Public Health Safeguarding: A Review into Bioactive Molecules and Advanced Technologies for Microorganism Management

1. Introduction

Multidrug-resistant organisms and antimicrobial-resistant diseases threaten the healthcare system and public health. Thousands die annually from resistant diseases (O'Neill et al., 2014). As antimicrobial resistance increases, their effectiveness becomes increasingly uncertain [2]. Consequently, it is crucial to investigate potential replacements for current antimicrobials. They account for over two-thirds of all newly authorized pharmaceutical applications [3].

In contrast to antibiotics produced by microorganisms, plant-based antimicrobials have attracted many researchers due to their promising effects with fewer side effects. Bioactive chemicals with antibacterial, antifungal, and cytotoxic bioactivity are produced by microorganisms [4,5,6,7]. Once again, their ability to produce functionally rich secondary metabolites contributes to their success in various habitats. Due to their unique biological features, microorganisms have recently attracted researchers' interest as a potential source of new antimicrobial drugs [8]. Modern molecular biology, genetic, genomic, and computational approaches make mining microbial structure systems for drug discovery easier [9,10,11].

Viruses, bacteria, archaea, fungi, and protists are all examples of microorganisms, which are biotic, ubiquitous, diverse entities. Novel antimicrobial agents are primarily discovered in bacteria and fungi. Clinically resistant germs like S. aureus, MRSA, M. luteus, B. subtilis, and E. faecalis are toxic to cyclic peptides, polyketides, and alkaloids found in bacteria, such as mathiapeptide A, destotamide B, Marfomycins A, B, and E; spirotetronates polyketides, abyssomycin C, Lobophorin F, and H; and sesquiterpene derivatives, alkaloids, and mafuraquinocins A and D,which are derived from bacterial sources (Table 1).

Inhibitors of Gram-negative and Gram-positive pathogenic bacteria include fungus-derived xanthones, emerixanthones, and engyodontiumsones [12]. Analogues of ambuic acid, such as penicyclone and depsidone, have this property. Yeast [16,17], microalgae[14,15], and cyanobacteria[13,14] are among the other groups whose compounds have demonstrated anti-infective potential in both animal and laboratory studies.

In order to combat the rise of antibiotic-resistant bacteria, modern scientific research has prepared the way for the development of synthetic antimicrobials by altering the chemical and structural makeup of naturally occurring goods. Novel antimicrobials have been fabricated and visualized using X-ray crystallography, structured-guided designs, and components based on primogenitor cell lines [18]. Oxepanoprolinamide is a derivative of lincosamide that has a higher therapeutic efficacy against resistant bacterial strains and an increased probability of overcoming multidrug resistance caused by ATP binding cassette (ABC) F-, Erm-, and Cfr genes [18]. Multiple resistant microbial strains have emerged recently, and new technologies have become available (Figure 1).

2. Bacterial Sources of Antimicrobials

Bacteriocin, organic acids, diacetyl, hydrogen peroxide, and other antimicrobials are produced by lactic acid bacteria (LAB). The growth of some bacteria can be inhibited by the bacteriocin produced by the boza-isolated Lactobacillus pentosus ST712BZ [19,20]. Bacteriocin was discovered by Andre Gratias in 1925 when he realized that colicin V hindered E. coli growth [21].

Klaenhammer categorized bacteriocins based on their molecular weight, susceptibility to enzymes, capacity to withstand high temperatures, presence of modified amino acids after translation, and their mechanism of action. [22]. Class I separates lantibiotics into Ia and Ib by chemical structure and charge. Bacteriocin classes IIa, IIb, and IIc exist [22]. Some heat-stable peptides weigh less than 10 kDa. Class IV consists of large peptides and carbohydrates or lipids, whereas Class III includes thermo-labile peptides with a high molecular weight, often exceeding 30 kDa, such as Helveticin J. [23]. Despite competing theories by researchers such as Cotter et al. [24] and Drider et al. [25], there is still no one-size-fits-all solution.

There are currently three main categories into which bacteriocins are classified based on their biochemical and genetic characteristics. Everything else is in Class III, Class II is non-lantibiotic, and Class I contains lantibiotics [25]. The antimicrobial effect of bacteriocins is determined by their main structure [26, [Table 2]. Some bacteriocins enter cells and interfere with protein synthesis and gene expression [26], while others interfere with the proton motive force of electrified membrane vesicles [27]. Antibiotics have a dual action against bacteria [26,28,29].

