomyces boulardii, and Lactobacillus plantarum. In addition to its effect on cellular biomass growth, a kinetic model for both strains growth is also presented. This model includes factors such as the S. boulardii capability to hydrolyze saccharose into their respective monosaccharides and the inhibition effect of lactic acid production on L. plantarum growth. Finally, a mixed culture production scheme is proposed for both microorganisms, in order to take advantage of the yeast's capability to hydrolyze saccharose, in order not to require the addition of extra glucose to the culture medium.

Modification of media using food-grade components for the fermentation of Bifidobacterium and Lactobacillus strains in large-scale bioreactors

Probiotic bacteria continue to receive increasing attention in the food and feed industries. However, the production of Bifidobacterium and Lactobacillus at an industrial scale is challenging because of specific nutrient requirements and conditions, which are complicated and costly. We developed low-cost culture media by modifying the carbon and nitrogen sources for Bifidobacterium animalis subsp. lactis KMP-H9-01 and Lactobacillus reuteri KMP-P4-S03 from available food grade components. Sucrose (15 g/l) was selected as a suitable carbon source for both strains because it was the most economic and facilitated bacterial growth that was equal to that of glucose. The Bifidobacterium strain required beef extract as a nitrogen source to multiply. The fermentation of both strains using the modified media formula in 5-L and 50-L bioreactors showed that the highest cell counts of L. reuteri and B. animalis subsp. lactis were 9 and 9.8 log CFU/ml after 12–15 h, respectively. The concentration (g/l) ratio between lactate and acetate obtained from B. animalis subsp. lactis was 7:7.4 at 12 h and 11.4:10.6 at 40 h; the ratio was similar at both time points (6.9: 1.1) for L. reuteri. Thus, this economically modified food-grade medium for the large-scale fermentation of two probiotic bacteria was efficient.

TEXTURIZED SOY PROTEIN AS AN ALTERNATIVE LOW-COST MEDIA FOR BACTERIA CULTIVATION

Most of the culture media used in bacterial growth is composed of complex ingredients, increasing the value of the product. This makes its acquisition unavailable by educational institutions without sufficient funding, making even more difficult the practical teaching of microbiology. Therefore, the development of an alternative medium of simple composition and low-cost becomes necessary. This work aimed to use texturized soy protein (TSP) as a low cost culture medium that allows the bacterial growth. For the composition of the broths, concentrations between 0.5% and 10% were prepared. Thirty-eight bacteria, including important pathogens associated with food, were inoculated and the concentration of 7.5% TSP allowed the growth of 100% of the tested bacteria, with a production cost of approximately 86% and 68% lower than tryptic soy broth and agar, respectively. This work demonstrates that the use of a culture medium of easy acquisition and low cost is feasible and has good results.

Hydrogen production by dark fermentation using a new low-cost culture medium composed of corn steep liquor and cassava processing water: Process optimization and scale-up

The use of effluents for hydrogen production through dark fermentation is promising because it results in the generation of value-added products and reduction of the effluent’s organic load. A low-cost medium using agroindustrial effluents, corn steep liquor (CSL) and cassava processing wastewater (CPW) was evaluated for hydrogen production with microbial consortia (Vir and Gal). Four variables were evaluated for their impact on biohydrogen production through a Plackett Burman design. Subsequently, the significant variables were optimized using a central composite design, resulting in two mathematical models with regression coefficients R2 > 0.92. The maximum yields were validated and resulted in 107 and 83.1 mL of biohydrogen/g COD removed for Vir and Gal, respectively. The lower medium cost for biohydrogen production was 81.5 USD/m3, approximately 80% more economical than some supplemented media. Finally, the scale-up of the biohydrogen production by consortia to 5L resulted in an increase of more than 40%.

First report on the production of phytotoxic metabolites by Mycoleptodiscus indicus under optimized conditions of submerged fermentation

ABSTRACT An alternative to controlling weeds resistant to conventional herbicides is the isolation of new active principles. Fungi can produce phytotoxic metabolites that may be used in the development of new herbicides. The objectives of this study were: (1) isolate, select, and identify a fungus producer of phytotoxic metabolites and (2) optimize the culture conditions of this fungus in a low-cost culture medium, with the aim of increasing the phytotoxic effects of their metabolites in weeds and commercial plants. Fungi were isolated from the leaves of Conyza sp. with disease symptoms and selected according to the production of phytotoxic metabolites in solid and submerged fermentation in a low-cost culture medium. A Plackett–Burman Design and Central Composite Rotational Design were used to optimize the conditions of temperature, agitation, pH, and concentrations of glucose and yeast extract in submerged fermentation. The phytotoxic metabolites produced under optimal conditions were tested on 10 commercial plants and weeds that are difficult to control. Of the nine fungi isolated, Mycoleptodiscus indicus UFSM54 produced higher leaf lesions. The production of phytotoxic metabolites was optimized when the fungus was cultivated at 35°C, 50 rpm, and 1.5 g L−1 of glucose in submerged fermentation. The metabolites of M. indicus caused severe phytotoxic effects on germination and seedling growth, and enhanced lesion development on detached plant leaves. The present study is the first to report on the production of phytotoxic metabolites by M. indicus, a potential producer of bioherbicides.

