REVIEW | doi:10.20944/preprints202004.0528.v1
Online: 30 April 2020 (11:09:28 CEST)
Sugarcane is an important industrial crop of semitropical and tropical areas. Due to the importance of this crop it is cultivated on nearly 20 million hectares by more than 90 countries. This crop belongs to family Poaceae, a grass family which is economically vital tracheophyte family that has properties much like that of wheat, maize, rice and sorghum. The Poaceae family is globally vital for providing dietary macromolecules, carbohydrates, and different nutrients. The most important item of sugarcane for the consumption is sucrose that gets accumulated in the stalk internodes. Sucrose is taken out and refined by different mills which is used as a sweetener in human food industries and in the fermentation industry. Large production of alcohol is attained by Brazilian sugarcane industries.
REVIEW | doi:10.20944/preprints202006.0133.v1
Online: 11 June 2020 (11:53:18 CEST)
Tree members of the genus Acacia have benefits that are obvious for enhancing soil fertility in farming, forestry and agroforestry in regions with nutrient-poor soils, and for restoring degraded ecosystems and lands. Nevertheless, the species of the genus Acacia have got the potential to bring about significant adverse impacts on biodiversity or ecosystem functioning when it gets invasive. The ecology of the species in nearly all areas of its created range remains poorly understood. Here we have compiled the information regarding the importance, cultivation and the production of important species of genus Acacia. We hope this information will be useful to get awareness about the crucial trees in the genus Acacia.
ARTICLE | doi:10.20944/preprints202008.0541.v1
Subject: Life Sciences, Biotechnology Keywords: antioxidant activity; dietary supplements; microalgal cultivation
Online: 25 August 2020 (08:41:14 CEST)
Microalgae production is expensive and demands high volumes of water and energy to meet all market demands and yet the production of secondary metabolites using alternative media needs deeper studies. Among alternative media, the use of sugar cane vinasse has gained attention for microalgae cultivation. In this work, we’ve compared the biomass yield and secondary metabolites of Chlorella sorokiniana grown in a commercial medium (Sueoka) and in alternative medium prepared with cane vinasse (0.1%) supplemented with N, P2O5 and K2O (commercial formula 20-5-20 g.L-1). The production of microalgae biomass grown in an alternative medium was 2.2% lower, but reached its maximum growth point 14 days faster than the commercial medium. The average level of Phenolic compounds on the commercial medium were 6.02 ± 0.13 mg GAE .g-1 and increased to 15.28 ± 0.32 mg GAE.g-1when C. sorokinianawere grown in the vinasse medium. Same how, Flavonoids contents varied from 13.12 ± 1.33 to 72.30 ± 5.28 mg QE. g-1 to commercial and vinasse medium, respectively. The maximum AOA of C. sorokiniana grown in vinasse medium was 88.05% at a concentration of 1500 µg.mL-1 of extract, with IC50 at 357.7 ± 27.35 µg.mL-1. Different factors seem to induce variances on secondary metabolites synthesis, mostly stress due to QOD and ions added to the medium by vinasse. The authors recognize there are more investigations to be done, but increasing a natural and low cost pathway for flavonoids yield open up an interesting field for the bioprospection of microalgae.
ARTICLE | doi:10.20944/preprints202003.0023.v1
Subject: Earth Sciences, Environmental Sciences Keywords: shifting cultivation; Philippines; biodiversity; carbon; REDD+
Online: 2 March 2020 (02:27:00 CET)
The Philippines is both a biodiversity hotspot and a megadiverse country. The country also has experienced one of the highest rates of deforestation in Southeast Asia and is among the first countries to introduce a massive reforestation program to address the country’s rapid biodiversity and forest loss. Drawing upon an empirical study from the Leyte island and other relevant case studies from the Philippines, in this chapter, we demonstrate that recovering secondary forests following shifting cultivation, locally known as kaingin have the high potentials for biodiversity and carbon co-benefits. Based on our empirical study, we also found that secondary forest regrowing after kaingin use can potentially be used as a cost-effective reforestation measure with multiple benefits to people and the environment in upland areas of the Philippines. We also discuss measures that are essential for such programs to be successful.
