Production Of Bioproducts Research Articles

Halophilic microalga-based circular economy producing functional food by reclaiming high-salinity seafood processing sewage

The high-salinity and nutrient-rich feature of seafood processing wastewater (SPW) challenges current treatment technologies. Microalgae-based circular economies offer promising solutions to reduce environmental pollution and increase bioproducts production. In this study, we employed industrial capacity demonstrated halophilic microalga Chlorella MEM25 as a model to demonstrate a closed-loop circular bioeconomy. The approach mutually achieves clean water via the reclamation of high-salinity SPW and the production of valuable bioproducts suitable for functional food. With experimentally and computationally optimized parameters, MEM25 grows robustly in SPW-based culture and produces high amounts of essential amino-acid-rich proteins (52.89 ± 0.65%) and polyunsaturated fatty acid-rich lipids (33.20 ± 0.59%), which are ideal ingredients for functional food. Moreover, the removal efficiency of phosphate was as high as 98.21%, further demonstrating the advantages of using microalgae to produce functional food and reclaim SPW as an eco-friendly circular model.

Microalgal co-cultivation -recent methods, trends in omic-studies, applications, and future challenges.

The burgeoning human population has resulted in an augmented demand for raw materials and energy sources, which in turn has led to a deleterious environmental impact marked by elevated greenhouse gas (GHG) emissions, acidification of water bodies, and escalating global temperatures. Therefore, it is imperative that modern society develop sustainable technologies to avert future environmental degradation and generate alternative bioproduct-producing technologies. A promising approach to tackling this challenge involves utilizing natural microbial consortia or designing synthetic communities of microorganisms as a foundation to develop diverse and sustainable applications for bioproduct production, wastewater treatment, GHG emission reduction, energy crisis alleviation, and soil fertility enhancement. Microalgae, which are photosynthetic microorganisms that inhabit aquatic environments and exhibit a high capacity for CO2 fixation, are particularly appealing in this context. They can convert light energy and atmospheric CO2 or industrial flue gases into valuable biomass and organic chemicals, thereby contributing to GHG emission reduction. To date, most microalgae cultivation studies have focused on monoculture systems. However, maintaining a microalgae monoculture system can be challenging due to contamination by other microorganisms (e.g., yeasts, fungi, bacteria, and other microalgae species), which can lead to low productivity, culture collapse, and low-quality biomass. Co-culture systems, which produce robust microorganism consortia or communities, present a compelling strategy for addressing contamination problems. In recent years, research and development of innovative co-cultivation techniques have substantially increased. Nevertheless, many microalgae co-culturing technologies remain in the developmental phase and have yet to be scaled and commercialized. Accordingly, this review presents a thorough literature review of research conducted in the last few decades, exploring the advantages and disadvantages of microalgae co-cultivation systems that involve microalgae-bacteria, microalgae-fungi, and microalgae-microalgae/algae systems. The manuscript also addresses diverse uses of co-culture systems, and growing methods, and includes one of the most exciting research areas in co-culturing systems, which are omic studies that elucidate different interaction mechanisms among microbial communities. Finally, the manuscript discusses the economic viability, future challenges, and prospects of microalgal co-cultivation methods.

Leveraging Milk Permeate Fermentation to Produce Lactose-Free, Low-In-Glucose, Galactose-Rich Bioproducts: Optimizations and Applications

Previous studies highlighted Brettanomyces claussenii as a versatile yeast that produces ethanol or acetic acid from lactose, or selectively metabolizes glucose while leaving behind galactose, depending on different operational conditions. This flexibility enables the production of galactose-rich bioproducts from liquid dairy residues. The purpose of this study is to: (i) optimize the anaerobic fermentation of milk permeate (MP) by B. claussenii to maximize the yields of galactose and ethanol and minimize leftover glucose, and (ii) combine the optimized process with distillation and drying and characterize its multiple products. Response surface methodology was used for the optimization. Three fermentation parameters were chosen as input factors: temperature (25–35 °C), inoculation level (7.0–8.5 log cfu/mL), and time (4–40 days), with three metabolites as responses: galactose, glucose, and ethanol. The optimal combination of parameters resulted in temperature, 28 °C; inoculation level, 7.6 log cfu/mL; and time, 33 days. Under these conditions, the fermented product was predicted to have 63.6 g/L galactose, 4.0% v/v ethanol, and 0 g/L residual glucose. The optimal parameters were used to run 18 L fermentations followed by distillation and freeze-drying. As a result, four product streams were obtained and characterized for relevant physicochemical and nutritional attributes. Our results show that the partial fermentation of MP by B. claussenii can be the first step to develop lactose-free, low-in-glucose, galactose-rich bioproducts, which improve the value of this residue and broaden its applications in the food supply chain.

