Microalgae is a sustainable protein source that has been widely applied in animal feeds, functional foods, pharmaceutical, and cosmeceutical industries. Waste products could be a potential cost-saving and nutrient-rich substrate in the cultivation of microalgae for protein production. This study aims to investigate the cultivation condition of Haematococcus pluvialis for protein synthesis using synthetic brewery wastewater (BW). H. pluvialis was cultivated in the Bold's Basal Medium (BBM) mixed with synthetic BW at different concentrations. Various cultivation conditions including brewer's spent grain hydrolysate (BSGH) concentrations, pH, and light sources were studied. The molecular weight, amino acids profile and antioxidant activity of synthesized protein were determined. Fed-batch cultivation using different percentages of fresh medium replacement for enhancing protein production was investigated. The 20% fed-batch cultivation reached 27 × 105 ± 0.42 cells/mL, and 4-fold of the protein content of 64.93 ± 5.30% of dry weight was recorded on day-13. Seven essential amino acids (lysine, threonine, histidine, phenylalanine, isoleucine, leucine, methionine) were produced in fed-batch cultivation. Red LED obtained the highest DPPH radical scavenging activity of 27.47 ± 0.98%. The findings suggested that BW is a promising substrate in the cultivation of H. pluvialis to commercially produce protein for numerous industrial applications.

In various natural ecosystems, bacteria most often live in a sessile state enchased in a self-produced extracellular matrix forming biofilms. Due to their either negative or positive impact on different aspects of our daily life, the number of studies devoted to biofilms is increasing. Most research is based on biofilms formed by a single bacterial species. These simple models allowed the understanding of the mechanisms involved in biofilms formation and regulation. This likewise helped the development of several means to control the biofilms formation. However, these models do not closely mimic the natural biofilms known as biochemically and microbiologically heterogeneous and dynamic structures. For this reason, current studies focus more on multispecies biofilms using complex models to best approximate the natural environment. In this review, we addressed on available examples of multispecies biofilms in different domains to illustrate the complexity and organization of life within a consortium. Finally, we review the most used analytical techniques to study multispecies biofilms highlighting the need of multi-scale strategies to better decipher this complex lifestyle.

Astaxanthin (AX) is a potent antioxidant with increasing biotechnological and commercial potential as a feed supplement, and gives salmonids and crustaceans their attractive characteristic pink color. The red yeast Phaffia rhodozyma naturally produces AX as its main fermentation product but wild-type strains and those previously generated through classical random mutagenesis produce low yields of AX. Existing strains do not meet commercial economic requirements, fundamentally due to a lack of understanding of the underlying mechanisms and genotype-phenotype associations regarding AX production in P. rhodozyma. In the present study, screening of P. rhodozyma CBS 6938 mutant strains generated through chemical and ultra violet radiation mutagenesis delivered increased AX production yields that were then maximized using culture media optimization and fed-batch culture kinetic modeling. The whole genomes of the wild-type and eight increased production strains were sequenced to identify genomic changes. The selected strains produced 50-fold more AX than the wild-type strain with a total biomass of around 100 gDCW/L and a carotenoid production of 1 g/L. Genomic variant analyses found 368 conserved mutations across the selected strains with important mutations found in protein coding regions associated with regulators and catalysts of AX precursors in the mevalonate pathway, the electron transport chain, oxidative stress mechanisms, and carotenogenesis.

Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, "yeast-like" ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5'-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.

Process sustainability of biocatalytic processes is significantly empowered with the use of continuous-flow technologies that offer high productivity, minimal wastes and low volumetric consumption. Combining microreactor design with 3D printing technology can broaden the engineering potentials. This work proposes a protocol to modify the surface of 3D-printed PLA scaffolds, based on chitosan deposition. Mimicking the concept of microplates, multi-well plates were designed to facilitate parameter testing. Immobilization of laccase from Trametes versicolor was successfully performed, while chitosan and cross-linker concentration and incubation time were optimized. Τhe developed protocol was applied for the continuous flow bioconversion of hydroxyyrosol, yielding a TTN of 438.6 × 103 for a total of 10 h continuous use. Also, a peristaltic flow pattern seemed to favor the system performance, reaching 95% bioconversion efficiency in a total of 1 h reaction time. The potential of the developed system was further evaluated for the biotransformation of different biophenols from dietary sources, proving the efficiency of the system as a versatile biotechnological tool.

