Fast-growing Strains Research Articles

Evolutionary engineering of methylotrophic E. coli enables fast growth on methanol

As methanol can be derived from either CO2 or methane, methanol economy can play an important role in combating climate change. In this scenario, rapid utilization of methanol by an industrial microorganism is the first and crucial step for efficient utilization of the C1 feedstock chemical. Here, we report the development of a methylotrophic E. coli strain with a doubling time of 3.5 hours under optimal conditions, comparable or faster than native model methylotrophs Methylorubrum extorquens AM1 (Td~4hr) and Bacillus methanolicus at 37°C (Td~5hr). To accomplish this, we develop a bacterial artificial chromosome (BAC) with dynamic copy number variation (CNV) to facilitate overcoming the formaldehyde-induced DNA-protein cross-linking (DPC) problem in the evolution process. We track the genome variations of 75 cultures along the evolution process by next-generation sequencing, and identified the features of the fast-growing strain. After stabilization, the final strain (SM8) grows to 20 g/L of cell mass within 77 hrs in a bioreactor. This study illustrates the potential of dynamic CNV as an evolution tool and synthetic methylotrophs as a platform for sustainable biotechnological applications.

Microbial Community Structure in the Taklimakan Desert: The Importance of Nutrient Levels in Medium and Culture Methods.

Although the Taklimakan Desert lacks the necessary nutrients and conditions to support an extensive ecosystem, it is a treasure trove of extremophile resources with special structures and functions. We analyzed the bacterial communities using oligotrophic medium and velvet cloth replicate combined with an extended culture duration. We isolated numerous uncultured microorganisms and rare microorganisms belonging to genera not often isolated or recently described, such as Aliihoeflea, Halodurantibacterium, and Indioceanicola. A total of 669 strains were isolated from the soil of the Taklimakan Desert, which were classified into 5 phyla, 7 classes, 25 orders, 42 families, 83 genera, and 379 species. Among them, 148 strains were potential new species. Our data show that even when working with samples from extreme environments, simple approaches are still useful for cultivating stubborn microbes. Through comparing the isolation effects of different nutrient levels on microbial diversity and abundance, the results show that reducing the nutrient level of the medium was more conducive to improving the culturability of microorganisms in low-nutrient environments, while the high-nutrient medium was more suitable for the isolation of dominant fast-growing strains. This study helps to better reflect the diversity of microbial resources and lays a foundation for the further research and utilization of soil microbial resources in the Taklimakan Desert.

Comparative growth, survival, and feed conversion factor of the F1 generation of two new strains of tilapia Oreochromis niloticus produced by mass selection

Growth, survival, and use of feed of the F1 generation of two new strains of tilapia Oreochromis niloticus (GenBank Strain Y = OK078792 to OK078796 and Strain A = OK078797 to OK078807) produced by mass selection was compared with a known fast-growing commercial strain in Mexico (GenBank Strain Q = OR744820 to OR744838), during a grow-out phase of 165 days. Genetic variability including observed (Ho) and expected (He) heterozygosity, inbreeding coefficient (Fis), and paired comparisons of strain fixation index (Fst) of the three strains were also compared, and an additional analysis of molecular variance (AMOVA) was performed. Twelve hapas of 8.2 m3 (four for each strain) were stocked at a density of 25 org m−3 of 4.58 ± 0.67 g, which were fed with 45 to 25% crude protein feed under a dynamic feeding scheme of 8 to 3% live weight. Growth was estimated by growth rates in weight and length, and by the weight-specific growth rate (SGR). The use of feed was estimated by the total feed consumption rate and the feed conversion factor (FCF). Results indicate no significant differences (P > 0.05) in growth parameters of the three strains, except in SGR which was significantly higher in the control strain (Q) (2.75% day−1) as compared with strains Y and A (2.51 and 2.59% day−1 respectively). The average final weight and size of the fish were 314.6 ± 11.8 g and 25.5 ± 0.4 cm respectively. No significant differences (P > 0.05) were found in survival and feed utilisation among strains, with average values of 96.4% survival, feed consumption of 3.91 g day−1, and FCF of 2.08. Results of genetic diversity indicate that there were no significant differences (P > 0.05) in Ho and He of the three strains, with average values of 0.310 and 0.280 respectively. Fis values were negative and similar for the three strains, with a mean value of −0.109 which indicates no consanguinity in their populations. Finally, the paired comparisons of Fst were significantly different (P < 0.001), showing relatively low values but different from 0 (Y vs. A = 0.025, A vs. Q = 0.041 and Y vs. Q = 0.055), indicating slight but significant population differentiation between strains. In conclusion, the performance and genetic variability of the F1 generation of the two new strains suggest that valuable broodstock is now available for mass production of fry, with similar performance as to one of the best strains of tilapia in Mexico.

