Fernando Méndez-González

Water hyacinth is an invasive weed that can valorize through the production of hydrolytic enzymes via solid-state culture. This study explores the application of Trichoderma harzianum in producing xylanases and endoglucanases on water hyacinth beds. Laboratory-scale packed-bed column bioreactors (PBCBs) with a capacity of 8 grams of dry mass (g_dm) were used to evaluate the effects of temperature (28–36 °C) and initial moisture content (65–80%) on microbial growth and enzyme production. High yields of biomass and enzymes were produced at 30 °C. Moreover, xylanase activity was enhanced in cultures with a moisture content of 65% (~71.24 U/g_dm), and endoglucanase activity at 75–80% moisture (~20.13 U/g_dm). The operational conditions identified for xylanase production were applied to 6 L bench-scale cross-flow internally stirred bioreactors, packed to 40% capacity with 450 g_dm. Two stirring regimes were tested: intermittent and continuous. The results showed that continuous stirring promotes both microbial growth and xylanase activity. In fact, xylanase activity in continuous stirring conditions was comparable to that achieved in PBCBs. Consequently, continuous stirring enables a 56-fold increase in bioreactor capacity without compromising xylanase production. The approaches developed in this study can support the design of large-scale bioprocesses for the valorization of water hyacinth.

Accurate quantification of spore concentration is essential in producing, analyzing, and applying diverse biotechnological products. Typically, spore concentration is estimated using a Neubauer hemocytometer, which is labor-intensive, time-consuming, and often imprecise. Methods based on spore optical properties can be explored as an alternative to using the Neubauer hemocytometer, improving the practicality and precision of analysis. This study aimed to establish the analysis conditions for quantifying the spore concentration using spectrophotometry and colorimetry (using L*a*b color space). For this study, spores from entomopathogenic fungi Beauveria bassiana, Hirsutella thompsonii, Cordyceps fumosorosea, Cordyceps javanica, Akanthomyces dipterigenus, Metarhizium acridum, Metarhizium anisopliae, and Metarhizium robertsii were produced and analyzed. The results indicate that both spectrophotometry and colorimetry analysis accurately estimate spore concentration. The quantification ranges achieved with both methods directly depend on the spore volume. Spores with a 15 µm³ or less volume exhibit a wider quantification range than those with larger volumes. Moreover, the L*a*b* color space analysis allows quantifying spores in a concentration range from 4.5 × 10⁶ to 3 × 10⁸ spores/mL, which is wider than the range obtainable through spectrophotometry. This extended concentration range provided by colorimetry offers a practical advantage for producing and applying entomopathogenic fungi spores.

Food production and processing generates a large amount of waste. A small fraction of agricultural waste is used locally to produce composts and biofuels. However, agricultural waste can be used as substrates in solid-state culture (SSC) processes to obtain biopesticides, biofertilizers, enzymes, and biosurfactants. These products are obtained in bioreactors with different configurations, ranging from laboratory-scale to industrial-scale. The adaptation and implementation of these biotechnological processes allow the comprehensive use of agricultural waste, contributing to agricultural sustainability. Therefore, this chapter analyzes the bioreactors, microorganisms, and agricultural waste used to produce biopesticides, biofertilizers, enzymes, and biosurfactants by SSC.

Corn, a widely cultivated cereal, is a rich source of several antioxidant compounds, including phenolic compounds. Some of these compounds are bound within the components of the corn cell wall, requiring specific treatments to release them. Solid-state fermentation (SSF) using Rhizopus oryzae enhanced the antioxidant capacity and phenolic content in legumes and grains. However, there is limited information regarding its application in blue corn. This study aimed to evaluate the effect of R. oryzae growth on the phenolic content and antioxidant capacity of blue corn kernels. The results demonstrated that R. oryzae used blue corn kernels for growth, which led to an increase in free phenolic compounds, reaching their highest concentration at 60 h of culture. Compared to the unfermented control (0 h), SSF increased by up to 161% in total phenolic content and a 94% increase in antioxidant capacity. The main phenolic compounds identified in the blue corn extracts at 60 h using RP-HPLC-ESI-MS included caffeic acid 4-O-glucoside, rosmanol, p-coumaroyl tartaric acid glucosidic ester, 5-heptadecylresorcinol, and caffeoyl tartaric acid. The SSF represents an eco-friendly method for enhancing the antioxidant capacity and phenolic compounds of blue corn grain.