REVIEW | doi:10.20944/preprints202110.0390.v1
Subject: Life Sciences, Biotechnology Keywords: Biological contaminants; grazers; microalgae; open cultivation; biopesticides
Online: 26 October 2021 (14:36:19 CEST)
Microalgae biomass is a budding raw material for the origination of food, fuel, and other value-added products. However, bulk production of microalgal biomass at commercial level is a herculean task for the current microalgal mass production technologies due to the undesirable contaminations by biological pollutants. These contaminants hamstring the production of microalgae biomass by debilitating the growth of cultures, crumble the quality of biomass and sometimes may crash the whole culture. The best utilization of the microalgae biomass at industrial level could be attained by avoiding various possible biological contaminations in mass cultivation system, understanding the contamination mechanisms, and the complex interactions of algae with other microorganisms. This review explores the various types of biological pollutants, their possible mode of infection along with mechanisms, different controlling methods to maintain desired microalgae culture.
ARTICLE | doi:10.20944/preprints202104.0453.v1
Subject: Life Sciences, Biochemistry Keywords: high-pressure microbiology; deep biosphere; cultivation devices; decompression
Online: 17 April 2021 (08:46:55 CEST)
High temperatures (HT) and high hydrostatic pressures (HHP) are characteristic of deep-sea hydrothermal vents and other deep crustal settings. These environments host vast and diverse microbial populations, yet only a small fraction of those populations have been successfully cultured. This is due, in part, to the difficulty of sampling while maintaining these in situ conditions and also replicating those high-temperature and high-pressure conditions in the laboratory. In an effort to facilitate more HT-HHP cultivation, we present two HT-HHP batch culture incubation systems for cultivating deep-sea vent and subsurface (hyper)thermophilic microorganisms. One HT-HHP system can be used for batch cultivation up to 110 MPa and 121°C, and requires sample decompression during subsampling. The second HT-HHP system can be used to culture microorganisms up to 100 MPa and 160°C with variable-volume, pressure-retaining vessels that negate whole-sample decompression during subsampling. Here, we describe how to build cost effective heating systems for these two types of high-pressure vessels, as well as the protocols for HT-HHP microbial batch cultivation in both systems. Additionally, we demonstrate HHP transfer between the variable-volume vessels, which has utility in sampling and enrichment without decompression, laboratory isolation experiments, as well as HHP filtration.
ARTICLE | doi:10.20944/preprints202002.0050.v1
Subject: Earth Sciences, Other Keywords: potato cultivation soil suitability; agricultural landscape categorisation; GIS
Online: 5 February 2020 (02:52:46 CET)
Growing potato demands considerable external inputs of pesticides due to its susceptibility to various pests and pathogens. Here we present an attempt to differentiate the Slovak rural landscape with respect to the possibility of effective potato cultivation and to characterise soil parameters of current potato cultivation areas with the aim to increase the sustainability of the potato production. The selection was based on soil climatic, production and economic parameters. By using the GIS tools and existing databases on soil characteristics in Slovakia, maps of soil suitability categories for potato cultivation were generated. In Slovakia, it was found that 12.3% of farmland is very suitable for potato cultivation and that as much as 43.1% is not suitable. Later the specified categories were characterised in detail and specified with respect to geographic, soil, climatic, production and economic parameters. Currently, most potato crops are cultivated on Eutric Cambisols (27%), Chernozems (20%) and Mollic Fluvisols (18%). Loamy soils (65%), soils without gravel (62%), deep soils (74%) and soil situated on plains (55%) are dominant in these regions. We suggest that potato cultivation should be concentrated on the most suitable areas, thereby increasing the economic profitability, improving the ecological stability of the country and supporting the sustainability of the agriculture.
ARTICLE | doi:10.20944/preprints201903.0241.v1
Subject: Social Sciences, Other Keywords: vegetable intake; vegetable cultivation; qualitative study; Japanese, adults
Online: 26 March 2019 (10:49:20 CET)
Objective: We examined the reasons why vegetable cultivation increases or does not increase vegetable intake among adult Japanese vegetable growers.Materials and Methods: A qualitative cross-sectional study using a self-completed anonymous questionnaire was sent to participants (aged 20–74 years residing in three areas of a city in Gunma Prefecture, Japan) in September 2016. The questionnaire addressed perceptions of whether vegetable cultivation would increase vegetable intake, with four possible answers: strongly disagree, disagree, agree, and strongly agree. Respondents were then asked reasons for their view, with free-text responses. We also asked about participants’ characteristics and whether they found that growing vegetables had changed their vegetable intake and access to vegetables. We categorized the free-text answers by content.Results: We analyzed 442 answers, and reasons for vegetable growing increasing vegetable intake were grouped into five categories: “availability,” “purpose of cultivation,” “quality,” “increased positive emotions toward vegetables,” and “unconsciousness”; for it not increasing intake were also grouped into five categories: “limited quantities,” “negative emotions toward vegetables,” “cultivation for a purpose other than eating vegetables,” “access to vegetables from other sources,” and “limits associated with self-cultivation.”Conclusion: We found five main reasons why vegetable growing may and may not increase vegetable intake.
