The 1-aminocyclopropane-1-carboxylate deaminase (ACCD) enzyme plays an important role in stress alleviation of both biotic and abiotic stressors in plants and thereby enhances their growth under harsh environmental conditions. In-depth analysis of AcdS gene encoding for ACC deaminase reveals its presence in diverse microorganisms including bacteria and fungi. Particularly, plant growth-promoting bacteria (PGPB) containing ACCD supports plant growth by modulating the level of 'stress ethylene' and cleaving its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) into α-ketobutyrate and ammonia, enabling PGPB to utilize ACC as a carbon and nitrogen source. The reduced synthesis of ethylene in plants further relieves the ethylene inhibition of plant growth and development, and improves plant resistance to various stressors. Therefore, the dual role of microbial ACCD makes it a cost-effective and eco-friendly biocatalyst for sustainable agricultural productions. The inducible ACCD encoding gene AcdS is differentially regulated by varying environmental conditions. Successful generation of transgenic plants with microbial AcdS gene enhanced biotic and abiotic stress tolerance in plants. In the present review, we discuss the importance of ACCD-producing PGPB for their ability to reduce ethylene production and the promotion of plant growth under stress conditions. We also highlighted the development of transgenic plants by overexpressing bacterial AcdS gene to improve their performance under stress conditions.

Nearly 15 billion metric tons of fossil fuels are consumed each year all over the world resulting in the depletion of non-renewable energy resources day by day therefore in the coming years, the shortage and price hikes of these fuels are inevitable. On the other hand, the global energy demand is projected to rise by almost 28% by 2040 compared to current levels. Thus, there is a dire need for developing the alternatives to meet these energy needs. Biomass is seen as a significant alternative energy source to fossil fuels from this standpoint. The main bottle neck in utilizing the biomass for this purpose is the lack of an efficient conversion or pretreatment technology which prompted scientists to delve into novel stepwise biomass conversion technologies. This review article encompasses various methods for the processing the algal biomass to generate potential biobased products such as algal crude oil, biogas, and fuel alcohols. Among the various techniques of thermochemical conversion of algal biomass, hydrothermal liquefaction and gasification are the most sustainable ones. Furthermore, anaerobic digestion of lignocellulosic biomass is the commercially workable technique providing biogas and biohydrogen. Future generations may find algal biofuels to be low-cost and ecologically benign alternative to fossil fuels. This review is a connotative illustration of the conversion technologies for algal biomass, which includes both thermochemical and biochemical processes. It also highlights the salient features along with the limitations of each of these technologies and bearing in mind the expansion of a superstructure depiction to capture the various biomass feedstocks and employment techniques for the generated bioenergy through various biomass conversion technologies.

Fossil fuels such as petroleum resources continue to be a significant fraction of the energy portfolio. Oil sludge or slops have become an unavoidable waste in the petroleum industries, and their improper disposal measures have led to environmental pollution. Various conventional and traditional practices for disposal and recovery are practiced, but the efficiency of MEOR has led to more promising and efficient ways to recover oil from oil reservoirs and waste sludges. Among the microbial bioactives, biosurfactants are the key players in the whole process. Although, MEOR using biosurfactants has been largely practiced for recovering oil from oil reservoirs, microbial metabolites are also proving to be effective in recovering oil from the waste oily sludges generated as a part of the petroleum production cycle. MEOR stands out as a economically sound alternative over other conventional methods that require large capital investment, heavy energy consumption and varying recovery efficiency. This review describes the scope of MEOR to be useful in reducing and reusing the waste oily sludge accumulation that when untreated causes environmental pollution, allowing sustainable use of natural resources. However, lack of reproducibility at field scale, large scale production of bioactive compounds are the major reasons leading to its incompatibility. The review aims to address the gaps and possible strategies to help speed up the efficiency by thoroughly focusing on the biosurfactant mediated MEOR process dynamics and use of various non-renewable substrates as measure of waste utilization for the production of metabolites. The molecular makeup of these significant molecules after extracting them from the contaminated niche will help discover microbial diversity. Furthermore, the limitations to this biosurfactant assisted MEOR can be solved by using genomic and high throughput approaches to execute an economical implementation at field scale level.

A novel magnet-driven rotary mixing aerator (MDRMA) was proposed to reduce the diameter and rising rate of bubbles while improving CO2 mass transfer during microalgae growth in a column photobioreactor. Visualization results showed that the bubble number density increased significantly with the increasing rotation rate of MDRMA. As the rotation rate increased from 0 to 300 r min-1, the average bubble diameter decreased from 3.8 mm to 2.0 mm (47.4% reduction), while the corresponding average bubble rising rate decreased from 0.32 m s-1 to 0.22 m s-1 (31.3% reduction). Notably, when the rotation rate of MDRMA exceeded 300 r min-1, a significant number of microbubbles with diameters below 0.5 mm were generated. When synthetic flue gas containing 12% CO2 (v/v) was supplied for microalgae cultivation, a maximum biomass concentration of 1.75 g L-1 was obtained with rotating MDRMA at 200 r min-1, showing a 57.7% increase compared to the control aerated with a silicone tube aerator. Meanwhile, the pigment content reached 14.48 mg L-1, 22.7% higher than that of the control.