The transition to sustainable agriculture requires strategies that replace the use of harmful chemical substances for human health and the environment. Among them is the production and application of microorganisms with the capacity to agricultural pest populations control. Among these microorganisms are species of filamentous fungi capable of successfully combating the disappearance of insects, weeds, and phytopathogenic fungi. However, the industrial production of high-quality propagules is considered the bottleneck that limits meeting the demand for pesticides. Due to their resistance to environmental conditions and infective capacity, conidia are the preferred 228propagules to apply in the field. The conidia are produced by solid-state culture in bioreactors with different configurations and technological degrees. Bioreactors with a low level of technology, such as plastic bags, present a simple and low-cost process but with some reproducibility problems. On the contrary, bioreactors with trays, columns, and shakers allow a controlled process, which improves the production, productivity, and quality of the conidia. Therefore, in this chapter, the characteristics of some fungal classifications used in biological control and the different types of bioreactors involved in propagule production will be analyzed.

Pistachio green hull (PGH) represents the non-edible fraction obtained after the seed is harvested and is an important source of phenolic compounds. Solid-state fermentation (SSF) is a viable biotechnological and economical technique for extracting phenolic compounds. This study aimed to evaluate the SSF with Aspergillus niger GH1 to recover total phenolic compounds (TPC) with antioxidant capacity (AC) from PGH. For this, the time of higher TPC and AC (DPPH [2,2-diphenyl-1-picrylhydrazyl], ABTS [2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate)], FRAP [ferric reducing antioxidant power]) was selected. Then, moisture, inoculum concentration, and aeration rate were evaluated. A. niger GH1 was able to grow and colonize the PGH, with the higher value of TPC (23.83 mg/g of dry mass (gdm)) obtained after 24 h of culture, which significantly correlated with AC (Pearson’s R = 0.69). Moisture and aeration rate were the main factors influencing TPC. The highest values for both TPC and AC were achieved in treatment 8 (60% moisture, 5 × 10⁶ spores/mL, and 1 L/Kgwm min), resulting in a 129% and 1039% increase, respectively. Gallic acid 4-O-glucoside and geranine were identified in the PGH extracts using high-performance liquid chromatography coupled with mass spectrometry. The SSF provides eco-friendly alternatives for releasing bioactive compounds from PGH, adding value to this waste.

Entomopathogenic fungi (EF) offer a sustainable alternative to chemical substances for combating insects that are harmful to agriculture. The efficacy of these fungi has resulted in a growing demand for products derived from EF propagules. Among these propagules, spores generated through solid-state culture (conidia) have gained popularity due to their unique composition and structures that intensify their effectiveness during application. Simple processes were designed in the mid-20th century to produce conidia using bags or low-capacity containers as bioreactors. These traditional methods remain popular today due to their low investment costs and independence from the technological level of the region. However, the increasing demand for bioinsecticides based on fungal conidia necessitates the implementation of more sophisticated processes with greater capacity and industrialization. For this purpose, trays, packed bed columns, and stirred bioreactors equipped with instrumentation for monitoring and controlling the process can be operated on a large scale. Previous studies have shown that the operating characteristics of these high-tech bioreactors surpass the production and productivity of infectious conidia obtained in traditional bioreactors. However, the bottleneck for implementing high-tech bioreactors in the industry lies in the scale-up, where issues such as overheating and poor gas exchange arise with increasing bioreactor dimensions. The problem to be solved is overheating and poor gas exchange that occur when the dimensions of the bioreactor increase. Nevertheless, significant progress has been made in recent years towards successfully integrating high-tech bioreactors into the industry. Developing these industrialized conidia production processes will facilitate the intensification of sustainable agricultural practices, leading to the production of chemical-free foods.