A critical review of multiple alternative pathways for the production of a high-value bioproduct from sugarcane mill byproducts: the case of adipic acid

Biobased fuels, chemicals, and materials can replace fossil fuel products and mitigate climate change. Sugarcane mills have the potential to produce a wider range of biobased chemicals in a similar approach to bioethanol production, including adipic acid. Multiple alternative pathways for converting simple sugars into adipic acid have been described, with the potential for integration into a sugar mill. The economics and expected greenhouse gas emissions reductions compared to fossil-based adipic acid were investigated in the present study to identify preferred pathways for implementation in sugarcane biorefineries. Nine biobased pathways for adipic acid production were screened for technical performances, resulting in the selection of four preferred options for rigorous comparison, i.e., direct microbial conversion of sugars, and production via cis,cis-muconic acid, glucaric acid, and glycerol as intermediate, obtained from sugars. The minimum selling prices of adipic acid for an attractive return on investment were determined for these pathways, using either A-molasses or a combination of A-molasses and pretreated sugarcane lignocelluloses in biorefineries designed to be energy-self-sufficient. Adipic acid production from A-molasses via cis,cis-muconic acid was the best overall performing scenario with the lowest minimum selling price of USD 2,538/Mt and lowered greenhouse gas emissions (2,325 g CO2 eq/kg wet) compared to fossil-based adipic acid production. The scenarios with combined A-molasses and lignocellulosic feedstock had increased minimum selling prices by 29 to 101% compared to adipic acid production from A molasses via cis,cis-muconic acid.

Natural and recombinant bioactive compounds from Schizochytrium sp.: Recent advances and future prospects

Schizochytrium sp. is a unicellular heterotrophic marine protist, with global distribution used at an industrial scale for saturated fatty acid and polyunsaturated fatty acid production. Diverse methodologies for the genetic transformation of Schizochytrium sp. have been developed to express heterologous proteins, such as through Agrobacterium tumefaciens and electroporation. Culture optimization has been pursued using the nitrogen starvation strategy that shifts the metabolism of Schizochytrium sp. to lipid production, but the over-compensation has been demonstrated could improve biomass and protein biosynthesis. This review paper discusses several studies focused on maximizing the production of high-valued bioproducts, highlighting culture media optimization and genetic/metabolic engineering in Schizochytrium sp. with a biorefinery approach for biopharmaceutical and recombinant protein production.

Bacillus amyloliquefaciens: Harnessing Its Potential for Industrial, Medical, and Agricultural Applications-A Comprehensive Review.

Bacillus amyloliquefaciens, a Gram-positive bacterium, has emerged as a versatile microorganism with significant applications in various fields, including industry, medicine, and agriculture. This comprehensive review aims to provide an in-depth understanding of the characteristics, genetic tools, and metabolic capabilities of B. amyloliquefaciens, while highlighting its potential as a chassis cell for synthetic biology, metabolic engineering, and protein expression. We discuss the bacterium's role in the production of chemicals, enzymes, and other industrial bioproducts, as well as its applications in medicine, such as combating infectious diseases and promoting gut health. In agriculture, B. amyloliquefaciens has demonstrated potential as a biofertilizer, biocontrol agent, and stress tolerance enhancer for various crops. Despite its numerous promising applications, B. amyloliquefaciens remains less studied than its Gram-negative counterpart, Escherichia coli. This review emphasizes the need for further research and development of advanced engineering techniques and genetic editing technologies tailored for B. amyloliquefaciens, ultimately unlocking its full potential in scientific and industrial contexts.