Precipitation and flocculation pretreatments promise improved clarification of cell culture fluids (CCF) to intensify the production of monoclonal antibodies (mAb). However, such pretreatments pose the risks to alter the mAb and damage cells. This can be additionally exacerbated by the subsequent clarification process, for example by high shear forces during disk stack centrifugation, resulting in a release of host cell impurities. To overcome these limitations and enhance the clarification particularly of cell cultures with low viability and thus a high level of impurities, this study investigated low-pH precipitation and cationic polymer flocculation, each in combination with a mild fluidized bed centrifuge (FBC) separation and a subsequent filtration step. Therefore, low-viable CCF´s were pretreated and characterized to investigate the effects of additives on CCF composition and stability. In clarification experiments, both pretreatments achieved similar FBC throughput compared to an untreated reference but increased the maximum filter throughput up to four times. Furthermore, high mAb recoveries (> 91%), low turbidities (< 3.1 NTU) and high DNA removals (> 91%) were achieved. Similar glycan profiles and dimer ratios suggest consistent mAb quality. These findings have a great potential to intensify mAb downstream processing with both CCF pretreatments using a FBC clarification approach.

Previously, we showed that the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) could produce and secrete the beta-propeller phytase FTEII in an active form under the control of the AOX1 promoter and methanol as the inductor. In this work, we engineered P. pastoris strains to construct a constitutive P. pastoris expression system (GAP promoter) and extracellularly produce the phytase FTEII. We optimized the culture conditions to increase the extracellular volumetric phytase productivity (Qp) and evaluated the impact of the optimization process on the physiological response of the host. Moreover, we analyzed the expression levels of the FTEII gene and endogenous genes for P. pastoris cells in cultures with the lowest and highest Qp to understand which processes (from heterologous gene expression to protein secretion) might be responsible for the increase in Qp. The results indicate that a low specific growth rate and temperature in the fed-batch phase increases the Qp, which was correlated with an upregulation of the KAR2 and PSA1-1/MPG1 genes rather than increased heterologous gene transcription.

Stenotrophomonas maltophilia is a global emerging pathogenic bacillus that is highly drug resistant and known to cause nosocomial infections in immunocompromised hosts. Because of their novel modes of action, bacteriocins are being proposed as alternatives to antibiotics for the treatment of infections caused by multidrug resistant bacteria. This study is the first report of modular bacteriocins called stenocins, which were discovered in the genomes of S. maltophilia. These two novel peptidoglycan-degrading bacteriocins were identified, cloned, and expressed in plants. We demonstrate that plant-expressed stenocins are functional and inhibit the growth of Stenotrophomonas strains in vitro.

Fossil fuels are sharing a large portion of energy demand. Conventional energy sources emit a huge amount of greenhouse gas into the atmosphere, which creates energy and environmental challenges for the ecosystem. To fulfill the world energy demand and to support environmental as well as economic development in a sustainable way, with the utilization of technological advancement of renewable energy resources, algae are presently believed as most adaptable feedstock materials for bioenergy production. Algae has a high fixation rate of atmospheric carbon dioxide which supports to fast growth rate with high productivity per unit area in the form of renewable algal biomass. The present article aims to elaborate on the three generations of biofuels, sustainable microalgae biomass production, cultivation systems, and a wide range of growth parameters. The microalgae harvesting methods and their challenges are also discussed, with a special focus on lipid extraction methods and future r recommendations. The upstream and downstream processes of microalgae could help to harness the microalgae energy in an eco-friendly manner and will help in achieving overall sustainable development.

Malic acid is one of the organic acids which is used in various industries including food and pharmaceuticals. Biotechnological production of malic acid by an efficient microorganism is highly desirable as the process will be eco-friendly and cost-effective. In this study, malic acid synthesis by Zymomonas mobilis was studied by expressing Escherichia coli malic enzyme gene under Pchap, Ptac and Ppdc promoters. The mae+ recombinants were obtained by recombineering-based genomic integration of Pchap-mae, Ptac-mae and Ppdc-mae sequences. The Ppdc promoter showed the highest expression of malic enzyme and the Pchap the lowest. However, cell growth was limited in mae+ recombinant containing Ppdc promoter. The metabolic analysis showed the highest level of malic acid in Ppdc-mae recombinant (2.84 g/L), which was about eight times higher than that in the wild type strain. The study showed that these three promoters can be used to produce organic acids in Z. mobilis.