When non-lantibiotics bind to the mannose phosphotransferase permease (Man-PTS) subunits C and D, they establish an intra-membrane channel that allows ions to move, ultimately leading to cell death [29,30]. Because of their high net positive charges, circular bacteriocins electrostatically attract bacterial membranes that are negatively charged. Cell death, alterations to the membrane potential, ion efflux, and holes are all results of this interaction [31]. The cell wall hydrolyzes more quickly due to bacteriolysins, which gradually causes the cell to disintegrate [32,33]. Target cells' ability to take up glucose is inhibited by bacteriolysis, leading to their death from starvation [34,35,36].

Synergistic or antagonistic interactions can occur between antimicrobial drugs and the microorganisms they are effective against [37]. Nisin and other bancteriocins kill E. faecalis and other bacteria that cause periodontal disease, preventing gingivitis and dentobacterial plaque in dogs [26]. Some Pseudomonas species produce the anionic biosurfactant rhamnolipid [38,39]. Antimicrobial, antibiofilm, antitumor, and antioxidative properties have all been observed for these compounds [40,41,42].

They can modify viral membrane glycoproteins and interact with viral lipid membranes, especially in light of their use in combating bovine coronaviruses, HSV-1 and HSV-2 [43,44]. The rhamnolipids (M15RL) produced by the Antarctic bacterium Pseudomonas gessardii (P.gessardii) M15 have been shown to inhibit SARS-CoV-2 [45].

3. Bacterial Sources of Antifungal Compounds

The red pradimicins A, B, and C are produced by Actinomadura hibisca [56]. These pradimicins are effective against Aspergillus and Candida [57, [Table 3]. Pratimimins are benzonapthacenequinones that transport D-alanine and carbohydrates, as revealed by spectral analysis and chemical breakdown [56]. By forming a compound with the cell walls of vulnerable microbes, pradimicins damage fungal cells by binding to terminal D mannosides [57]. Many species of Actinoplanes create antifungal metabolites as well.

The Actinoplanes ianthinogenes-produced purpuromycin kills Trycophyton mentagrophytes [58]. Produced by Octamycini [58]. Streptomyces species produce branching bacilli that are aerobic and Gram-positive Chemical antifungals. The chemicals nystatin, phoslatomycins, phthoxazolin A, faeriefungin, butyrolactols, sultriecin, polyoxin, and dunaimycins are examples.

Aspergillus species, Saccharomyces spp., and Candida albicans can be effectively targeted by azobacilin, bacereutin, cispentacin, and mycocerein, which are generated from Bacillus cereus compounds, as stated by Kerr. [58] Bacillus licheniformis produces both the peptide A12-C and fungicin M-4. [63,64]. Enterobacter agglomerans becomes antibacterial against Aspergillus niger, Candida species, phytopathogenic fungi, and dermatophytes, when treated with pyrrolnitrin, according to research by Chernin et al.

A and B, two antifungal herbicolins, are again produced by Enterobacter agglomerans [65,66,67]. The dipeptide CB-25-1 from Serratia plymuthica [68], Dihydroaeruginoic acid [69], pyocyanin [70], and 1-hydroxyphenazine [71] are compounds created by P. aeruginosa, which is present in a healthy human stomach. Pseudomonas also produces the peptide family pseudomycin [72], the caryoynencins [73], and the cyclic hydroxamic acid, G1549 [74], among other antibacterial chemicals.

Antimicrobial chemicals can also be found in Burkholderia species, which are bacteria [75]. Cepalycin [76], xylocandins [77], and heptylmethylquinolinone [78] are some of the other compounds that B. cepacia can create. The antibacterial enacyloxcins are thought to have originated in the Burkholderia species [79]. There are eight different antibacterial chemicals in the enacyloxcin family [76,77]. Maltophilin gives the Rhizosphere strain of Stenotrophomonas maltophilia antifungal characteristics. Antifungal action against Neurospora crassa, but not yeast, has been observed for polyenic antibiotics from the genus Gluconobacter [80].

4. Fungal Sources of Antimicrobials

Since penicillin G was first isolated from a fungus species in 1928 [81], because of their prolific secondary metabolite production, they are promising candidates for use as starting materials in developing new antimicrobial drugs. Enfumafungin, a triterpenoid extracted from a Hormonema species, was first identified more than ten years ago and has since been demonstrated to be particularly efficient against Candida spp. and Aspergillus spp. [82]. Phase II clinical trials are underway for SCY-078, a semisynthetic derivative of enfumafungin [83,84].