On farm Performance Evaluation of Exotic Chickens in Central Tigray, Northern Ethiopia

The study was carried out on two purposively selected districts of central zone of Tigray, viz., Mereb Leke and Tahtay Michew. The aim of the study was to evaluate the on-farm performance of three exotic chicken strains under the farmer condition. A total of 96 households (48 household per district) were participated and then the selected strains (Sasso Rhode Island Red, Kuroiler and Koekoek) were distributed. GLM procedure was used for the on-farm data analysis. Data collected during the entire study were, growth data, fertility and hatchability, egg production, weight and age at point of lay. Better overall average daily body weight gains were achieved from 12-16 and 16-20 weeks by kuroiler chicken strains with value of 10.29, 13.68 gram respectively. Egg at first lay of SRIR and Kuroiler strains were 23.69 and 25.25 weeks respectively. The overall average egg production in the study area was 63.36% which was scored by SRIR strain. According to this study, based on their fast-growing kuroiler strain and based on their egg production potential SRIR strain were recommended to the study area and like agro ecologies.

Growth characteristics and molecular identification of indigenous Limnospira strains from Ethiopian soda lakes as a protein source

Malnutrition poses a significant global challenge in regions such as Ethiopia, particularly in children with limited dietary diversity. Arthrospira, a protein-rich cyanobacterium known for its rapid growth even under extreme conditions, is a promising solution for biomass production. This study addresses knowledge gaps in the morphological characterization and taxonomic classification of Arthrospira by focusing on precise species identification and strategic strain selection for optimal biomass production. Cyanobacterial strains were cultivated in a modified Spirulina Ogawa Terui medium at 35 °C under the photosynthetically active radiation of 160 μmol photons m−2 s−1 with a 12/12 h light/dark period in laboratory conditions. One hundred Arthrospira-like strains were isolated and characterized based on their morphological attributes according to accepted criteria, from Ethiopian soda lakes. Systematic evaluation of the growth rate comparison test identified four exceptionally fast-growing strains, with LC-30 exhibiting the highest dry weight (3.27 ± 0.51 g L˗1) and specific growth rate (1.63 ± 0.05 d˗1) among them. DNA analysis confirmed that all four examined strains belong to the Limnospira genus, specifically identified as Limnospira fusiformis. LC-30 and LA-08 demonstrated protein and phycocyanin levels (582.80 ± 0.07 and 557.90 ± 0.09 mg g˗1, respectively) comparable to those reported for Arthrospira platensis, and surpassing conventional protein sources, thus suggesting their potential for nutritional supplementation. The study highlights the importance of bioprospecting indigenous strains from soda lakes, revealing promising candidates for large-scale biomass production.

Recent advances in engineering fast-growing cyanobacterial species for enhanced CO2 fixation

Atmospheric CO2 removal (CDR) is a fundamentally endergonic process. Performing CDR or Bioenergy with Carbon Capture and Storage (BECCS) at the gigatonne scale will produce a significant additional burden on the planet’s limited renewable energy resources irrespective of the technology employed. Harnessing photosynthesis to drive industrial-scale CO2 fixation has been of significant interest because of its minimal energy requirements and potential low costs. In this review, we evaluated the thermodynamic considerations of performing atmospheric carbon removal using microalgae and cyanobacteria versus physicochemical processes and explore the implications of these energetic costs on the scalability of each respective solution. We review the biomass productivities of recently discovered fast-growing cyanobacterial strains and discuss the prospects of genetically engineering certain metabolic pathways for channeling the fixed carbon into metabolic ‘carbon sinks’ to further enhance their CO2 capture while concurrently extracting value. We share our perspectives on how new highly productive chassis strains combined with advanced flux balance models, essentially coupling synthetic biology with industrial biotechnology, may unlock more favorable methods for CDR, both from an economic and thermodynamic perspective.