Evaluation of agro-industrial residues produced in Costa Rica for a low-cost culture medium using Bacillus subtilis 168

Agro-industrial residues correspond to all the materials generated from activities that involve the transformation of both crops and livestock to obtain processed or semi-finished products. In Costa Rica, the primary sector of economy generates more than 6.3 trillion tons of organic residues per year. The daily generation of this residues pose environmental and economic problems. In recent years, biotechnological-based alternatives have emerged with the purpose of taking advantage of the high nutritional content of these residues to cultivate microorganisms capable of producing compounds with high demand at a commercial level. The present study evaluates six agro-industrial residues produced in Costa Rica, in order to growth Bacillus subtilis 168. An optimization of the culture medium was carried out under a complete factorial design 23, where the variables evaluated were carbon, nitrogen and phosphorus sources. Molasses at 10% m/v, wheat bran at 0.5% m/v, and K2HPO4 at 0.01%, as a carbon, nitrogen, and phosphorus sources, respectively, were identified as optimal for the growth of Bacillus subtilis 168.

Generation of cortical neurons through large-scale expanding neuroepithelial stem cell from human pluripotent stem cells

BackgroundConsiderable progress has been made in converting human pluripotent stem cells (hPSCs) into cortical neurons for disease modeling and regenerative medicine. However, these procedures are hard to provide sufficient cells for their applications. Using a combination of small-molecules and growth factors, we previously identified one condition which can rapidly induce hPSCs into neuroepithelial stem cells (NESCs). Here, we developed a scalable suspension culture system, which largely yields high-quality NESC-spheres and subsequent cortical neurons.MethodsThe NESC medium was first optimized, and the suspension culture system was then enlarged from plates to stirred bioreactors for large-scale production of NESC-spheres by a stirring speed of 60 rpm. During the expansion, the quality of NESC-spheres was evaluated. The differentiation potential of NESC-spheres into cortical neurons was demonstrated by removing bFGF and two pathway inhibitors from the NESC medium. Cellular immunofluorescence staining, global transcriptome, and single-cell RNA sequencing analysis were used to identify the characteristics, identities, purities, or homogeneities of NESC-spheres or their differentiated cells, respectively.ResultsThe optimized culture system is more conducive to large-scale suspension production of NESCs. These largely expanded NESC-spheres maintain unlimited self-renewal ability and NESC state by retaining their uniform sizes, high cell vitalities, and robust expansion abilities. After long-term expansion, NESC-spheres preserve high purity, homogeneity, and normal diploid karyotype. These expanded NESC-spheres on a large scale have strong differentiation potential and effectively produce mature cortical neurons.ConclusionsWe developed a serum-free, defined, and low-cost culture system for large-scale expansion of NESCs in stirred suspension bioreactors. The stable and controllable 3D system supports long-term expansion of high-quality and homogeneous NESC-spheres. These NESC-spheres can be used to efficiently give rise to cortical neurons for cell therapy, disease modeling, and drug screening in future.

Development of a low-cost culture medium for the rapid production of plant growth-promoting Rhodopseudomonas palustris strain PS3.

Rhodopseudomonas palustris PS3 is one of the purple phototrophic non-sulfur bacteria (PNSB), which have plant growth-promoting effects on various plants. To expand the scale of PS3 fermentation in a time- and cost-effective fashion, the purpose of this work was to evaluate the use of low-cost materials as culture media and to optimize the culture conditions via response surface methodology. Corn steep liquor (CSL) and molasses were identified as potential materials to replace the nitrogen and carbon sources, respectively, in the conventional growth medium. The optimum culture conditions identified through central composite design were CSL, 39.41 mL/L; molasses, 32.35 g/L; temperature, 37.9°C; pH, 7.0; and DO 30%. Under the optimized conditions, the biomass yield reached 2.18 ± 0.01 g/L at 24 hours, which was 7.8-fold higher than that under the original medium (0.28 ± 0.01 g/L). The correlation between the predicted and experimental values of the model was over 98%, which verified the validity of the response models. Furthermore, we verified the effectiveness of the R. palustris PS3 inoculant grown under the newly developed culture conditions for plant growth promotion. This study provides a potential strategy for improving the fermentation of R. palustris PS3 in low-cost media for large-scale industrial production.