However, in recent years, favolon, a metabolite of strobilurins generated by Favolaschia calocera (F. calocera), has been found and demonstrated to be less toxic while still displaying substantial antifungal action against human pathogens [85]. By disrupting quorum sensing, fungal metabolites prevent biofilm formation. Coprinuslactone, isolated from C. comatus, inhibits P. aeruginosa biofilm formation [86]. A Kenyan basidiomycete's microporenic acid A is lethal to biofilms of Staphylococcus aureus and Candida albicans [87]. Antibiotics that also work against biofilms hold great promise.

Influenza Rubylide S, produced by the marine fungus Aspergillus terreus OUCMDZ-1925, inhibited the activity of the H1N1 virus [88]. Cladosporium sphaerospermum 2005-01-E3 yielded a new hybrid polyketide called Cladosin C. Penicillium chrysogenum PJX-17 is the source of the potent H1N1-fighting sorbicillinoids sorbicatechols A and B [89].

Trypilepyrazinol has demonstrated antiviral activity against several viruses, including HIV and HCV [90]. Aspernigrin C and malformin C, which A. niger SCSIO Jcsw6F30 produced, had strong antiviral activity against HIV-1 [91]. The Streptomyces kaviengensis (F7E2f) isolate antimycin A blocks mitochondrial electron transport and pyrimidine synthesis, making it effective against WEEV [92] [Table 4].

5. Advances in Micro-Culturing Technology

In 1951, Hungarian scientist Dr. Gyula Takatsy invented microwell plates, the first to minimize laboratory sample sizes. Figure 2 from Banks [94] illustrates how his idea completely changed laboratory titrations and serial dilutions. There are several drawbacks to using 96-well culture plates. It is not genuinely high-throughput, and evaporation makes it difficult to keep a constant volume in a culture over time. Furthermore, waste product accumulation within wells and restrictions in oxygen transmission restrict the expansion of confluent cells. Finally, Ingham et al. [95] point out that the low picture resolution of microwell plates makes it hard to visualize qualitative data.

The complicated interactions between microorganisms and high-throughput screening required 50 years of "micro-culturing" progression. Miniaturizing culture conditions allows for the isolation and compartmentalization of microorganisms. Fast-growing species often obscure slower-growing ones due to this type of competition. However, although they are relatively rare, slow-growing organisms account for a sizable fraction of microbial diversity [96]. Microenvironments are highly understudied, and the microtechnologies discussed here allow researchers to do so on spatiotemporal scales relevant to microorganisms [97].

6. Microarrays

The transition from mL-scale to mL-scale cell culture begins with microarray technology: automated and manual arrays print cell droplets in liquid or alginate gel onto microscope slides. The microarray has several possible uses, including but not limited to studying phenotypic, morphological, proliferative, and cell-cell communication outcomes of cell-culture miniaturization [98].

The "nano-biofilms" of Candida albicans were printed in a single study utilizing microarray printing techniques to create 1,200 tiny droplets of 30 nL each. When compared to normally grown Candida albicans, the "nano-biofilms" formed by Candida albicans were determined to be genetically and morphologically identical. Subsequently, 28 combinatorial synergistic antifungals were found to be effective against the microdroplets when tested for drug sensitivity.

In addition, microarrays are a useful tool for speeding up the discovery of novel metabolic pathways and developing novel drugs against well-established ADROs. Liquid droplets are easily damaged in studies, and secondly, they can only be interrogated in a static position on a Petri plate. Gel droplets protect cells from damage and provide a stable experimental environment, but the optimal conditions for investigating flow remain to be determined for microarray technology [99].

7. Antimicrobial Peptides

Various naturally occurring compounds known as antimicrobial peptides (AMPs) protect hosts from pathogens, including bacteria, fungi, parasites, and viruses [100]. Small in size, most AMPs are cationic because they contain several lysine or arginine residues. This positive charge allows AMPs to interact with the predominantly negative bacteria membranes. However, some AMPs have anionic properties [101,102].