A toolbox to engineer the highly productive cyanobacterium Synechococcus sp. PCC 11901.

Synechococcus sp. PCC 11901 (PCC 11901) is a fast-growing marine cyanobacterial strain that has a capacity for sustained biomass accumulation to very high cell densities, comparable to that achieved by commercially relevant heterotrophic organisms. However, genetic tools to engineer PCC 11901 for biotechnology applications are limited. Here we describe a suite of tools based on the CyanoGate MoClo system to unlock the engineering potential of PCC 11901. First, we characterized neutral sites suitable for stable genomic integration that do not affect growth even at high cell densities. Second, we tested a suite of constitutive promoters, terminators, and inducible promoters including a 2,4-diacetylphloroglucinol (DAPG)-inducible PhlF repressor system, which has not previously been demonstrated in cyanobacteria and showed tight regulation and a 228-fold dynamic range of induction. Lastly, we developed a DAPG-inducible dCas9-based CRISPR interference (CRISPRi) system and a modular method to generate markerless mutants using CRISPR-Cas12a. Based on our findings, PCC 11901 is highly responsive to CRISPRi-based repression and showed high efficiencies for single insertion (31% to 81%) and multiplex double insertion (25%) genome editing with Cas12a. We envision that these tools will lay the foundations for the adoption of PCC 11901 as a robust model strain for engineering biology and green biotechnology.

Faster-growing parasites threaten host populations via patch-level population dynamics and higher virulence; a case study in Varroa mites (Mesostigmata: Varroidae) and honey bees (Hymenoptera: Apidae).

Honey bee parasites remain a critical challenge to management and conservation. Because managed honey bees are maintained in colonies kept in apiaries across landscapes, the study of honey bee parasites allows the investigation of spatial principles in parasite ecology and evolution. We used a controlled field experiment to study the relationship between population growth rate and virulence (colony survival) of the parasite Varroa destructor (Anderson and Trueman). We used a nested design of 10 patches (apiaries) of 14 colonies to examine the spatial scale at which Varroa population growth matters for colony survival. We tracked Varroa population size and colony survival across a full year and found that Varroa populations that grow faster in their host colonies during the spring and summer led to larger Varroa populations across the whole apiary (patch) and higher rates of neighboring colony loss. Crucially, this increased colony loss risk manifested at the patch scale, with mortality risk being related to spatial adjacency to colonies with fast-growing Varroa strains rather than with Varroa growth rate in the colony itself. Thus, within-colony population growth predicts whole-apiary virulence, demonstrating the need to consider multiple scales when investigating parasite growth-virulence relationships.

Comparative analysis on growth performance, survival and harvest traits between offspring from wild and mass-selected populations of Crassostrea nippona

Crassostrea nippona has recently been recognized as a preferred alternative to C. gigas in summer due to its firm flesh and high glycogen content. To promote industrial development, the mass selective breeding program was initiated to improve growth performance of C. nippona in 2014. Although a steady genetic gain of approximately 10% for growth traits was achieved in each of the first three generations of the selected strains, little is known about the cumulative genetic progress in desired traits contributed by the selective breeding in the genetic improvement program. In this study, the aquaculture performance of the selected population was comprehensively evaluated by comparing the production performance and reproductive traits of the progeny (WF1 and SF4) from the wild population and the third-generation selected line in commercial farming environments. The results showed that the diameters of eggs from both populations were at similar levels, but the fertilization and hatching rates of the progeny from the selected lines were significantly higher than those of the wild population (P < 0.05). At larval stages, the shell height and cumulative survival rate (CSR) of individuals from SF4 were consistently superior to that of WF1, especially on day 6 post-fertilization, in which CSR of the improved C. nippona was increased by 25.08% compared to that of wild oysters. Meanwhile, a significant 12.10% enhancement in the metamorphosis rate was observed in SF4 when compared with WF1. During the grow-out stage, SF4 displayed distinct advantages in growth performance and survival compared to the wild strain, with 23.19, 33.48, 34.18 and 79.11% improvement in shell height, body weight, CSR and final yield at harvest on day 800, respectively. Meanwhile, relative brood amount (RBA) associated with initial development of offspring was found to be significantly higher in SF4 compared with WF1 (P < 0.05). Remarkably, the gonadal development, sex ratios and biochemical composition of oysters from both populations were at similar levels at final harvest, implying that high-intensity artificial selection had no significant effect on reproductive characteristics and nutritional quality of C. nippona. The encouraging results in the present study suggest that the overall genetic progress contributed by genetic breeding is considerable in the fast-growing strain of C. nippona, and such gains are expected to continue to increase with further selective breeding.