Protein engineering and iterative multimodule optimization for vitamin B6 production in Escherichia coli

Vitamin B6 is an essential nutrient with extensive applications in the medicine, food, animal feed, and cosmetics industries. Pyridoxine (PN), the most common commercial form of vitamin B6, is currently chemically synthesized using expensive and toxic chemicals. However, the low catalytic efficiencies of natural enzymes and the tight regulation of the metabolic pathway have hindered PN production by the microbial fermentation process. Here, we report an engineered Escherichia coli strain for PN production. Parallel pathway engineering is performed to decouple PN production and cell growth. Further, protein engineering is rationally designed including the inefficient enzymes PdxA, PdxJ, and the initial enzymes Epd and Dxs. By the iterative multimodule optimization strategy, the final strain produces 1.4 g/L of PN with productivity of 29.16 mg/L/h by fed-batch fermentation. The strategies reported here will be useful for developing microbial strains for the production of vitamins and other bioproducts having inherently low metabolic fluxes.

Cultivation of Chlorella sorokiniana in indoor and outdoor raceway ponds under glyphosate stress for bioproduct production

Cultivation of Chlorella sorokiniana in indoor and outdoor raceway ponds under glyphosate stress for bioproduct production

New nutrients evaluation in Spirulina maxima growth for phycocyanin, carbohydrate, and biochar production

Purpose The purpose of this study is to investigate the influence of light intensity and sources of carbon and nitrogen on the cultivation of Spirulina maxima. Design/methodology/approach Cultures were carried out in a modified Zarrouk medium using urea, sodium acetate and glycerol. A Taguchi experimental design was used to evaluate the effect on the production of biocompounds: productivities in biomass, carbohydrates, phycocyanin and biochar were analyzed. Findings Statistical data analysis revealed that light intensity and sodium acetate concentration were the most important factors, being significant in three of the four response variables studied. The highest productivities in biomass (46.94 mg.L−1.d−1), carbohydrates (6.11 mg.L−1.d−1), phycocyanin (3.62 mg.L−1.d−1) and biochar (22, 48 mg.L−1.d−1) were achieved in experiment 4 of the Taguchi matrix, highlighting as the ideal condition for the production of biomass, carbohydrates and phycocyanin. Practical implications Sodium acetate and urea can be considered, respectively, as potential sources of carbon and nitrogen to increase Spirulina maxima productivity. From the results, an optimized cultivation condition for the sustainable production of bioproducts was obtained. Originality/value This work focuses on the study of the influence of light intensity and the use of alternative sources of nitrogen and carbon on the growth of Spirulina maxima, as well as on the influence on the productivity of biomass and biocompounds. There are few studies in the literature focused on the phycocyanin production from microalgae, justifying the need to deepen the subject.

Influences of Water Deficit Stress and Symbiosis with Growth-Promoting Bacteria on Seed Biochemistry of Camelina sativa

Camelina sativa, being a flowering plant in the family Brassicaceae, is traditionally cultivated as an oilseed crop due to its exceptionally high level (up to 45%) of omega-3 fatty acids, which is uncommon in vegetable sources. In the recent years camelina has been growing more than before because of its potentiality in the production of biodiesel and bioproduct. An aforementioned experiment was designed in three irrigation levels (100%, 75% and 50% of field capacity) and Micrococcus yunnanensis was used as plant growth-promoting bacteria during the reproductive phase.
 The bacteria were incubated in a nutrient broth medium at the temperature of 28°C for 24h on shaker ceaselessly. When sowing, seeds were treated with 1.0 ml bacteria suspension in the inoculated groups. Water stress was applied to each pot in the levels of 75 and 50 % FC from the budding stage to full maturity in generative phase. Camelina silique yield was harvested at the maturity stage for analysis of seed quality and biochemical responses. Total soluble carbohydrate was extracted thrice from 100 mg of mature seed using extraction soluble including glacial acetic acid, methanol, and water. Oil and protein content were measured using Near-Infrared Reflectance spectrometer. Seed fatty acid contents were determined using gas chromatography. The results showed a significant relation between the highest proportion of fatty acid and the polyunsaturated fatty acid (55.12 to 65.66%) in particular linolenic acid. The increase of polyunsaturated fatty acid and saturated fatty acid was coincided with the decrease of monounsaturated fatty acid under water deficit stress. The application of plant growth-promoting bacteria is proven to increase protein with 50% of field capacity. In general, water deficit stress and plant growth-promoting bacteria have significant effects on the remobilization of nutrients from the soil to developing seed and following metabolism synthesis.