The synergistic action with traditional antibiotics, low resistance risk, great efficacy at low concentrations, and broad spectrum activity of AMPs have all attracted attention [103,104]. They act on multiple plasma membrane and intracellular targets to destroy pathogenic organisms. Antimicrobial peptides (AMPs) are antibacterial, antifungal, antiviral, antiparasitic, and immunomodulatory [100]. While treating patients with VRE, Acinetobacter baumannii, or MRSA; S. Staphylococcus aureus, Listeria monocytogenes, and the bacterium E. Foodborne E. coli, Salmonella, and Vibrio parahaemolyticus infections can be prevented with the use of AMPs.

Defensins, nisin, and cecropins are AMPs that kill Gram-positive and Gram-negative bacteria. Both the Aristicluthys nobilia interferon-I inspired AMP P5 (YIRKIRRFFKKLKKILKK-NH2) and AMP P9 (SYERKINRHFKTLKKNLKKK-NH2) have been demonstrated to inhibit MRSA [105]. As a result of AMR, there has been a dramatic increase in demand for AMPs, which are used in both human healthcare and agricultural settings.

8. Antiviral Peptides

Viral illnesses have been documented ever since recorded history began. Scientists did not begin isolating viruses until the nineteenth century. Viruses are still a leading source of human illness, possibly related to the lengthy process of finding and developing new vaccines [106]. Although antiviral medication is being used, it is not without risk. Because viral epidemics like H1N1, Ebola, and Zika develop and reappear quickly, several antivirals have little efficacy. There is a growing demand for more effective antiviral medications. Antiviral peptides (AVPs) have been studied extensively recently as a potential first line of defense against viruses [106].

One antiviral peptide (AVP), clavain, is sourced from the tunicate Styela clava [107]. Viruses other than HIV have been inhibited by clavanin A and clavanin B, respectively [108,109]. Some HIV drugs include enfuvirtiude, dermaseptin-S1, S4, dermaseptin-S2, magainin 2, dermaseptin-LL-37, carin 1, maximin 3, gramicidin D, and siamycin-I and -II [110]. Antiviral peptides against the coronavirus are being developed in response to the recent COVID-19 epidemic.

9. Microorganisms Affect Climate Change

Phytoplankton in the oceans reproduce far more quickly than tree species (days vs. decades)30, are more distributed, and are less affected by seasonal shifts. Phytoplankton are among the first organisms that respond rapidly to climate shifts. The cyclical structure of phytoplankton blooms makes it difficult to forecast how climate change will affect primary productivity. Grazing and viruses are examples of top-down variables, while the availability of key nutrients and vertical mixing are examples of bottom-up variables that affect phytoplankton blooms. [111,112,113,114,115,116]

Chemolithoautotrophic bacteria and archaea fix CO2, supplementing the role of marine phytoplankton in CO2 sequestration [119,120,121] in both deep-sea waters [117] and surface waters in polar winter [118]. Archaea and Marine bacteria are crucial for the ocean's surface respiration and cycling of many elements. We do not know much about how methanogens and methanotrophs add to the release of this greenhouse gas into the air. Marine viruses, for example, affect the efficiency with which carbon is stored and deposited in the deep ocean, and Eukaryotic grazers are integral components of the microbial food chain. Global biogeochemical cycles [122] are affected by climate change because of its effect on predator-prey relationships, particularly virus-host interactions.

Dissemination is typically simpler for bacteria than it is for larger creatures. Many microbial species vary biogeographically due to dispersal, lifestyle (including the influence of host association), and environmental variables that impact the composition and functioning of communities. [123,124,125,126]. Temperature and latitudinal gradients are particularly significant for marine ecosystems [127,128]. The adaptive potential [129] of microorganisms like bacteria, archaea, and microalgae is greatly enhanced by their vast population densities and fast asexual generation rates. Ocean acidification and other environmental variables related to climate change have received very little attention in evolutionary studies [129,130]. Little is known about the molecular mechanisms underlying physiological reactions or their biogeochemical consequences [131].

10. Terrestrial Biome

About half of the world's net primary production [120] comes from land plants. More carbon than is found in the atmosphere and plants is stored in the world's soils—a total of 2,000 billion tonnes. Land microorganisms number about 1029, like aquatic ones. Soil bacteria regulate the amount of organic carbon kept in soil and the amount released into the atmosphere. [133,134], in addition to controlling productivity with macronutrients like phosphate and nitrogen. Mycorrhizal fungi help plants get nitrogen and phosphate in various environments [134]. Plants provide their fungal symbionts with lots of carbon.