Non-Targeted Metabolomics Reveals the Metabolic Alterations in Response to Artificial Selective Breeding in the Fast-Growing Strains of Pacific Oyster

Non-Targeted Metabolomics Reveals the Metabolic Alterations in Response to Artificial Selective Breeding in the Fast-Growing Strains of Pacific Oyster

An analysis of the welfare of fast-growing and slower-growing strains of broiler chicken

Due to concerns about the welfare of fast-growing (FG) strains of broiler chicken, animal welfare organisations have advocated the use of certain slower-growing (SG) strains that meet key welfare targets under test conditions. However, a widespread transition to SG strains could negatively affect sustainability because these birds tend to have higher feed conversion ratios and longer production cycles. It is important therefore to review the extent and limits of SG welfare improvements under test conditions and on commercial farms, to support the best policy decisions. Following a systematic literature search, 63 source papers were identified. Most reported comparative welfare outcomes for at least one SG strain with at least one FG counterpart, whilst a minority examined the suitability of various SG strains for niche production. The literature review considered different types of study design and accounted for confounding factors such rearing environment and diet. Additionally, a quantitative analysis of effect size for mortality, gait score and contact dermatitis was conducted across studies that had compared birds under similar rearing conditions and that had used SG strains with an average daily growth rate of at least 40g/day. Modern, commercial SG strains performed better on most relevant welfare traits than FG strains. This was the case even when the ‘fairest’ comparisons were made at equivalent bodyweights (when SG birds were older), under matched-environmental conditions, experimentally or on farm. The quantitative analysis found that FG strains had a higher estimated incidence rate ratio for mortality (risk of death over a given period of time) of between 1.69 and 2.16, contact dermatitis affecting 15-25% more birds, and a mean gait score 0.65 points higher than SG strains. FG strains were also less active but other differences in behaviour were inconsistent with some behaviours (e.g. dustbathing) often absent altogether. Growth rate was generally, but not always, predictive of welfare problems. Alternative strategies, such as the slaughter of birds prior to the onset of any welfare decline, could be evaluated in future in terms of welfare improvement and efficiency of production.

Ciceribacter sichuanensis sp. nov., a plant growth promoting rhizobacterium isolated from root nodules of soybean in Sichuan, China.

The fast-growing rhizobia-like strains S101T and S153, isolated from root nodules of soybean (Glycine max) in Sichuan, People's Republic of China, underwent characterization using a polyphasic taxonomy approach. The strains exhibited growth at 20-40°C (optimum, 28°C), pH 4.0-10.0 (optimum, pH 7.0) and up to 2.0% (w/v) NaCl (optimum, 0.01%) on Yeast Mannitol Agar plates. The 16S rRNA gene of strain S101T showed 98.4% sequence similarity to the closest type strain, Ciceribacter daejeonense L61T. Major cellular fatty acids in strain S101T included summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C19:0 cyclo ω8c. The predominant quinone was ubiquinone-10. The polar lipids of strain S101T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethyl ethanolamine, phosphatidyl ethanolamine, amino phospholipid, unidentified phosphoglycolipid and unidentified amino-containing lipids. The DNA G + C contents of S101T and S153 were 61.1 and 61.3mol%, respectively. Digital DNA-DNA hybridization relatedness and average nucleotide identity values between S101T and C. daejeonense L61T were 46.2% and 91.4-92.2%, respectively. In addition, strain S101T promoted the growth of soybean and carried nitrogen fixation genes in its genome, hinting at potential applications in sustainable agriculture. We propose that strains S101T and S153 represent a novel species, named Ciceribacter sichuanensis sp. nov., with strain S101T as the type strain (= CGMCC 1.61309T = JCM 35649T).

