Alternative methods for gibberellic acid production, recovery and formulation: A case study for product cost reduction.

The aim of this work was to develop a new production, recovery and formulation process of gibberellic acid (GA3). Low-cost byproducts - citrus pulp (CP) and soybean hulls (SH) - were employed as substrate for GA3 production by Gibberella fujikuroi in semisolid fermentation. A CP/SH mixture (70%/30%) promoted high productivities both in bubble column reactor (1.66 mg L/h), and in stirred tank reactor (2.13 mg L/h). GA3 production medium cost (US$ 6.70/m3) was reduced by 85% when compared to previously reported synthetic media (US$ 44.96/m3). It was described that GA3 fermented extract has low stability, and that liquid and powder formulation of the fermented extract maintained the biomolecule activity over 6 months. Alginate and alginate/kefiran beads containing GA3 showed encapsulation efficiency of 70% and 60%, respectively. This work supports good perspectives for GA3 production using cheap substrates and simple formulation of clarified extract to favour its use in agricultural countries.

Bacillus lipopeptides as powerful pest control agents for a more sustainable and healthy agriculture: recent studies and innovations.

MAIN CONCLUSION: Lipopeptides could help to overcome a large concern in agriculture: resistance against chemical pesticides. These molecules have activity against various phytopathogens and a potential to be transformed by genetic engineering. The exponential rise of pest resistances to different chemical pesticides and the global appeal of consumers for a sustainable agriculture and healthy nutrition have led to the search of new solutions for pest control. Furthermore, new laws require a different stance of producers. Based on that, bacteria of the genus Bacillus present a great agricultural potential, producing lipopeptides (LPs) that have high activity against insects, mites, nematodes, and/or phytopathogens that are harmful to plant cultures. Biopesticide activity can be found mainly in three families of Bacillus lipopeptides: surfactin, iturin, and fengycin. These molecules have an amphiphilic nature, interfering with biological membrane structures. Their antimicrobial properties include activity against bacteria, fungi, oomycetes, and viruses. Recent studies also highlight the ability of these compounds to stimulate defense mechanisms of plants and biofilm formation, which is a key factor for the successful colonization of biocontrol organisms. The use of molecular biology has also recently been researched for continuous advances and discoveries of new LPs, avoiding possible future problems of resistance against these molecules. As a consequence of the properties and possibilities of LPs, numerous studies and developments as well as the attention of large companies in the field is expected in the near future.

Update and Revalidation of Ghose's Cellulase Assay Methodology.

New studies on cellulolytic enzymes aiming to improve biofuels production lead to a concern over the assaying methods commonly applied to measure their activity. One of the most used methods is Ghose's cellulase and endoglucanase assay, developed by the International Union of Pure and Applied Chemistry in 1987. Carrying out this method demands high volumes of reagents and generation of high amounts of chemical residues. This work aimed to adapt Ghose's methodology to reduce its application cost and residue generation and validate the adjustments. To do so, International and Brazilian laws were applied to validate methodologies. Method's modifications were successfully validated according to all institutions and were considered linear, accurate, precise, and reproducible. It was possible to reduce the volume of reagents and residues in 12 times. Considering the routine work of most laboratories, it is a great reduction on material costs and residue treatment, which reflects in sustainability and environmental impacts.

Biotechnological approaches for cocoa waste management: A review.

Cocoa beans provide raw materials for global food industries valued in excess of $47 billion in world exportations. Through on-farm processing, about 80% of cocoa fruit is discarded as residual biomass, including cocoa pod husks, cocoa bean shells and cocoa sweatings. Farmers routinely discard these residues/by-products during the initial cocoa bean processing steps, occupying vast areas and raising social and environmental concerns. Alternatively, this residual biomass is used as cocoa tree fertilizer. However, its disposal is performed without proper treatment, resulting in putrid odors and plant diseases. Recently, some studies have reported the use of cocoa by-products in the production of high-value-adding molecules with potential applications in the food, pharmaceutical and cosmetic industries. In this aspect, biotechnological approaches have been shown to be a viable alternative for the transformation of this residual biomass into fine products. This article reviews the biotechnological approaches implemented for the management and exploitation of cocoa by-product. Related topics on cocoa production and residual biomass generation, sustainability and valorization of cocoa chain are addressed and discussed.