Up to 25% of all man-made carbon dioxide (CO2) is stored in forests [135,136], and forests account for 50% of all terrestrial primary productivity. Grasslands account for about 29% of Earth's surface area. These dry and semiarid regions respond differently to human-caused climate change than their forested counterparts [137,138], and they are crucial to the carbon budget. Lakes comprise around 4% of the Earth's non-glaciated surface area, and many methane gas emissions come from shallow lakes [139,140].

11. Climate Change Affects Microorganisms

Climate change affects microbial community structure and diversity through seasonality, temperature, plant composition, litter, and root exudates [141,142]. Natural geothermal warming in the lab and the long run (more than 50 years) boosted respiration and biomass of soil microbes, which released net carbon dioxide, while substrates were depleted, decreasing biomass and microbial activity [143]. That some microbial groups find it difficult to adjust to warmer environments is clear from this.

12. Infectious Diseases

Climate change impacts several environmental characteristics [144], which impacts illnesses in marine and terrestrial organisms [145]. (Figure 3). Modifying their defensive systems and nutrient cycling routes may make corals more vulnerable to bleaching and disease. [146] due to changes in their microbiome brought about by warming oceans. Damage to fish tissue from ocean acidification may impair their immune systems, allowing germs to colonize their bodies more easily [147]. Pathogens [148] potentially contribute to forest mortality due to drought and heat stress.

As a result of changes in host and parasite acclimation [149], climate change can raise disease risk. Temperature might increase susceptibility to infection for ectotherms (like amphibians), potentially by perturbing immune responses [149,150]. The Cuban tree frog is more vulnerable to Batrachochytrium dendrobatidis when temperatures fluctuate weekly and daily. Assessing host-pathogen reactions directly is of utmost importance rather than making assumptions based on research solely focused on the growth rates of individual microorganisms in order to determine the significance of climate change accurately. [149], as there is a discrepancy between the effect of rising temperatures on infection and the fungus' reduced growth capacity in pure culture. Recent research indicates that antibiotic resistance among human infections is projected to increase due to climate change [151].

Due to climate change, the transmission season may last longer, the rate at which diseases replicate in the vector may speed up, and the number and distribution of mosquitoes may grow. El Nio/Southern Oscillation (ENSO) and other large-scale climate phenomena significantly impact the spread of many infectious diseases, especially in vector-borne or aquatic environments. ENSO is associated with several significant human and animal illnesses, including malaria, dengue fever, Zika virus disease, cholera, the plague, African horse sickness, and many more. [152,153,154,155,156].

13. Tools and Techniques

Used for Identifying New Microorganism-Based Antimicrobial Drugs Finding new bioactive chemicals that are effective against disease-causing microorganisms is important because of the spread of antimicrobial resistance. The challenges encountered over the years include the inherent low concentrations of antimicrobials in natural sources, the absence of advanced and inventive drug discovery tools, and the substantial costs and limited investment in drug development and discovery. [157,158]. The following are examples of conventional methods for extracting useful chemicals from bacteria:

• Examples of diffusion processes include Agar discs, antimicrobial gradients, wells, plugs, cross streaks, and poisoned food. The agar disc diffusion method was created in 1940 to determine bacterial resistance [159]. Detecting some bacterial infections is difficult [160]. This comprises Streptococci, H. influenzae, N. gonorrhoea, N. meningitidis, and H. parainfluenzae. Agar-grown bacteria are exposed to a predefined dosage of the test chemical in this assay. The test compound's antimicrobial properties permeate into the agar, stopping the growth of sensitive bacteria. The zone's diameter is measured when a growth inhibitor is used [161,162]. Agar disc diffusion is a simple and cost-effective method for determining the minimum inhibitory concentration (MIC). Nevertheless, the outcomes are imprecise. [161].
• The antimicrobial gradient technique (Etest) may be utilized to calculate the minimum inhibitory concentration (MIC) of antibacterials, antifungals, and other antimicrobials, which combines dilution and diffusion techniques. This technique can also be utilized to analyze medication interactions [161,163,164,165].
• Depending on the type of analysis being done, either broth or agar dilution might be utilized. Typically, agar dilutions are used to investigate the efficiency of combinations of antifungal pharmacological drugs against Candida sp., Aspergillus, Fusarium, and dermatophytes [166,167].
• Several methods exist for screening and determining the susceptibility of microorganisms to antimicrobial medications. These include the time-kill test [168], the ATP bioluminescence assay [169,170,171,172], and the flow-cytofluorometric approach [173]. ATP bioluminescence has allowed for the evaluation of cellular ATP levels. The luciferin-luciferase bioluminescent test is popular due to its high sensitivity. A high quantum yield chemiluminescent reaction with MgATP2+ oxidizes luciferin catalytically via the luciferase. Light intensity correlates with ATP levels under ideal conditions. Stimulating ATP release from a disintegrating cell to react with luciferin-luciferase and produce light can determine cellular ATP. A luminometer is used to quantify the brightness of an object.
• A flow cytometer and flow cytofluorometric approach can identify antimicrobial resistance and predict the chemical's effect on microbe cell damage and viability [174].