During a recent manufacturing campaign for a monoclonal antibody using a fed-batch process, poor cell culture performance was observed across two manufacturing sites with similar scales and equipment. Root cause analysis indicated that the poor cell culture performance was linked to the production basal media. Genealogy of the precursor raw materials used in the media revealed that a particular lot of Poloxamer 188 (P188) was the common link to the poor-performing media lots. P188 serves a critical role in protecting cells against shear in cell culture bioprocesses. However, the small-scale studies suggested that the poor cell culture performance was cytostatic in nature rather than being caused due to lack of shear protection. Several P188 lots were tested analytically using SEC-MS and RP-LC-MS methods and a unique low molecular weight species was identified in the suspect lot of poloxamer. The impurity was identified to be polypropylene oxide (PPO), a reaction intermediate in P188 synthesis. Spiking studies with PPO further confirmed its cytostatic nature. This case study highlights yet another scenario where lot-to-lot variability continues to impact bioprocesses and re-emphasizes the need for robust analytical and cell-culture raw material screening methods.

Over expression of recombinant proteins triggers a cellular stress response (CSR) that down-regulates numerous genes that have a key role in sustaining expression. Instead of trying to individually up-regulate these genes we hypothesized that a superior strategy would be to modulate the expression of global regulators that control the expression of many such downstream genes. Transcriptomic profiling of post induction cultures expressing recombinant asparaginase in Escherichia coli showed the down-regulation of several critical genes many of which were under the control of the global regulator lrp which is known to have a significant impact on both amino acid metabolism and protein translation. Therefore, to ameliorate the deleterious effects of the CSR we decided to supplement the activity of lrp using plasmid-based co-expression. We observed that the test culture containing an additional plasmid expressing lrp under the arabinose promoter gave a 50% higher yield of recombinant L-Asparaginase after 32 h in batch culture compared to the control, which had only one plasmid expressing the recombinant protein. This approach helped us design a better performing strain, which could sustain expression rates for a significantly longer time period. This work illustrates that modifying the expression of regulatory genes could serve as a better strategy to prevent the reprogramming of the cellular machinery which is the hallmark of the CSR and help in the design better hosts for recombinant protein expression.

Euglena gracilis is a freshwater flagellate possessing secondary chloroplast of green algal origin. This protist has numerous biotechnological applications such as production of biofuels and pharmaceuticals, and it can be also used for bioremediation of polluted water and wastewater. One of the highest limitations for its large-scale cultivation is that it cannot synthesize vitamins B1 and B12 which are expensive and they have to be added to media. This study revealed that E. gracilis can be grown for long time periods without the addition of vitamins B1 and B12 in the co-culture containing filamentous fungus Cladosporium westerdijkiae, and bacteria Lysinibacillus boronitolerans and Pseudobacillus badius. Growing of E. gracilis in such co-cultures without the addition of vitamins can dramatically reduce large scale cultivation costs. Moreover, C. westerdijkiae could be used in biotechnology for immobilization and effective harvesting of E. gracilis from big cultivation containers by bioflocculation.

Microalgae is one of the most potential materials for biofuels and dietary supplements. However, the high cost of cultivation has always restrained its commercial application. Static magnetic fields (SMF), with the advantages of low operational cost and non-toxic secondary pollution, exhibits great potential in the promotion to the microalgal growth and metabolism. In this study, the dynamic patterns on the biomass and metabolites including pigment, protein, carbohydrate, lipid and fatty acids of C. pyrenoidosa and T. obliquus under 30 mT SMF for 15 days at 24 h·d-1 were explored. Results demonstrated that SMF triggered the growth of C. pyrenoidosa and T. obliquus by 32.8% and 31.5%, respectively. SMF significantly stimulated protein synthesis by 44.3%, whereas decreased carbohydrate by 19.7% and lipid by 23.4% in C. pyrenoidosa (p < 0.05), indicating that SMF was a promising approach for inducing intracellular carbon partition to the protein synthetic pathway. The carbohydrate content exhibited a significant lower by 43.7% in T. obliquus under SMF than that of the control (p < 0.05), while no significant changes were observed in either the protein or the lipid. SMF applied for the two microalgae had negative effects on the fatty acids (MUFAs, PUFAs, and TFAs). The results indicated that SMF could not only significantly accelerate the growth of the two microalgae, but also influence their metabolites.