TECHNICAL NOTE | doi:10.20944/preprints202202.0341.v1
Subject: Biology, Animal Sciences & Zoology Keywords: Ctenophora; breeding; comb jelly; cultivation; husbandry; protocol; sea walnut
Online: 25 February 2022 (15:26:33 CET)
Ctenophores are marine organisms attracting significant attention from evolutionary, molecular biology and ecological research. Here we describe an easy and affordable set-up to maintain a stable culture of the ctenophore Mnemiopsis leidyi. The challenging delicacy of the lobate ctenophores can be met by monitoring the water quality, providing the right nutrition, and adapting the handling and tank set-up to their fragile gelatinous body plan. Following this protocol allows stable laboratory lines, a continuous supply of embryos for molecular biological studies, and independence from population responses to environmental fluctuations.
ARTICLE | doi:10.20944/preprints202111.0574.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: Zantedeschia aethiopica; X-ray mutagenesis; cultivation; Pectobacterium; mutation breeding
Online: 30 November 2021 (14:42:05 CET)
The development of new cultivars is important for the profitability of the floriculture industry. There are a limited number of cultivars of Zantedeschia aethiopica, an iconic ornamental cut flower, garden plant, and potted plant, due to the incompatibility of interspecific crossings within the genus. Most present-day varieties are the result of spontaneous mutations or classical breeding within the species, followed by a long selection process. Breeders are very interested in the development of a time- and cost-effective method for producing new Z. aethiopica cultivars with novel characteristics. Here, Z. aethiopica mutants were generated by treating seeds with 100 Gy of X-ray radiation. The resulting putative mutants were selected based on particular flowering parameters and compared to non-irradiated, control plants. Over two growing seasons, characteristics such as early flowering, flower size and shape, yield and response to soft-rot disease were monitored and considerable variation was observed among the mutated lines. Out of 319 mutants, 20 lines were selected based on their phenotypes and then propagated and further analyzed. Within this group, only two phenotypes displayed at least five improved flowering properties under natural, Mediterranean conditions. The rest displayed two to four desired combinations of flowering traits, some with great commercial potential.
ARTICLE | doi:10.20944/preprints202010.0395.v1
Subject: Social Sciences, Economics Keywords: Keywords: farm contracts, firms, farmers, cultivation production, Thai Binh.
Online: 19 October 2020 (15:51:04 CEST)
In an age of market liberalization, globalization, and expanding agribusiness, there is a danger that small-scale farmers will find difficulty in fully participating in the market economy. One of the solutions to solve the above situation is to strengthen linkages between farmers and firms through contract farming. The purpose of this study is to assess the factors affecting the level of linkages between businesses and farmers in crop development in Thai Binh province, Vietnam. The two main research methods used were exploratory factor analysis (EFA) and regression analysis with survey data which was conducted from 300 farmers, 10 firms, and 15 factors related to contract-farming. The results indicated that there are 5 factors that significantly affect the level of participation of farmers in the linking process, the order of impacts from strong to weak is as follows: (1) Commitment and sharing of benefits and risks between farmers and firms in the linking process, (2) Issues related to management skills and enterprises capacity of link firms, (3) Price issues that farmers are interested in in the process of implementing links with firms, (4) Policy environment - Institutions related to farmer contracts between firms and farmers, (5) Household awareness about the benefits brought about by farmer contracts with firms. Based on results, a number of specific solutions have been proposed to enhance the efficiency of farming contracts between firms and farmers in cultivation production.