14. Micromachined Devices

The grid layouts used in microfabrication allow for predictability and the use of readily available automated software for scoring growth, both of which contribute to the technology's high level of accuracy [95]. There is a wide range in mechanical strength, permeability, biocompatibility, and surface charge among the materials utilized to make these gadgets [175].

An unanticipated advantage of these microdevices is their portability, allowing in situ incubation in locations other than a laboratory. Hence, rather than creating and managing artificially altered growth circumstances, we may rely on the inherent microenvironment to facilitate the cultivation of previously unattainable species. [176]. Kaeberlein et al. [177] proposed in the early 2000s that it would be possible to isolate and culture microorganisms without understanding the precise components necessary to sustain the establishment of a given ecosystem by just employing their natural milieu. Since this was the case, they created diffusion chambers by placing an agar matrix containing an ambient sample between two membranes with pores of 0.03 m [178].

Inoculation of the ichip is accomplished by dilution-to-extinction of the ambient sample, resulting in the development of monocultures without the need for manual picking and isolation [179]. Many phylogenetically unique species, including those discovered in human mouths [180] and saltwater [181], have never been cultured using conventional methods. However, since the ichip was introduced, they have been cultivated and isolated.

Recently, novel antibiotics such as Novo10 and Neocitreamicin I and II have been identified thanks to the successful cultivation of rare environmental species utilizing the specified diffusion chips and chips [182,183]. Figure 4: Teixobactin was identified during the isolation and passage of Eleftheria terrae, a novel -proteobacteria [184]. It effectively killed Gram-positive bacteria, even those that had developed resistance to antibiotics.

Cultures of previously unknown species were established in a soil sample from the environment using the ichip [184]. Antibiotic activity against S. aureus was tested by culturing various species onto agar plates. The in vitro efficacy of bioactive compounds against a wide range of pathogens was determined after their extraction, purification, and characterization. Using this method, researchers found teixobactin, a new antibiotic molecule that prevented any mutants from becoming resistant in early testing. After initial discovery, innovative medications undergo extensive testing for safety and efficacy before being developed for consumer use.

15. Single Emulsion Droplet Microfluidics

Because each droplet functions as a miniature bioreactor, droplet microfluidics has recently gained importance for studying cells. The culture medium often has length scales similar to prokaryotic and eukaryotic cells. Therefore, droplets ranging in size from pico- to microliters facilitate the fast passage of gasses, nutrients, metabolic waste, and similar substances. [97].

Beneyton et al. [185] employed a microfluidic system to conduct a screening process for the enzymatic production of -amylase by the filamentous fungus Aspergillus niger. The droplets were generated at a frequency of 80-90 droplets per second and had a volume in the nanoliter range (10-18 nL). Agar droplets have also been widely used for cell confinement, in addition to the liquid, aqueous phase often associated with single emulsions. During encapsulation, the agar syringe is kept liquid by submerging it in a water bath that is heated to a regulated temperature [186]. Alkayyali et al. [187] created the Microbe Domestication Pod (MD Pod) to cultivate marine samples separated within agarose beads in a single emulsion. After the gel beads are injected inside the Pod, the device may be placed back in its natural home to continue its development.

Silicon molds can be etched, embossed, or lithographed using various methods available due to microelectromechanical system (MEMS) microfabrication processes. Soft photolithography, pioneered by Xia and Whitesides [188], is currently one of the most popular fabrication processes. In soft lithography, a master mold of the necessary microfluidic device is made of photo-cross-linkable polymers rather than silicon wafers. Commercially available chips may not be ideal for exploratory research, especially with environmental materials. Several university-based public foundries have been established to facilitate customization and ease of use in the fabrication process by enabling users to submit their designs for microfabricated chips without needing specialized training or financial outlay [189].