WHEY BASED CULTURE MEDIA FOR THE PRODUCTION OF SELENIUM NANOPARTICLES RICH PRODUCT BY THREE LACTIC ACID BACTERIAL STRAINS.

The ability of three LAB strains namely, L. brevis, L. plantarum and P. acidilactici to grow and produce SeNPs in MRS or whey media supplemented with 100 or 200 ppm of Se(IV) as sodium selenite (Na2SeO3) was investigated. The presence of Se(IV) in the media retarded the bacterial growth in the first six hours of incubation thereafter, the bacterial growth gradually increased up to 72 hr. P. acidilactici was more tolerable to the inhibitory effect of Se(IV) than the other two strains. Although MRS media was more suitable for the growth of the three LAB strains, as estimated by the reduction of media pH and increase of absorbance compared to whey media, both media showed significantly similar conversion rate (98-100%) of Se(IV) to SeNPs at the end of the incubation period (72 hr). L. brevis, produced smaller SeNPs (125.7 nm) followed by L. plantarum (140.7 nm) and P. acidilactici (176.6 nm). The obtained data demonstrate the possibility of using the whey as a source of low-cost culture media for the production of milk whey based product rich in SeNPs with the assistant of the environmentally friendly LAB for different usage, i.e. as a feed supplement for livestock production.

Differentiation of Human Induced Pluripotent Stem Cells into Male Germ Cells.

Infertility is defined as not being able to become pregnant or to conceive a child after one year or longer of regular unprotected intercourse. Male infertility refers to a male's inability to cause pregnancy that can result from deficiencies in semen quality, sperm concentration, or abnormal sperm function. Till now, there are few effective methods for the treatment of a couple with male infertility. In the past few years, stem cell-based therapy as a promising strategy has emerged for the treatment of male infertility. Human Pluripotent Stem Cells (hPSCs) can self-renew and differentiate into any type of cell. Human Embryonic Stem Cells (hESCs) and induced Pluripotent Stem Cells (hiPSCs) are two pluripotent populations that can proliferate and give rise to ectodermal, mesodermal, endodermal, and germ cell lineages. Both undifferentiated hiPSCs and hESCs are powerful candidates for the treatment of male infertility. Generation of male germ cells from hPSCs can provide new mechanistic insights into the regulation of spermatogenesis and have a great opportunity for families with infertility. Therefore, a robust, reproducible, and low-cost culture method that supports hPSCs differentiation into male germ cells is necessary. However, very few studies have focused on the derivation of sperm-like cells from hiPSCs and the details of hPSCs differentiation into male germ cells have not been fully investigated. Therefore, in this review, we focus on the in vitro differentiation potential of hiPSCs into male germ cells.

Technological variables influencing the growth and stability of a silage inoculant based on spray-dried lactic acid bacteria.

To investigate the influence of different culture media and fermentation conditions on growth performance and viability of three lactic acid bacteria (LAB) strains with potential as silage inoculants, and to optimize spray-drying in order to enhance survival to dehydration and storage stability. In house-formulated MRS was a suitable low-cost culture medium for Lactobacillus plantarum Hv75, Pediococcus acidilactici 3903 and L. buchneri B463. Uncontrolled pH biomass production conferred enhanced stability during storage at 4°C after spray-drying. The use of whey protein concentrate 35 (WPC)-maltodextrin (M) as matrix, inlet temperature of 145-150°C and air flow rate of 601lh-1 was adequate for the production of dehydrated LAB. According to the desirability function, at this optimized condition, moisture content, yield and solubility were predicted to be 3·96, 73·68 and 90·36% respectively. Those conditions also showed a decrease of 0·855 log CFU per gram after drying, no loss in viability at 4°C for 6months and 1 log CFU per gram reduction at 25°C. Stable and economically feasible dehydrated LAB cultures were obtained using alternative culture media, fermentation under uncontrolled pH and optimizing spray-drying process conditions through the desirability function method. Our results can be utilized for efficient production and commercialization of several dry LAB.