Influence of culture media composition on the rheology of microalgae concentrates on a large scale

The role of microalgae in the production of bioproducts and biofuels, along with their ability to provide a sustainable pathway for wastewater treatment, makes them promising alternatives to conventional processes. Nevertheless, large-scale downstream processing requires an understanding of biomass rheology that needs to be addressed further. This study aimed to characterize microalgal concentrates rheologically in different culture media. The presence of bacteria was quantified by photorespirometry and plate counting techniques. The culture medium was found to significantly influence viscosity, with primary wastewater exhibiting the highest viscosity and seawater plus pig slurry the lowest. The concentration of heterotrophic bacteria was directly related to the viscosity. Extracellular polysaccharides (EPS) in supernatant exhibited an inverse viscosity trend compared to biomass concentrates, with pig slurry cultures having higher concentrations. These findings emphasize the profound influence of culture medium and EPS on the rheology of microalgal biomass, underscoring the need for continued research aimed at facilitating and optimizing large-scale downstream processes within the framework of a circular economy and the attainment of the Sustainable Development Goals (6,8, and 12).

Design a novel internally illuminated mirror photobioreactor to improve microalgae production through homogeneous light distribution

In this study, a novel internally illuminated mirror photobioreactor (IIM-PBR) was designed to improve microalgae biomass production through providing a homogenous light distribution in cultivation medium. The performance of the IIM-PBR was compared with internally illuminated control photobioreactor (IIC-PBR) and externally illuminated control photobioreactor (EIC-PBR) in terms of cell growth, wastewater treatment and bioproducts generation. Compared with the IIC-PBR and EIC-PBR, the IIM-PBR increased microalgae growth rate up to 60 % and 30%, respectively. Municipal wastewater treatment revealed that the IIM-PBR could significantly improve nutrients removal as the final removal efficiencies of 90%, 95% and 90% were obtained for nitrate, phosphate and COD, respectively. Moreover, the IIM-PBR increased the total bioproducts production by 89% and 46% compared to in the IIC-PBR and EIC-PBR, respectively. Based on the energy consumption calculation, the mirror's light-reflective properties of the IIM-PBR resulted in a significant reduction of total energy consumption (∼10 times).

Biological characteristics and ability of Streptomyces rochei BT02 to inhibit the Ralstonia solanacearum causing bacterial wilt disease

In the agricultural field, actinomycetes are outstanding biological control agents to inhibit the growth of pathogenic microorganisms. Based on the manual of bacterial classification of the International Streptomyces Project (ISP) and the 16S-rRNA gene sequence, the actinomycete BT02 strain was named Streptomyces rochei BT02. This strain contains two polyketide synthase type I (pks-I) and polyketide synthase type II (pks-II) genes related to antibiotic synthesis. The result showed that the actinomycetes S. rochei BT02 grow well at 31οC, 1% salt, pH 7.0, and assimilate different carbon and nitrogen sources. The S. rochei BT02 showed antibacterial activity against three tested strains of Ralstonia solanacearum (Minimum inhibitory concentration =15 mg/ml). In greenhouse conditions, this strain reduced 43.5% the wilt disease index. That demonstrated that S. rochei BT02 could be a promising source for the production of bioproducts.

Functional and sequence-based metagenomics to uncover carbohydrate-degrading enzymes from composting samples

The renewable, abundant, and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples’ microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited β-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples.Key points• Sequence- and function-based metagenomics were used to find CAZymes in compost samples.• Thermophilic composts proved to be rich in bacterial GH3, GH5, and GH9 enzymes.• Compost-derived fosmid libraries are enriched in clones with β-glucosidase activity.