16. Double Emulsion Droplet Microfluidics and Polymer-Based Nanocultures

Microfluidic channels with hydrodynamic pressure flow and coflowing geometry can create double-emulsion droplets, which function similarly to single-emulsion droplets. The cell inoculum is in an aqueous phase at the system's center. The hydrophobic, hydrophilic intermediate phase produces double emulsion droplets, which are stabilized in solution by a surfactant in a continuous watery (hydrophilic) phase.

Because double-emulsion droplets partition chemical reactions into nanoliter bioreactors, droplet microfluidics enables high-throughput research. Products that have passed the chemical test can be separated using fluorescence-activated cell sorting. Zinchenko et al. [190] demonstrated the platform's efficacy by identifying enhanced cellular clones capable of producing catalytically active enzymes from 106 inactive enzymes in 10 m droplets. The researchers demonstrated that the assay can be heat-inactivated to prevent catalytic activation and that droplets may be frozen at 80°C and thawed for discontinuous workflows.

Modifying the polymeric membrane to fit the specific structural requirements of different uses is possible. PDMS's high mechanical toughness and elasticity make it an important material; its Young modulus is around 0.5 to 3 MPa [191]. While this aids in producing robust capsules for cell encapsulation, it complicates subsequent processing steps, such as emptying the capsules.

17. Applications of Microbial-based Microsystems and Perceived Challeanges

Optical transparency allows for the study of phylogenetic responses in isolated colonies that have been stimulated chemically. Observing increasingly complex consortia to examine intra- and inter-species interactions is vital in differentiating symbiotic and antagonistic behaviors across species and pathogenic switching in opportunistic illnesses.

Many difficult-to-manage species, however, have unidentified secondary metabolite needs [192]. Microorganisms can be cultivated in engineered microsystems, even in extreme environments that would have previously been impossible. Clostridium difficile was deemed an "Urgent Threat" [193], according to the 2019 report on Antimicrobial Resistance Threats from the United States Centres for Disease Control and Prevention (CDC). Monocultures provide a new perspective for studying the intricate interactions between gut microbiota and C. difficile in a low-oxygen environment by allowing optical access under a microscope.

The purpose of these ecosystems is not confined to the production of so-called "unculturable" species. Many applications that previously were impossible to accomplish with the level of control, accuracy, and resolution required to identify study targets have been made possible thanks to the development of miniaturizing culturing techniques [194]. Low-throughput, expensive approaches are often connected with drug and enzymatic metabolite screening in the biotechnology industry [195]. Microfluidics is superior because it streamlines the process by shrinking everything to a microscopic size. Rapid turnaround time (a few hours instead of days) reduces the overall expense of screening procedures. Diagnostic tools, biological assays, and analytical tools in the pharmaceutical business can greatly benefit from the replicability and automation provided by microfluidics [196].

Materials design is an example of a more specialized difficulty encountered during gadget production. According to Aleklett et al. [97], unlike naturally occurring habitats, PDMS, an essentially hydrophobic elastomer, does not offer cells a heterogeneous setting where they can be mechanically manipulated. However, the hydrophilic surface modifications induced by oxygen plasma treatment of PDMS are only transitory, as the surface quickly reverts to its lower free energy state due to the migration of oligomers inside the PDMS network [197].

18. Conclusions

In this day and age of antimicrobial resistance, the diversity of chemicals found in bacteria and other microorganisms and their powerful action is important. Expanding our search to include these organisms will lead us to potentially game-changing antimicrobials with mechanisms of action not yet seen in existing drugs. Antimicrobial resistance can thus be drastically decreased in this way. In order to fully understand the influence of microbial diversity and activity on various levels of interactions and system fluxes, it is necessary to perform experiments across several scales, ranging from individual organisms to communities and entire ecosystems. To accurately portray the Earth as a whole, models of the biosphere must account for both the biotic (consisting of bacteria, plants, and organic matter substrates) and abiotic (including mineral surfaces, ocean physics, and chemistry) environments in which microbes operate. There are now fascinating and unique ways to explore "microbial dark matter" and the accompanying microbial dynamics because of the downsizing of culturing technologies. Precise instruments created by microfabrication techniques are used to investigate the microenvironment of cells. Instead of using conventional nutrient-rich liquids, these devices utilize natural surroundings that exist everywhere.