How to Perform a Microfluidic Cultivation Experiment—A Guideline to Success

As a result of the steadily ongoing development of microfluidic cultivation (MC) devices, a plethora of setups is used in biological laboratories for the cultivation and analysis of different organisms. Because of their biocompatibility and ease of fabrication, polydimethylsiloxane (PDMS)-glass-based devices are most prominent. Especially the successful and reproducible cultivation of cells in microfluidic systems, ranging from bacteria over algae and fungi to mammalians, is a fundamental step for further quantitative biological analysis. In combination with live-cell imaging, MC devices allow the cultivation of small cell clusters (or even single cells) under defined environmental conditions and with high spatio-temporal resolution. Yet, most setups in use are custom made and only few standardised setups are available, making trouble-free application and inter-laboratory transfer tricky. Therefore, we provide a guideline to overcome the most frequently occurring challenges during a MC experiment to allow untrained users to learn the application of continuous-flow-based MC devices. By giving a concise overview of the respective workflow, we give the reader a general understanding of the whole procedure and its most common pitfalls. Additionally, we complement the listing of challenges with solutions to overcome these hurdles. On selected case studies, covering successful and reproducible growth of cells in MC devices, we demonstrate detailed solutions to solve occurring challenges as a blueprint for further troubleshooting. Since developer and end-user of MC devices are often different persons, we believe that our guideline will help to enhance a broader applicability of MC in the field of life science and eventually promote the ongoing advancement of MC.

Chemotypes and Their Stability in Mentha longifolia (L.) L.—A Comprehensive Study of Five Accessions

Mentha longifolia (L.) L. is the most widespread wild-growing mint species found, and its chemical composition is extremely diverse. We studied the essential oil (EO) yield, composition, and chemotaxonomy of five, northern Hungarian accessions of the species in a cultivation experiment covering two vegetation years at two parallel sites. The long-term goal is to establish the cultivation of this stress-tolerant species in Hungary as a source of flavoring and preservative agents for commercial use. Essential oil yield (1–2 mL/100 g) was observed to be dependent on both the accession and the year. Accession HV1 is assumed to be a new, presumably rare chemotype containing carvacrol (19.28–20.56%), 1,8-cineole (14.87–17.45%), thymol (13.36–13.90%), carvacryl acetate (8.81–10.40%), and para-cymene (7.24–8.01%). Only minor fluctuations occurred in concentrations of these constituents due to habitats and years. A radical change in essential oil composition was observed in accession HV2, as one batch was based on thymol (19.79%) and 1,8-cineole (14.93%), while the others were rich in dihydrocarvone isomers (up to 69%). Although this needs further investigation, it does explain the coexistence of limonene-oxo and γ-terpinene pathways in horsemint. According to the literature, the pathway leading to thymol isomers and/or esters may be rare in the entire Mentha genus. We also demonstrated that known chemotypes of horsemint may differ in variability of their EO composition. Our results also led to the conclusion that any declaration on chemotype needs detailed examination and is not realistic on the basis of a single sample. Assumptions were made about the potential areas of utilization: beside fragrance and flavoring uses of essential oils free from pulegone and menthofurane, thymol-based ones may be used as antioxidative and anti-spoilage agents.

Divergent Adaptation Strategies of Vascular Facultative Epiphytes to Bark and Soil Habitats: Insights from Stoichiometry

Understanding the stoichiometric traits of plants is critical for studying their ecological adaptation strategies. Facultative epiphytes (which can also live on the ground) are an important component of epiphytic flora of montane forest ecosystems. However, a key gap persists in our understanding how facultative epiphytes can adapt different nutritional conditions of ground and canopy habitats? To study adaptive strategies of facultative epiphytes and the characteristics of the content and stoichiometric homeostasis of C, N, and P elements, we conducted a field experiment and a greenhouse N and P additions cultivation experiment. We found that epiphytic individuals of facultative epiphytes showed lower C:N and C:P ratios, higher variation in elemental composition, and more pronounced N limitation than terrestrial individuals. Moreover, facultative epiphytes showed strong control over the elemental composition of leaves, and their stoichiometric homeostasis of leaves and stems were stronger than roots. Furthermore, the homeostasis of facultative epiphytes decreased in the order N > P. Our results indicated that epiphytic and terrestrial individuals of facultative epiphytes have difference in nutrient limitation, and they use plastic strategies in different habitats. Epiphytic individuals survive in the intermittent habitat through luxury consumption of nutrient while terrestrial individuals were relatively conservative nutrient users. Furthermore, our results implied that facultative epiphytes maintain stable metabolic leaf activity via variable element concentrations of roots to adapt to highly heterogeneous forest habitats.