Arthrospira maxima OF15 biomass cultivation at laboratory and pilot scale from sugarcane vinasse for potential biological new peptides production.

An environmental friendly process was developed to produce Arthrospira maxima's biomass from sugarcane vinasse, which was generated in a bioethanol production chain, at laboratory and pilot scale. Peptides fractions were than obtained from enzymatically hydrolyzed biomass. High microalgae biomass productivities were reached (0.150 g L-1 day-1) coupled with a significant reduction of BOD and COD (89.2 and 81%, respectively). Three peptide fractions were obtained from microalgae biomass through single or sequential enzymatic hydrolysis. Antioxidant, antimicrobial, anti-inflammatory, and/or anti-collagenase activities of biopetides' fractions were observed. The PHS showed multi-biological activities. The three peptides fractions could be potential candidates for different applications in pharmaceutical, cosmetic and food industry.

Exploring the impacts of postharvest processing on the aroma formation of coffee beans - A review.

The aim of this review is to describe the volatile aroma compounds of green coffee beans and evaluate sources of variation in the formation and development of coffee aroma through postharvest processing. The findings of this survey showed that the volatile constituents of green coffee beans (e.g., alcohols, aldehydes, and alkanes) have no significant influence on the final coffee aroma composition, as only a few such compounds remain in the beans after roasting. On the other hand, microbial-derived, odor-active compounds produced during removal of the fruit mucilage layer, including esters, higher alcohols, aldehydes, and ketones, can be detected in the final coffee product. Many postharvest processing including drying and storage processes could influence the levels of coffee aroma compositions, which remain to be elucidated. Better understanding of the effect of these processes on coffee aroma composition would assist coffee producers in the optimal selection of postharvest parameters that favor the consistent production of flavorful coffee beans.

Current advances in gibberellic acid (GA3) production, patented technologies and potential applications.

MAIN CONCLUSION: Gibberellic acid is a plant growth hormone that promotes cell expansion and division. Studies have aimed at optimizing and reducing production costs, which could make its application economically viable for different cultivars. Gibberellins consist of a large family of plant growth hormones discovered in the 1930s, which are synthesized via the terpenes route from the geranylgeranyl diphosphate and feature a basic structure formed by an ent-gibberellane tetracyclic skeleton. Among them, only four have biological activity, including gibberellic acid (GA3), which acts as a natural plant growth regulator, especially for stem elongation, seed germination, and increased fruit size. It can be obtained from plants, fungi, and bacteria. There are also some reports about microalgae GA3 producers. Fungi, especially Gibberella fujikuroi, are preferred for GA3 production via submerged fermentation or solid-state fermentation. Many factors may affect its production, some of which are related to the control and scale-up of fermentation parameters. Different GA3 products are available on the market. They can be found in liquid or solid formulations containing only GA3 or a mixture of other biological active gibberellins, which can be applied on a wide variety of cultivars, including crops and fruits. However, the product's cost still limits its large and continuous application. New low-cost and efficient GA3 production alternatives are surely welcome. This review deals with the latest scientific and technological advances on production, recovery, formulation, and applications of this important plant growth hormone.

Gibberellic Acid Production by Different Fermentation Systems Using Citric Pulp as Substrate/Support.

Gibberellic acid (GA3) is an important phytohormone, a member of gibberellins family, which acts as a promoter and regulator of plant growth. This study aimed to evaluate GA3 production by Fusarium moniliforme LPB03 and Gibberella fujikuroi LPB06 using different techniques of fermentation, solid state fermentation (SSF), submerged fermentation (SmF), and semisolid state fermentation (SSSF), and different types of bioreactors. In all techniques, citric pulp (CP), a subproduct obtained from the extraction of orange juice, was employed as the substrate/support. GA3 production by SSF reached 7.60 g kg-1 and 7.34 g kg-1 in Erlenmeyer flasks and column bioreactors, respectively. For SmF, the highest concentration of GA3 obtained was 236.00 mg L-1 in Erlenmeyer flasks, 273.00 mg L-1 in a 10 L stirred tank reactor (STR), and 203.00 mg L-1 in a 1.5 L bubble column reactor (BCR). SSSF was conducted with a CP suspension. In this case, GA3 concentration reached 331.00 mg L-1 in Erlenmeyer flasks and 208 mg L-1 in a BCR. The choice of the fermentation technique is undoubtedly linked to the characteristics and productivity of each process. The methods studied are inexpensive and were found to produce good proportions of GA3, making them suitable for several applications.