Fungal-assisted immobilization of microalgae for simultaneous harvesting and product customization: Effects of geometry, loading, and microalgae concentration

The production of sustainable foods and other bioproducts is expected to increase to achieve key circular bioeconomy goals. In this study, Haematococcus pluvialis microalgae cells were simultaneously harvested and immobilized in the hyphal matrix of the edible fungal strain Aspergillus awamori. The study investigated the impact of fungal loading, pellet geometry, and initial microalgae cell concentration on the immobilization performance and product characteristics. It was found that higher fungal loading and larger fungal pellets contributed to increased immobilization performance. High initial microalgae concentrations led to substantial inhibition of the immobilization behavior, which could be partially overcome by increased specific loading rate of fungi (g/g algae). A proof-of-concept demonstration study of product protein content showed that manipulation of key operational parameters allowed the product composition to be tuned to desired criteria. This study provides new insights to the fungal-assisted microalgae immobilization process and demonstrates a novel application in bioproduct customization.

Spirulina and Its Potential in Bioproduct Production: a Review

The use of the microalgal biomass in biorefineries can produce different products, such as biofuel, food, feed, and bioactive compounds. In addition, the microalgae used in waste treatment can have its biomass exploited, such as Spirulina sp., which shows satisfactory results in the studies performed. Thus, the present study aimed to describe the production of Spirulina bioproducts in biorefineries, demonstrating its benefits and its potential for sustainable development. The study utilized an integrative literature review methodology, and the research question was "What bioproducts can be developed from a Spirulina biorefinery and how can they contribute to sustainability?". The articles were initially screened based on their title and abstract, and only those that fit the research question were selected for full reading. The reviewed studies suggest that the processing of microalgae within biorefineries has significant potential for synergy, sustainable processes can be developed that maximize the use of microalgae biomass and minimize waste and emissions. Spirulina can be used to treat wastewater from food processing plants, where microalgae could absorb nutrients and organic matter while producing biomass that could be harvested and converted into other bioproducts. However, challenges such as high operating costs, variability in biomass and effluent composition, and the need for scalable technologies must be addressed to fully realize the benefits of microalgae in biorefineries. Further research and investment in this area are necessary to develop cost-effective, scalable, and sustainable microalgae biorefineries, involving interdisciplinary collaborations between different fields and partnerships between academia, industry, and government.

Thermosynechococcus sp. CL-1 cultivation for Zn removal in swine wastewater with simultaneous CO2 fixation and macromolecules accumulation

Treatment of swine wastewater via cultivation of photosynthetic microorganism is highly promising due to the benefits in nutrient utilization, cost reduction, high efficiency, and production of various bioproducts. Besides, photosynthetic microorganisms are also acknowledged for their high tolerance toward extreme growth conditions, including the presence of heavy metals. This study assessed the tolerance of Thermosynechococcus sp. CL-1 (TCL-1) in the cultivation using swine wastewater for CO2 fixation, Zn removal, and production of lipid, protein, and carbohydrate. Addition of 113.2 mM dissolved inorganic carbon was also set for simulation of almost saturated CO2 content in a chemical scrubbing wastewater after CO2 absorption process. Experiment was conducted in a 1 L flat panel photobioreactor for 12 h periods, under illumination of various light intensities, and compared with the performance in modified Fitzgerald medium. TCL-1 cultivation under 1000 μE/m2/s light intensity provided the optimum growth condition in swine wastewater which led to high biomass productivity (55.69 mg/L/h) and CO2 fixation rate (91.8 mg/L/h). TCL-1 in this cultivation also removed 62.5 % Zn content from swine wastewater and bioadsorption route was detected as the major elimination process. The rapid utilization of nitrogen compounds in swine wastewater resulted in the dominant production of protein reached to 59.5 ± 3.1%dwc.

Trends on Chlamydomonas reinhardtii growth regimes and bioproducts.