Isolation of SAR11 Marine Bacteria from Cryopreserved Seawater

ABSTRACTWhile marine microorganisms are frequently studied in their natural environment, isolated strains are invaluable resources that can be used in controlled experiments to expand upon direct observations from natural systems. Here, we sought a means to enhance culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. Using a raw seawater sample collected from the tropical Pacific Ocean, a subsample was diluted in seawater growth medium to create 576 2-ml dilution cultures containing 5 cells each and incubated for a high-throughput culturing (HTC) experiment, while another portion was cryopreserved in 10% glycerol. After 10 months, a cryopreserved aliquot was thawed and used to create a second cultivation experiment of 480 2-ml cultures containing 5 cells each and 470 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from the raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (92%) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater environmental sample were isolated by both approaches.IMPORTANCE High-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing them in the presence of cryoprotectants, such as glycerol, has been shown to be an effective method of storing viable cells over long time periods (i.e., years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as an inoculum for high-throughput cultivation experiments. We found that SAR11 and other abundant marine bacteria could be isolated from seawater that was previously cryopreserved for nearly 10 months at a rate of culturability similar to that of the same seawater used fresh, immediately after collection. Our findings (i) expand the potential of high-throughput cultivation experiments to include testing when immediate isolation experiments are impractical, (ii) allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and (iii) enable cultivation experiments across a wide range of other conditions that would benefit from having source inocula available over extended periods of time.

Performance Comparison of Experimental IoT Based Drip and Fibrous Capillary Irrigation Systems in The Cultivation of Cantaloupe Plants

The demand for freshwater and food is on the increase due to the rapid growth in the world’s population, while the effect of global warming and climate change poses a severe threat on water use and food security. Conventional irrigation system suffers due to an inefficient management of water and energy, while insufficient supply of water to plant increases their stress which often affects its growth and development. Hence, there is a need to increase research focus on water use efficiency in irrigation agriculture. This paper is aimed at investigating the performance of smart drip and subsurface fibrous capillary irrigation experiment for the cultivation of cantaloupe plant to increase the yield and quality of fruit while decreasing the water and energy usage. To achieve enhancement of subsurface fibrous capillary and drip irrigation system, an Internet of Things (IoT) approach was used to improve monitoring of soil, weather, plant and control of water application. The performance comparisons of both methods was evaluated in terms of water-saving in greenhouse cultivation experiment. The results obtained, shows that the smart fibrous capillary irrigation has water use efficiency of 19 g/Litre with average fruit sweetness of13.5 Brix. While, drip irrigation has 4.85 g/Litre and average sweetness of 10 Brix on the harvested fruit after 90 days of cantaloupe plant cultivation experiment. These have shown that precision irrigation through enhanced smart fibrous capillary irrigation can be used to achieve high water-saving and a good quality yield. It is expected that the research output will help to improve water-saving agriculture towards achieving food security.Keywords: Water Saving; Capillary Irrigation; Drip Irrigation; Internet of Things; Water Use Efficiency

Isolation of SAR11 marine bacteria from cryopreserved seawater

In this study, we sought a means to increase current culture collections of SAR11 marine bacteria by testing the use of seawater cryopreserved with glycerol as an inoculum. In July 2017, raw seawater was collected outside of Kāne‘ohe Bay, Hawai‘i, in the tropical Pacific Ocean. A portion of this sample was diluted in seawater-based growth medium to create 576 × 2 mL dilution cultures containing 5 cells each and incubated for a high-throughput cultivation experiment, while another portion was cryopreserved in 10% glycerol. After ten months, a cryopreserved aliquot of seawater was thawed, diluted in seawater-based growth medium, and distributed to create a second high-throughput cultivation experiment of 480 × 2 mL dilution cultures containing 5 cells each and 94 cultures containing 105 cells each. The raw seawater cultivation experiment resulted in the successful isolation of 54 monocultures and 29 mixed-cultures, while cryopreserved seawater resulted in 59 monocultures and 29 mixed cultures. Combined, the cultures included 51 SAR11 isolates spanning 11 unique 16S rRNA gene amplicon sequence variants (ASVs) from raw seawater inoculum and 74 SAR11 isolates spanning 13 unique ASVs from cryopreserved seawater. A vast majority (115 of 125) of SAR11 isolates from the two HTC experiments were members of SAR11 subclade Ia, though isolates of subclades IIIa and Va were also recovered from cryopreserved seawater and subclade Ib was recovered from both. The four most abundant SAR11 subclade Ia ASVs found in the initial seawater sample used to create both culture experiments were isolated by both approaches.ImportanceHigh-throughput dilution culture has proved to be a successful approach to bring some difficult-to-isolate planktonic microorganisms into culture, including the highly abundant SAR11 lineage of marine bacteria. While the long-term preservation of bacterial isolates by freezing in the presence of cryoprotectants such as glycerol has been shown to be an effective method of storing viable cells over long time periods (i.e. years), to our knowledge it had not previously been tested for its efficacy in preserving raw seawater for later use as inoculum for high-throughput cultivation experiments. We found that SAR11 and other abundant marine bacteria could be isolated from seawater that was previously cryopreserved for nearly 10 months, at a rate of culturability similar to that of the same seawater used fresh, immediately after collection. Our findings expand the potential of high-throughput cultivation experiments to include opportunities where immediate isolation experiments are impractical, allow for targeted isolation experiments from specific samples based on analyses such as microbial community structure, and enable cultivation experiments across a wide range of other conditions that would benefit from having source inoculum available over extended periods of time.