Milk kefir: composition, microbial cultures, biological activities, and related products.

In recent years, there has been a strong focus on beneficial foods with probiotic microorganisms and functional organic substances. In this context, there is an increasing interest in the commercial use of kefir, since it can be marketed as a natural beverage that has health promoting bacteria. There are numerous commercially available kefir based-products. Kefir may act as a matrix in the effective delivery of probiotic microorganisms in different types of products. Also, the presence of kefir's exopolysaccharides, known as kefiran, which has biological activity, certainly adds value to products. Kefiran can also be used separately in other food products and as a coating film for various food and pharmaceutical products. This article aims to update the information about kefir and its microbiological composition, biological activity of the kefir's microflora and the importance of kefiran as a beneficial health substance.

Bacillus atrophaeus: main characteristics and biotechnological applications - a review.

The genus Bacillus includes a great diversity of industrially important strains, including Bacillus atrophaeus (formerly Bacillus subtilis var. niger). This spore-forming bacterium has been established as industrial bacteria in the production of biological indicators for sterilization, in studies of biodefense and astrobiology methods as well as disinfection agents, in treatment evaluation and as potential adjuvants or vehicles for vaccines, among other applications. This review covers an overview of the fundamental aspects of the B. atrophaeus that have been studied to date. Although the emphasis is placed on recent findings, basic information's such as multicellularity and growth characteristics, spore structure and lifecycle are described. The wide biotechnological application of B. atrophaeus spores, including vegetative cells, is briefly demonstrated, highlighting their use as a biological indicator of sterilization or disinfection.

Life cycle and spore resistance of spore-forming Bacillus atrophaeus.

Bacillus endospores have a wide variety of important medical and industrial applications. This is an overview of the fundamental aspects of the life cycle, spore structure and factors that influence the spore resistance of spore-forming Bacillus. Bacillus atrophaeus was used as reference microorganism for this review because their spores are widely used to study spore resistance and morphology. Understanding the mechanisms involved in the cell cycle and spore survival is important for developing strategies for spore killing; producing highly resistant spores for biodefense, food and pharmaceutical applications; and developing new bioactive molecules and methods for spore surface display.

Effect of forced aeration on citric acid production by Aspergillus sp. mutants in SSF.

Citric acid (CA) is one of the most important products of fermentation in the world. A great variety of agro-industrial residues can be used in solid state fermentation. Aspergillus niger parental strain (CCT 7716) and two strains obtained by mutagenesis (CCT 7717 and CCT 7718) were evaluated in Erlenmeyer flasks and glass columns using citric pulp (CP) as substrate/support, sugarcane molasses and methanol. Best results using glass columns (forced aeration) were found in the fourth day of fermentation: 278.4, 294.9 and 261.1 g CA/kg of dry CP with CCT 7716, CCT 7718 and CCT 7717, respectively. In Erlenmeyer flasks (aeration by diffusion) CA reached 410.7, 446.8 and 492.7 g CA/kg of dry CP with CCT 7716, CCT 7718 and CCT 7717, respectively. The aeration by diffusion improved CA production by the three strains. A data acquisition system specially developed for biotechnological processes analysis was used to perform the respirometric parameters measurement.

Glycerol-based sterilization bioindicator system from Bacillus atrophaeus: development, performance evaluation, and cost analysis.