Engineering Saccharomyces cerevisiae for fast vitamin-independent aerobic growth

Chemically defined media for cultivation of Saccharomyces cerevisiae strains are commonly supplemented with a mixture of multiple Class-B vitamins, whose omission leads to strongly reduced growth rates. Fast growth without vitamin supplementation is interesting for industrial applications, as it reduces costs and complexity of medium preparation and may decrease susceptibility to contamination by auxotrophic microbes. In this study, suboptimal growth rates of S. cerevisiae CEN.PK113-7D in the absence of pantothenic acid, para-aminobenzoic acid (pABA), pyridoxine, inositol and/or biotin were corrected by single or combined overexpression of ScFMS1, ScABZ1/ScABZ2, ScSNZ1/ScSNO1, ScINO1 and Cyberlindnera fabianii BIO1, respectively. Several strategies were explored to improve growth of S. cerevisiae CEN.PK113-7D in thiamine-free medium. Overexpression of ScTHI4 and/or ScTHI5 enabled thiamine-independent growth at 83% of the maximum specific growth rate of the reference strain in vitamin-supplemented medium. Combined overexpression of seven native S. cerevisiae genes and CfBIO1 enabled a maximum specific growth rate of 0.33 ± 0.01 h−1 in vitamin-free synthetic medium. This growth rate was only 17 % lower than that of a congenic reference strain in vitamin-supplemented medium. Physiological parameters of the engineered vitamin-independent strain in aerobic glucose-limited chemostat cultures (dilution rate 0.10 h−1) grown on vitamin-free synthetic medium were similar to those of similar cultures of the parental strain grown on vitamin-supplemented medium. Transcriptome analysis revealed only few differences in gene expression between these cultures, which primarily involved genes with roles in Class-B vitamin metabolism. These results pave the way for development of fast-growing vitamin-independent industrial strains of S. cerevisiae.

Self-Assembly of Nanofilaments in Cyanobacteria for Protein Co-localization.

Cyanobacteria offer great potential as alternative biotechnological hosts due to their photoautotrophic capacities. However, in comparison to established heterotrophic hosts, several key aspects, such as product titers, are still lagging behind. Nanobiotechnology is an emerging field with great potential to improve existing hosts, but so far, it has barely been explored in microbial photosynthetic systems. Here, we report the establishment of large proteinaceous nanofilaments in the unicellular model cyanobacterium Synechocystis sp. PCC 6803 and the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973. Transmission electron microscopy and electron tomography demonstrated that expression of pduA*, encoding a modified bacterial microcompartment shell protein, led to the generation of bundles of longitudinally aligned nanofilaments in S. elongatus UTEX 2973 and shorter filamentous structures in Synechocystis sp. PCC 6803. Comparative proteomics showed that PduA* was at least 50 times more abundant than the second most abundant protein in the cell and that nanofilament assembly had only a minor impact on cellular metabolism. Finally, as a proof-of-concept for co-localization with the filaments, we targeted a fluorescent reporter protein, mCitrine, to PduA* by fusion with an encapsulation peptide that natively interacts with PduA. The establishment of nanofilaments in cyanobacterial cells is an important step toward cellular organization of heterologous pathways and the establishment of cyanobacteria as next-generation hosts.

Culture-free identification of fast-growing cyanobacteria cells by Raman-activated gravity-driven encapsulation and sequencing

By directly converting solar energy and carbon dioxide into biobased products, cyanobacteria are promising chassis for photosynthetic biosynthesis. To make cyanobacterial photosynthetic biosynthesis technology economically feasible on industrial scales, exploring and engineering cyanobacterial chassis and cell factories with fast growth rates and carbon fixation activities facing environmental stresses are of great significance. To simplify and accelerate the screening for fast-growing cyanobacteria strains, a method called Individual Cyanobacteria Vitality Tests and Screening (iCyanVS) was established. We show that the 13C incorporation ratio of carotenoids can be used to measure differences in cell growth and carbon fixation rates in individual cyanobacterial cells of distinct genotypes that differ in growth rates in bulk cultivations, thus greatly accelerating the process screening for fastest-growing cells. The feasibility of this approach is further demonstrated by phenotypically and then genotypically identifying individual cyanobacterial cells with higher salt tolerance from an artificial mutant library via Raman-activated gravity-driven encapsulation and sequencing. Therefore, this method should find broad applications in growth rate or carbon intake rate based screening of cyanobacteria and other photosynthetic cell factories.