Biosurfactants are capable of meeting the challenges of the oil industry by reducing its social, economic, and environmental impacts. The aim of the present study was to produce a biosurfactant from Pseudomonas cepacia CCT6659 in 2.0-L and 5.0-L bioreactors and evaluate its long-term stability over 120 days of storage. Ecotoxicological tests were performed with Artemia salina larvae during the use of the biosurfactant to increase in solubilisation of heavy oil in seawater compared to the use of a chemical surfactant. The biosurfactant was also applied as a bioremediation agent for sand contaminated with a petroleum product and as an inhibitor of corrosion on metallic surfaces. A concentration of 35.0 g/L of the biosurfactant was achieved in the 5.0-L reactor and low toxicity to the bioindicator was found, with an approximate 40% reduction in the mortality rate compared to the chemical surfactant. The stability of the biosurfactant was demonstrated by the maintenance of its tensioactive properties throughout the entire storage period. Besides its advantageous bioremediating capacity, with the removal of 94.5% of oil from sand, the biosurfactant proved to be an effective inhibitor of both metallic corrosion and microbial biofilm, with minimal loss of mass (15.7%) compared to the control condition, demonstrating its potential for industrial applications.

Anaerobic digestion (AD) is an attractive bioprocess for waste treatment and energy recovery through methane-rich biogas production. Under temperate to cold climate, the implementation of AD for low-organic load wastewater treatment has been limited to date, due to the energetic and economic cost of maintaining optimal mesophilic temperature. Hence, we aim at (i) exploring the biotechnological potential of a microbial inoculum from Antarctic soils and sediments to run AD at low temperatures; and (ii) evaluating the effect of temperature over a psychrophilic-mesophilic range on both methane production rates and microbial community composition. Methane production stimulated by acetate amendment was detected from 5 to 37 °C, with a maximum at 25 °C, corresponding to the highest relative abundance of methanogenic archaea (c. 21.4% of the total community). From 5 to 25 °C, the predominant methanogen was Methanosaeta, while it shifted to Methanocorpusculum at 30 °C. Compared with an industrial mesophilic sludge, the relative methane production rate at 5 °C (compared to the maximum) was 40% greater in the Antarctic inoculum. Microbial communities from permanently cold Antarctic sediments efficiently produce methane at low temperatures revealing a biotechnological potential for the treatment of low-organic load residues in cold regions.

The effects of Indole-3-butyric acid (IBA) supplementation in combination with nitrogen limitation on the biomass, biochemical and fatty acid composition of three selenastracean microalgae were studied. In the algae tested, nitrogen deficiency alone resulted in lower biomass output, whereas supplementation with 0.075 mM IBA resulted in increased biomass production on Selenastrum capricornutum (679 mg L-1), Ankistrodesmus falcatus (521 mg L-1) and Kirchneriella obesa (361 mg L-1) than their respective controls. The chlorophyll content of the algae showed a negative correlation with IBA dosage. Whilst it could significantly stimulate carotenoid biosynthesis in S. capricornutum and nitrogen limited (NL) alone, it influenced carotene production in both A. falcatus and K. obesa. Among the treatments, NL+ 0.075 mM IBA markedly increased the total lipid content to 1.2-fold in S. capricornutum, 0.81-fold in A. falcatus and 0.35-fold in K. obesa, with higher lipid productivity than control. NL+ 0.05 mM IBA significantly increased the amount of saturated fatty acids and NL+ 0.075 mM IBA significantly increased the amount of unsaturated fatty acids in all members of the Selenastraceae family under study. Thus, the study promises the importance of Selenastraceae members as a valuable feedstock for various process industries.

Metagenomics sequencing has generated millions of new protein sequences, most of them with unknown functions. A relatively quick first step for function assignment is to use the existing public protein databases and their scanning tools. However, to date these tools are not able to identify all sequence features like conserved motifs or patterns. In this study we evaluated the capability of several protein public databases (e.g., InterPro, PROSITE, ESTHER, pfam, AlphaFold etc) and their scanning tools for identifying lipolytic features in 78 putative cold-adapted bacterial lipase sequences. Novel lipases that can tolerate extreme conditions have great biotechnological importance. We obtained the putative cold-adapted lipolytic sequences from the metagenomic study of anaerobic psychrophilic microbial community treating domestic wastewater at 4 and 15 ℃. Both newer and conventional protein classifiers failed to find lipolytic features for most of the putative lipases. InterProScan predicted lipase family membership for only 18 of the putative lipase sequences. For more than half of them (41 out of 78) InterProScan could not predict any protein family membership, let alone find lipolytic features in them. However, when the Lipase Engineering Database and AlphaFold were used, half of those sequences were classified. Conventional databases like PROSITE could find lipolytic patterns for 9 of the putative lipolytic sequences of which only one was identified by InterProScan as a lipase. Moreover, different scanning tools made different and inconsistent predictions for a certain putative lipase sequence. Even InterProScan, which integrates predictions from 13 protein member databases, did not have a consensus prediction for a certain lipase sequence. Our study shows that there is lack of information in public protein databases about bacterial lipase sequences and this limits their lipolytic feature prediction and biotechnological application. The integration of AlphaFold within the InterPro can improve the lipase identification and classification significantly.