The development of new value-added applications for glycerol is of worldwide interest because of the environmental and economic problems that may be caused by an excess of glycerol generated from biodiesel production. A novel use of glycerol as a major substrate for production of a low-cost sterilization biological indicator system (BIS; spores on a carrier plus a recovery medium) was investigated. A sequential experimental design strategy was applied for product development and optimization. The proposed recovery medium enables germination and outgrowth of heat-damaged spores, promoting a D (160 °C) value of 6.6 ± 0.1 min. Bacillus atrophaeus spores production by solid-state fermentation reached a 2.3 ± 1.2 × 10(8) CFU/g dry matter. Sporulation kinetics results allowed this process to be restricted in 48 h. Germination kinetics demonstrated the visual identification of nonsterile BIS within 24 h. Performance evaluation of the proposed BIS against dry-heat and ethylene oxide sterilization showed compliance with the regulatory requirements. Cost breakdowns were from 41.8 (quality control) up to 72.8 % (feedstock). This is the first report on sterilization BIS production that uses glycerol as a sole carbon source, with significant cost reduction and the profitable use of a biodiesel byproduct.

Study of the influence of sporulation conditions on heat resistance of Geobacillus stearothermophilus used in the development of biological indicators for steam sterilization.

Biological indicators are important tools in infection control via sterilization process monitoring. The use of a standardized spore crop with a well-defined heat resistance will guarantee the quality of a biological indicator. Ambient factors during sporulation can affect spore characteristics and properties, including heat resistance. The aim of this study is to evaluate the main sporulation factors responsible for heat resistance in Geobacillus stearothermophilus, a useful biological indicator for steam sterilization. A sequence of a three-step optimization of variables (initial pH, nutrient concentration, tryptone, peptone, beef extract, yeast extract, manganese sulfate, magnesium sulfate, calcium chloride and potassium phosphate) was carried out to screen those that have a significant influence on heat resistance of produced spores. The variable exerting greatest influence on G. stearothermophilus heat resistance during sporulation was found to be the initial pH. Lower nutrient concentration and alkaline pH around 8.5 tended to enhance decimal reduction time at 121 °C (D(121°C)). A central composite design enabled a fourfold enhancement in heat resistance, and the model obtained accurately describes positive pH and negative manganese sulfate concentration influence on spore heat resistance.

Relations between phenotypic changes of spores and biofilm production by Bacillus atrophaeus ATCC 9372 growing in solid-state fermentation.

Bacillus spp. spores are usually obtained from strains cultivated in artificial media. However, in natural habitats, spores are predominantly formed from bacteria present in highly surface-associated communities of cells. Solid-state fermentation (SSF) is the culture method that best mimetizes the natural environment of many microorganisms that grow attached to the surface of solid particles. This study aims to confirm that sporulation through SSF of Bacillus atrophaeus occurs by biofilm formation and that this model of fermentation promotes important phenotypic changes in the spores. Sporulation on standard agar and by SSF with sand and sugarcane bagasse as support was followed by a comparative study of the formed spores. Growth characteristics, metabolic and enzymatic profiles confirmed that sporulation through SSF occurs by biofilm formation promoting important phenotypic changes. It was possible to demonstrate that spores coat had different structure and the presence of ridges only on SSF spores' surface. The sporulation conditions did not affect the dry-heat spore resistance. The type of support evaluated also influenced in the phenotypic alterations; however, the used substrates did not cause interference. This work provides novel information about B. atrophaeus response when submitted to different sporulation conditions and proposes a new concept about bacterial biofilm formation by SSF.

Development of a low-cost sterilization biological indicator using Bacillus atrophaeus by solid-state fermentation.

The production of biological indicators involving bacterial sporulation and multi-step downstream processes has been described. The goal of the present work was to use fermented material as the final product in a biological indicator, thereby reducing processing steps and costs. The performance of three different inexpensive supports (vermiculite, sand, and sugarcane bagasse) was assessed by determining Bacillus atrophaeus sporulation during solid-state fermentation and by assessing the direct use of the fermentation products in the subsequent steps of the process. All three supports allowed spore production of between 10(7) and 10(9) CFU g(-1). Sand proved to be the best inert support enabling the direct use of the fermented product due to its easy homogenization, filling properties, and compatibility with recovery medium. Bacterial adhesion to the sand surface was supported by biofilm formation. The resistance to sterilization of the dried fermentation product was evaluated. For dry-heat resistance (160°C), the D value was 6.6 min, and for ethylene oxide resistance (650 mg/L), the D value was 6.5 min. The cost reduction of this process was at least 48%. No previous studies have been published on the application of sand as a support in solid-state fermentation for the production of biological indicators.