Herbisida natrium bispiribak dosis rendah terbukti efektif mengendalikan gulma pada sistem tanam benih langsung padi

AbstrakPengendalian gulma pada budidaya padi sawah sistem tanam benih langsung perlu dilakukan karena menyebabkan penurunan hasil. Penelitian dilakukan untuk mengetahui apakah herbisida Natrium Bispiribak dosis rendah dapat mengendalikan gulma pada sistem tanam benih langsung padi sawah. Percobaan dilaksanakan pada bulan April sampai bulan Agustus 2019, di Kecamatan Leuwimunding, Kabupaten Majalengka. Rancangan percobaan disusun dalam bentuk Rancangan Acak Kelompok dengan tujuh perlakuan dan empat kali ulangan. Perlakuan yang diuji yaitu dosis herbisida Natrium bispiribak 12, 18, 24, 30, dan 36 g/ha serta kontrol adalah penyiangan mekanis dan tanpa pengendalian gulma. Hasil percobaan menunjukkan bahwa perlakuan herbisida Natrium Bispiribak 12 g/ha efektif mengendalikan gulma, tidak menimbulkan keracunan pada tanaman padi, serta memberikan bobot gabah kering giling padi yang sama seperti pengendalian mekanis. Dosis Natrium Bispiribak yang rendah memberikan efisiensi biaya produksi dan tidak mencemari lingkungan.Kata Kunci: herbisida, hasil gabah, dosis rendah, lingkungan AbstractWeed control in rice cultivation must be implemented because of weeds can decrease rice yield through its competition of nutrient, water, and sunlight. The aim of this research was to find out whether the low-dose of Bispyribac Sodium herbicide could control weeds in the direct seeding system of lowland rice cultivation. Experiment was conducted in Leuwimunding District, Majalengka Regency, from April to August 2019. It used Randomized Block Design (RBD) that consisted of 7 treatments and 4 replications. Bispyribac Sodium dosages as the treatments were 12, 18, 24, 30, and 36 g/ha; rotary weeding and without weed control as treatment controls. The experimental results showed that Bispyribac Sodium 12 g/ha was effective in controlling weeds, did not cause toxicity in rice plants, and gave the same yield as mechanical control. Low dose of Bispyribac Sodium provided efficient production costs and did not pollute the environment..Keyword: herbicide, environment, low dosage, yield

Acid Rain Increases Impact of Rice Blast on Crop Health via Inhibition of Resistance Enzymes

Worldwide, rice blast (Pyricularia oryzae) causes more rice crop loss than other diseases. Acid rain has reduced crop yields globally for nearly a century. However, the effects of acid rain on rice-Pyricularia oryzae systems are still far from fully understood. In this study, we conducted a lab cultivation experiment of P. oryzae under a series of acidity conditions as well as a glasshouse cultivation experiment of rice that was inoculated with P. oryzae either before (P. + SAR) or after (SAR + P.) simulated acid rain (SAR) at pH 5.0, 4.0, 3.0 and 2.0. Our results showed that the growth and pathogenicity of P. oryzae was significantly inhibited with decreasing pH treatments in vitro culture. The SAR + P. treatment with a pH of 4.0 was associated with the highest inhibition of P. oryzae expansion. However, regardless of the inoculation time, higher-acidity rain treatments showed a decreased inhibition of P. oryzae via disease-resistance related enzymes and metabolites in rice leaves, thus increasing disease index. The combined effects of high acidity and fungal inoculation were more serious than that of either alone. This study provides novel insights into the effects of acid rain on the plant–pathogen interaction and may also serve as a guide for evaluating disease control and crop health in the context of acid rain.

Simulation and Experimental Analyses of Nitrate Absorption for Indicator Utilization in Soilless Culture

Background: Under intensive fertilization systems such as soilless cultures, a surplus nutrient supply could result in the excessive discharge of fertilizers. However, very few studies have developed nitrate absorption indicators for plants that can be used for decision-making under these systems. This study was conducted to develop indicators related to the absorption of nitrate that can be applied in online systems utilizing the monitored irrigation and drainage amount data, electrical conductivity (EC), and the nutrient analysis data of irrigation and drainage. Results: In the simulation, a stochastic change was generated for the nutrient absorption rate. The theoretical prediction was verified by the cultivation experiment and a higher correlation of tomato yield with the nitrate absorption indicator was confirmed than with the nitrate supply amount. Conclusions: The results of the simulation and cultivation experiments showed that the indicators related to nitrate absorption provide useful information regarding decision-making for efficient resource management through online monitoring.