Development of a low-cost polyculture system utilizing Hygroryza aristata floating grass in the coastal wetlands of Bangladesh

Worldwide the quality and high price of fish feed are an important issue for sustainable aquaculture that warranted the development of low-cost culture systems utilizing natural resources. An experiment was conducted in a tidally inundated wetland in the coastal region of Bangladesh utilizing naturally grown floating grass, Hygroryza aristata as a fish feed. The experiment was conducted under three treatments each with three replications for one year. In treatment one, the grass was planted and grown on the bottom soil before stocking of fish (T1; in-situ grass); in treatment two, the grass grown elsewhere was collected and supplied as fish feed in raw condition (T2; ex-situ grass); and in treatment three, the fishes were fed with commercial floating fish feed (T3; control). Grass carp, common carp, black carp and tilapia were reared at the ratio of 6:1:1:2 with a total stocking density of 12,350 fish ha−1. The survival rate, growth (weight gain, % weight gain and specific growth rate, SGR), production and cost and benefit of fish production were calculated. The survival of the fish varied from 76.00% to 85.14% irrespective of species. The weight gain, % weight gain and SGR of grass carp and common carp were highest in in-situ grass, followed by ex-situ grass and control, whereas tilapia showed the highest growth in control, followed by in-situ and ex-situ grass. The total production of fish was highest in in-situ grass (9060.70 kg ha−1 year−1), followed by control (8469.70 kg ha−1 year−1) and ex-situ grass (8001.23 kg ha−1 year−1). The net economic benefit and benefit-cost ratio were also highest in in-situ grass (US$ 17,994.96 ha−1 and 4.52), followed by ex-situ grass (US$ 14,278.30 ha−1 and 2.78) and control (US$ 4818.63 ha−1 and 0.32). The present findings indicate that coastal wetlands of Bangladesh can be used for commercial aquaculture using the H. aristata floating grass as fish feed, which may play an important role in the livelihood development of the people living in the coastal areas of the country.

Red Light Variation an Effective Alternative to Regulate Biomass and Lipid Profiles in Phaeodactylum tricornutum

Marine water diatom Phaeodactylum tricornutum is a photosynthetic organism that is known to respond to the changing light environment and adapt to different temperatures to prevent photoinhibition and maintain its metabolic functions. The objective of the present study was to test whether light shift variations in different growth phases impact the growth and lipid metabolism of P. tricornutum. Thus, we investigated R exposure in different growth phases to find the most effective light shift condition. The results showed that substituting white light (W) by red light (R) under autotrophic conditions, a condition called red shift (RS), increased biomass and lipid content compared to levels found under continuous W or R exposure alone. We observed an increase by 2-fold biomass and 2.3-fold lipid content in RS as compared to W. No significant change was observed in the morphology of lipid droplets, but the fatty acid (FA) composition was altered. Specifically, polyunsaturated FAs were increased, whereas monounsaturated FAs decreased in P. tricornutum grown in RS compared to W control. Therefore, we propose that a light shift during the beginning of the stationary phase is a low-cost cultivation strategy to boost the total biomass and lipids in P. tricornutum.

Low-cost media statistical design for laccase rPOXA 1B production in P. pastoris

Laccases (E.C. 1.10.3.2) are multicopper oxidases of great importance in the industry due to their non-specificity and high oxidative potential. Laccases are useful to bleach synthetic dyes, oxidize phenolic compounds and degrade pesticides, among others. Hence, the objective of this work was to optimize low cost culture media for recombinant (rPOXA 1B) laccase production from Pleurotus ostreatus in Pichia pastoris. To this end, low cost nitrogen sources were studied, such as malt extract, isolated soy protein and milk serum. Following, two central composite designs (CCD) were performed. In CCD-1 different concentrations of glucose USP (0–13.35 gL-1), protein isolated soy protein (5–25 gL-1), malt extract (3.5–17.5 gL-1) and (NH4)2SO4 (1.3–6.5 gL-1) were evaluated. In CCD-2 only different concentrations of glucose USP (7.9–22 gL-1) and isolated soy protein (15.9–44.9 gL-1) were evaluated. CCD-2 results led to a One Factor Experimental design (OFED) to evaluate higher isolated soy protein (20–80 gL-1) concentrations. In all designs, (CCD-1, CCD-2 and OFED) CuSO4 (0.16 gL-1) and chloramphenicol (0.1 gL-1) concentrations remained unchanged. For the OFED after sequential statistical optimization, an enzyme activity of 12,877.3 ± 481.2 UL−1 at 168 h was observed. rPOXA 1B activity increased 30.54 % in comparison with CCD-2 results. Final composition of optimized media was: 20 gL-1 glucose USP, 50 gL-1 isolated soy protein 90 % (w/w), 11.74 gL-1 malt extract, and 4.91 gL-1 (NH4)2SO4. With this culture media, it was possible to reduce culture media costs by 89.84 % in comparison with improved culture media previously described by our group.