Integrative proteome and metabolome analyses reveal molecular basis underlying growth and nutrient composition in the Pacific oyster, Crassostrea gigas

In order to comprehend the molecular basis of growth, nutrient composition, and color pigmentation in oysters, comparative proteome and metabolome analyses of two selectively bred oyster strains with contrasting growth rate and shell color were used in this study. A total of 289 proteins and 224 metabolites were identified differentially expressed between the two strains. We identified a series of specifically enriched functional clusters implicated in protein biosynthesis (RPL4, MRPS7, and CARS), fatty acid metabolism (ACSL5, PEX3, ACOXI, CPTIA, FABP6, and HSD17B12), energy metabolism (FH, PPP1R7, CLAM2, and RGN), cell proliferation (MYB, NFYC, DOHH, TOP2a, SMARCA5, and SMARCC2), material transport (ABCB1, ABCB8, VPS16, and VPS33a), and pigmentation (RDH7, RDH13, Retsat, COX15, and Cyp3a9). Integrated proteome and metabolome analyses indicate that fast-growing strain utilize energy-efficient mechanisms of ATP generation while promoting protein and polyunsaturated fatty acid synthesis, activating the cell cycle to increase cell proliferation and thus promoting their biomass increase. These results uncovered molecular mechanisms underlying growth regulation, nutrition quality, and pigmentation and provided candidate biomarkers for molecular breeding in oysters. SignificanceRapid growth has always been the primary breeding objective to increase the production profits of Pacific oyster (Crassostrea gigas), while favorable nutritional quality and beautiful color add commercial value. In recent years, proteomic and metabolomic techniques have been widely used in marine organisms, although these techniques are seldom utilized to study oyster growth and development. In this study, two C. gigas strains with contrasted phenotypes in growth and shell color provided an ideal model for unraveling the molecular basis of growth and nutrient composition through a comparison of the proteome and metabolome. Since proteins and metabolites are the critical undertakers and the end products of cellular regulatory processes, identifying the differentially expressed proteins and metabolites would allow for discovering biomarkers and pathways that were implicated in cell growth, proliferation, and other critical functions. This work provides valuable resources in assistance with molecular breeding of oyster strains with superior production traits of fast-growth and high-quality nutrient value.

Metabolic engineering of Synechococcus elongatus for photoautotrophic production of mannitol.

With multiple applications in food, pharmaceutical, and chemical industries as antioxidant or nonmetabolizable sweetener; the bioproduction of d-mannitol is gaining global attention, especially with photosynthetic organisms as hosts. Considering the sustainability prospects, the current work encompasses metabolic engineering of a widely used cyanobacterial strain, Synechococcus elongatus PCC 7942, and two newly isolated fast-growing cyanobacterial strains; S. elongatus PCC 11801 and S. elongatus PCC 11802, for mannitol production. We engineered these strains with a two-step pathway by cloning genes for mannitol-1-phosphate dehydrogenase (mtlD) and mannitol-1-phosphatase (mlp), where the mtlD expression was under the control of different promoters from PCC 7942, namely, Prbc225 , PcpcB300 , PcpcBm1 , PrbcLm17 , and PrbcLm15 . The strains were tested under the "switch conditions," where the growth conditions were switched after the first 3 days, thereby resulting in differential promoter activity. Among the engineered strains of PCC 11801 and PCC 11802, the strains possessing Prbc225 -mtlD module produced relatively high mannitol titers of 401 ± 18 mg/L and 537 ± 18 mg/L, respectively. The highest mannitol titer of 701 ± 15 mg/L (productivity 60 mg/L.d, yield 895 µM/OD730 ) was exhibited by the engineered strain of PCC 7942 expressing PcpcB300 -mtlD module. It is by far the highest obtained mannitol yield from the engineered cyanobacteria.