Acetobacter pasteurianus is an excellent cell factory for production of highly-strength acetic acid, and attracts an increasing attention in metabolic engineering. However, the available well-characterized constitutive and inducible promoters are rather limited to adjust metabolic fluxes in A. pasteurianus. In this study, we screened a panel of constitutive and acid stress-driven promoters based on time-series of RNA-seq data and characterized in A. pasteurianus and Escherichia coli. Nine constitutive promoters ranged in strength from 1.7-fold to 100-fold that of the well-known strong promoter Padh under non-acetic acid environment. Subsequently, an acetic acid-stable red fluorescent visual reporting system was established and applied to evaluate acid stress-driven promoter in A. pasteurianus during highly-acidic fermentation environment. PgroES was identified as acid stress-driven strong promoters, with expression outputs varied from 100% to 200% when acetic acid treatment. To assess their application potential, ultra-strong constitutive promoter Ptuf and acid stress-driven strong promoter PgroES were selected to overexpress acetyl-CoA synthase and greatly improved acetic acid tolerance. Notably, the acid stress-driven promoter displayed more favorable for regulating strain robustness against acid stress by overexpressing tolerance gene. In summary, this is the first well-characterized constitutive and acid stress-driven promoter library from A. pasteurianus, which could be used as a promising toolbox for metabolic engineering in acetic acid bacteria and other gram-negative bacteria.

The microbiologically induced calcite precipitation (MICP) can be an emerging approach that could tap onto soil bacterial diversity and use as a bioremediation technique. Based on the concept that bacteria with biomineralization capacity could be effective CaCO3 inductance agents, this study aimed to evaluate the simultaneous influence of 11 operational and environmental factors on the MICP process, for the first time. Therefore, Bacillus muralis, B. lentus, B. simplex, B. firmus, and B. licheniformis, isolated from alkaline soils, were used in the selection of the best performing bacterium compared with a well-known MICP bioagent Sporosarcina pasteurii DSM 33. Plackett-Burman's experimental design was labouring to screen all independent variables for their significances on five outputs (pH value, number of viable cells and spores, amount of urea and CaCO3 precipitate). According to experimentally obtained data, an artificial neural network model based on the Broyden-Fletcher-Goldfarb-Shanno algorithm showed good prediction capabilities, while differences in the relative influences were observed at the bacterial strain level. B. licheniformis turn out to be the most potent bioagent, with a maximum amount of CaCO3 precipitate of 3.14 g/100 mL in the optimal conditions.

Soil cementation based on microbially induced calcite precipitation (MICP) technology is a new research hotspot in the field of biogeotechnical engineering. However, due to the presence of only calcium bonds among particles, MICP-cemented soil shows obvious brittleness, which is the bottleneck of the application of MICP technology in geotechnical engineering. MICP-bonding technology can improve the shortcoming of high brittleness of the calcium bond by increasing the plastic bond among matrix particles. Based on MICP-bonding technology, this study isolated a high-efficiency strain named XR1#, which has the ability to produce both urease and extracellular polysaccharide. Gene sequencing identified the strain as Bacillus oceanicus. The superiority of XR1# bacteria is its ability to induce mineralization and polysaccharide production at the same time. Through the combination of calcium carbonate crystal filling and extracellular polysaccharide bonding, the strength and ductility of the sand column were improved, and the brittle characteristics of traditional MICP technology were reduced. By studying the effect of nutrient elements of the culture medium on calcium precipitation and polysaccharide yield, the two metabolites of XR1# bacteria could be artificially regulated. In an experiment with microbial-cemented fine sand, compared with the traditional Sporosarcina pasteurii, XR1#-cemented sand columns had high production and uniform distribution of calcium carbonate, high strength, and good ductility, and showed obvious advantages. This study provides a new strain with high efficiency and multiple functions for MICP-bonding technology and a new research idea for reducing MICP brittleness.

The nanobiotechnology, one heck of a biotechnology approach, is considered as a focal point in drug delivery systems. Although, plenty of novel approaches are reported in this area, it tackles with strict challenged which confine its broad application. Therefore, there is a need to dig deep into it along with all details. Here, a novel nanobiotechnology approach is described in detail for synthesis and characterization of phase shift dextran stabilized nanodroplets for ultrasound-induced cancer therapy. The aim of this work was to study the effect of parameters including homogenization speed and formulation of dextran and surfactant concentrations on particle size, entrapment efficiency and drug release kinetics of dextran stabilized perfluorohexane nanodroplets containing doxorubicin (Dox) drug. The obtained results showed that the particle size and encapsulation efficiency significantly increased by increasing polymer concentration. In addition, by increasing the homogenization speed, the particle size was increased while entrapment efficiency was decreased. On the other hand, by increasing the surfactant concentration, the particle size and entrapment efficiency reached to optimum values of 47.2 nm and 80.2%, respectively. In vitro release profile of doxorubicin was an apparently biphasic release process and 7-13% of drug was released after 24 h incubation in PBS with pH of 5.5, depending on the nanodroplets (ND) composition. Ultrasound exposure for 10 min resulted in triggered release of 82.95% of Dox from optimal formulation of sample C3 (0.1%w/v dextran, homogenization speed of 24000 rpm and Dox content of 500 µg).