The green microalga Chlamydomonas reinhardtii is a model microorganism for several areas of study. Among the different microalgae species, it presents advantageous characteristics, such as genomes completely sequenced and well-established techniques for genetic transformation. Despite that, C. reinhardtii production is still not easily commercially viable, especially due to the low biomass yield. So far there are no reports of scientometric study focusing only on C. reinhardtii biomass production process. Considering the need for culture optimization, a scientometric research was conducted to analyze the papers that investigated the growth regimes effects in C. reinhardtii cultivation. The search resulted in 130 papers indexed on Web of Science and Scopus platforms from 1969 to December 2022. The quantitative analysis indicated that the photoautotrophic regime was the most employed in the papers. However, when comparing the three growth regimes, the mixotrophic one led to the highest production of biomass, lipids, and heterologous protein. The production of bioproducts was considered the main objective of most of the papers and, among them, biomass was the most frequently investigated. The highest biomass production reported among the papers was 40gL-1 in the heterotrophic growth of a transgenic strain. Other culture conditions were also crucial for C. reinhardtii growth, for instance, temperature and cultivation process.

Assessing the potential of Chlorella sp. phycoremediation liquid digestates from brewery wastes mixture integrated with bioproduct production.

Digestates from different anaerobic digesters are promising substrates for microalgal culture, leading to effective wastewater treatment and the production of microalgal biomass. However, further detailed research is needed before they can be used on a large scale. The aims of this study were to investigate the culture of Chlorella sp. in DigestateM from anaerobic fermentation of brewer's grains and brewery wastewater (BWW) and to explore the potential use of the biomass produced under different experimental conditions, including diverse cultivation modes and dilution ratios. Cultivation in DigestateM initiated from 10% (v/v) loading, with 20% BWW, obtained maximum biomass production, reaching 1.36gL-1 that was 0.27gL-1 higher than 1.09gL-1 of BG11. In terms of DigestateM remediation, the maximum removal of ammonia nitrogen (NH4 +-N), chemical oxygen demand, total nitrogen, and total phosphorus reached 98.20%, 89.98%, 86.98%, and 71.86%, respectively. The maximum lipid, carbohydrate, and protein contents were 41.60%, 32.44%, and 27.72%, respectively. The growth of Chlorella sp. may be inhibited when the Y(II)-Fv/Fm ratio is less than 0.4.

Yield, Chemical Composition and Bioactivity of Essential Oils from Common Juniper (Juniperus communis L.) from Different Spanish Origins.

Essential oils (EOs) obtained from Juniperus communis L. are frequently used in the production of bioproducts. However, there are no studies regarding industrial crops' production, allowing for better control of the quality and production of juniper EOs. To select the plant material for developing future crops of this species in northern Spain, four locations where this shrub species grows in the wild were selected and samples of both genera were collected. The EOs were obtained by steam distillation, and their chemical composition and bioactivity were evaluated. The results showed that the yield of EOs from male and female samples were within the usual reported ranges, varying between 0.24 and 0.58% (dry basis, d.b.). However, limonene content in three locations varied between 15 and 25%, which is between 100% and 200% higher than the values usually reported for other European countries. The antibacterial activity was determined by broth microdilution and showed that gram-positive bacteria were more susceptible to the tested EOs since, in general, lower MIC values were obtained compared to gram-negatives. The EOs from location 1 (L1F) and 2 (L2M) inhibited the growth of six out of the eight clinical strains tested. Samples from location 1 were particularly effective, exhibiting MBC against two gram-negative (E. coli and P. mirabilis) and one gram-positive bacteria (E. faecalis). Moreover, the majority of the EOs tested showed anti-inflammatory activity. Cytotoxic effect has been demonstrated in tumor cell lines, with the best results observed against gastric carcinoma (AGS) cells (GI50 between 7 to 77 µg/mL). Although generally presenting higher GI50, most samples also inhibited the growth of non-tumoral cells, particularly hepatocytes (PLP2 cells). Therefore, its use for their anti-proliferative activity should consider specific conditions to avoid damaging normal cells. Finally, the results and conclusions obtained led to the selection of the female shrubs from location 1 (L1F) as the plant material to be propagated in order to produce plants for a future juniper crop.