Engineering the Metabolic Profile of Clostridium cellulolyticum with Genomic DNA Libraries

Clostridium cellulolyticum H10 (ATCC 35319) has the ability to ferment cellulosic substrates into ethanol and weak acids. The growth and alcohol production rates of the wild-type organism are low and, therefore, targets of metabolic engineering. A genomic DNA expression library was produced by a novel application of degenerate oligonucleotide primed PCR (DOP-PCR) and was serially enriched in C. cellulolyticum grown on cellobiose in effort to produce fast-growing and productive strains. The DNA library produced from DOP-PCR contained gene-sized DNA fragments from the C. cellulolyticum genome and from the metagenome of a stream bank soil sample. The resulting enrichment yielded a conserved phage structural protein fragment (part of Ccel_2823) from the C. cellulolyticum genome that, when overexpressed alone, enabled the organism to increase the ethanol yield by 250% compared to the plasmid control strain. The engineered strain showed a reduced production of lactate and a 250% increased yield of secreted pyruvate. Significant changes in growth rate were not seen in this engineered strain, and it is possible that the enriched protein fragment may be combined with the existing rational metabolic engineering strategies to yield further high-performing cellulolytic strains.

Mutagenesis and Selection Strategies of SCO and Carotenoid Producing Microorganisms

Biotechnologies of microorganisms for producing various industrially important products, such as single-cell oil (SCO), single-cell protein (SCP), carotenoid, enzymes, etc., have been studied since the last century and are of current actuality to the present day. The SCO and carotenoids are an alternative to oil of plant and animal origin and to pigment of plant and synthetic origin, respectively. These cellular components of microorganisms can be used in the food, aquaculture and livestock feed, pharmaceutical, and cosmetic industries. However, microorganisms-based technologies have not found ubiquitous practice due to a number of limitations. One of them is the threshold of the oil and carotenoids content in the biomass of microorganisms due to their nature. Therefore, the development of the fast-growing strains with a high level of these product accumulation is required. Random mutagenesis, adaptive laboratory evolution (ALE), and genetic engineering are used for strain improvement. This paper reviews random mutagenesis as a simple, cost-effective tool for improving single-cell oil and carotenoid synthesis in microorganisms, followed by the selecting of mutants with preferable characteristics. Nevertheless, it should be considered that random mutagenesis accelerates naturally occurring nonspecific mutations by exposure to a physical or chemical mutagenic agent. Despite the result of a large variety of created mutants, the characteristics of the mutants can be unstable and reversible. Therefore, different cultivation strategies for developing traits of interest and testing their persistence are reviewed in this study. Choosing effective mutagenesis techniques, screening, and selection methods is essential for creating suitable mutant strains. Various strengths and drawbacks of such tools are discussed in this review, and the main directions for further development are highlighted.

Effective and sustainable bioremediation of molybdenum pollutants from wastewaters by potential microalgae

Due to industrialization and growing population have greatly affected the quality of natural water resources globally. Molybdenum is a harmful metal that can cause serious health issues when exposed to >10 ppm levels. Several industries routinely discharged it, hence posing the environmental problems. For discharged industrial effluents, there are not any environmentally friendly and sustainable treatment method have developed for removing molybdenum hazards. The objective of this study was to design a green, sustainable approach for removing the molybdenum pollutant and to offset the treatment cost by obtaining values from microalgal by-products. Chlorella sorokiniana TU5 and Picochlorum oklahomensis, two fast-growing microalgal strains, were used for molybdenum treatment. The maximum removal efficiency was 115.65 mg L−1 with biomass and lipid yield of 2.35 g L−1 and 579.3 mg L−1, respectively, after 14 days of growth and further exposure to molybdenum pollutant. The molybdenum removal capacity was further increased by optimizing crucial factors, pH and temperature before removing biomass from the aqueous phase. Zeta potential study helped to determine the pH range that appropriately facilitate the strongest possible ionic bonds, leading to the enhanced molybdenum adsorption. In order to verify molybdenum adsorption and comprehend the dominating and quantitative interactions, FTIR was carried out to analyze the reactive groups in the algal cell surface. The current work analyses to control external parameters for improving the adsorption efficiency during harvesting of microalgal biomass, which efficiently improved the removal of molybdenum (V) from the liquid phase. The proposed technique aims to improve the bioremediation efficiency of molybdenum and other inorganic contaminants at an industrial scale by using microalgal treatment.