Use of soybean vinasses as a germinant medium for a Geobacillus stearothermophilus ATCC 7953 sterilization biological indicator.

A novel low-cost medium was developed from by-products and wastes from the ethanol agro-industry to replace commercial media in the production of a steam sterilization biological indicator (BI). Various recovery media were developed using soybean or sugarcane molasses and vinasse to prepare a self-contained BI. Media performance was evaluated by viability and heat resistance (D(121 °C) value) according to regulatory standards. A medium produced with a soybean vinasse ratio of 1:70 (1.4%) (w/v) produced the results, with D(121 °C)=2.9±0.5 min and Usk=12.7±2.1 min. The addition of 0.8% (w/v) yeast extract improved the germination of heat-damaged spores. The pH variation from 6.0 to 7.3 resulted in a gradual increase in the D(121 °C) value. The absence of calcium chloride resulted in a decrease in germination, while no significant differences were observed with starch addition. Soybean vinasses may thus be used as the main component of a culture medium to substitute for commercial media in the production of self-contained biological indicators. The use of ethanol production waste in this biotechnological process realized a reliable performance, minimized the environmental impact, and decreased BI production costs while producing a high quality product.

Application of the biorefinery concept to produce L-lactic acid from the soybean vinasse at laboratory and pilot scale.

Lactic acid is a product that finds several applications in food, cosmetic, pharmaceutical and chemical industries. The main objective of this work was the development of a bioprocess to produce L(+)-lactic acid using soybean vinasse as substrate. Among ten strains, Lactobacillus agilis LPB 56 was selected for fermentation, due to its ability to metabolize the complex oligosaccharides. Fermentation was conducted without need for supplementary inorganic nitrogen sources or yeast extract. Kinetic and yield parameters determined at laboratory scale were 0.864 and 0.0162 for YP/S and YX/S, 0.0145 g/L h (rx), 1.32 g/L h (rs) and 1.13 g/L h (rp). The use of vinasse enriched with soybean molasses provided higher lactic acid concentration (138 g/L), the best proportion of inoculum being 25% (v/v). After scale-up to a pilot plant, kinetic and yield parameters were 0.849 and 0.0353 for YP/S and YX/S, 0.0278 g/L h (rx), 0.915 g/L h (rs) and 0.863 g/L h (rp).

Bioindicator production with Bacillus atrophaeus' thermal-resistant spores cultivated by solid-state fermentation.

Bacillus atrophaeus' spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost-productivity relation. Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental designs and response surface methodology in which the influence of different variables--particle size, moisture content, incubation time, pH, inoculum size, calcium sources, and medium composition--was studied. The results have suggested that soybean molasses and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time. Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%), initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the control and no significant difference in dry heat resistance was observed.

Selection and optimization of Bacillus atrophaeus inoculum medium and its effect on spore yield and thermal resistance.

Bacillus atrophaeus's spores are used as biological indicators to monitor sterilization processes and as a Bacillus anthracis surrogate in the development and validation of biosafety methods. The regular use of biological indicators to evaluate the efficiency of sterilization processes is a legal requirement for health services. However, its high cost hinders its widespread use. Aiming at developing a cost-effective inoculum medium, soybean molasses and nutrient-supplemented vinasse were evaluated for their effectiveness in solid-state fermentation (SSF). In biomass production, the results demonstrated that all tested compositions favor growth by providing the nutritional demands of the microorganism. Optimum casein peptone and soybean molasses concentration (1.0%, 2.5%, or 4.0%) was determined by a 2((2-0)) factorial experimental design. The results have showed a positive influence of peptone on biomass production. In order to define peptone final concentration (4.0% or 6.0%), a 2(2) factorial experimental design was used. An optimized medium containing 4.0% soybean molasses and 4.0% casein peptone was similar in performance to a synthetic control medium (tryptone soy broth) in dry-heat thermal-resistant spore production by SSF. An experiment performed under optimum SSF conditions resulted in 1.9 x 10(10) CFU g(-1) dry matter with D (160 degrees C) = 5.2 +/- 0.2 min.