ARTICLE | doi:10.20944/preprints202105.0734.v1
Subject: Earth Sciences, Atmospheric Science Keywords: Land use change; soil quality; Lowland bamboo; Cultivation periods; Ethiopia
Online: 31 May 2021 (10:37:49 CEST)
In Ethiopia, bamboo thickets and woodlands play an important role in soil-water conservation and climate change mitigation in arid and semi-arid regions. However, bamboo mass flower-ing, rapid demographic changes and expansion of agricultural investments to bamboo domi-nated areas have led to deforestation and land degradation. In this study, we determined the effects of deforestation and subsequent cultivation on soil physical and chemical properties along a chronosequence of closely located agricultural lands with different ages (1, 3, 5 and 7 years) since converted from natural lowland bamboo forest. Hence, soil samples (n = 90) have been taken from both natural bamboo forests and adjacent agricultural lands at two soil depths (0-20 cm and 20-40 cm). Our result showed that CEC, K+, Ca+, Mg+ and available P were varied significantly with respect to cultivation periods and soil depth, while soil pH and Na+ varied with soil depth (P < 0.001). Soil C and total N contents (g/kg) in 0-20 cm soil layer declined significantly and exponentially with increasing years under cultivation. Conversion of natural bamboo forest to cropland during the past seven-year period significantly increased soil pH with soil depths, while CEC was declined throughout the cultivation period and soil depth. In general, the result revealed that conversion of natural lowland bamboo and subsequent cultivation of soil had negative effects on measured soil physicochemical properties.
ARTICLE | doi:10.20944/preprints202202.0267.v1
Subject: Life Sciences, Other Keywords: potato cropping systems; cultivation model; planting density; soil cover; sustainable productivity
Online: 22 February 2022 (10:06:43 CET)
Potato (Solanumtuberosum L.) is the world's largest non-cereal crop, occupying a position critical for global food security, the fourth largest crop food in China. Due to its location and weather conditions, Yunnan province is the fourth largest potato producer in China with twice yearly production system. However, compared to high number of consumers, potato yield is still low to satisfy the population need. In this study, we have analyzed impacts of three cultivation factors: (1) three cultivation models (T1, T2, and T3), (2) planting density (T1, T2, and T3), (3) soil covering (T1, T2, T3, T4) on plant growth, and yield production. The soil moisture content, temperature, roots growth, stems development, leaf area index, and number-weight of tubers per plant and per plot (g) have significantly increased with commercial yield average of 3tones/mu. This suggests that the combination of these three factors would be a great potato extension model in winter climate zones similar to Yunnan Province. This will contributes in satisfaction of the world population potato consumption need.
Subject: Life Sciences, Biochemistry Keywords: 16S rRNA gene; bacterial diversity; catabolic activity; cultivation; Pannonian steppe; pyrosequencing
Online: 22 June 2021 (14:26:55 CEST)
In this study, we examined the effect of salinity and alkalinity on the metabolic potential and taxonomic composition of microbiota inhabiting the sodic soils at different plant communities. The soil samples were collected in the Pannonian steppe (Hungary, Central Europe) under extreme dry and wet weather conditions. The metabolic profiles of microorganisms were analysed by MicroResp method, the bacterial diversity was assessed by cultivation and next generation amplicon sequencing based on the 16S rRNA gene. Catabolic profiles of microbial communities varied primarily according to the alkali vegetation types. Most members of the strain collection were identified as plant associated and halophilic/alkaliphilic species of Micrococcus, Nesterenkonia, Nocardiopsis, Streptomyces (Actinobacteria) and Bacillus, Paenibacillus (Firmicutes) genera. Based on the pyrosequencing data, the relative abundance of phyla Proteobacteria, Actinobacteria, Acidobacteria, Gemmatimonadetes and Bacteroidetes changed also mainly with the sample types, indicating distinctions within the compositions of bacterial communities according to the sodic soil alkalinity-salinity gradient. The effect of weather extremes was the most pronounced in the relative abundance of phyla Actinobacteria and Acidobacteria. The type of alkali vegetation caused greater shifts in both the diversity and activity of sodic soil microbial communities than the extreme aridity and moisture.
ARTICLE | doi:10.20944/preprints202203.0171.v1
Subject: Earth Sciences, Environmental Sciences Keywords: national park; social-ecological system; ecosystem services; tea cultivation; protected area management
Online: 11 March 2022 (14:47:43 CET)
A healthy park-people relation depends essentially on the fair and sustainable maintenance of rural livelihood. When protected area is designated, rural people may face restrictions of access to land and resource use for multiple ecosystem services. In Wuyishan of China, we analysed the role of traditional tea cultivation during consistent protected area management to find ways to maintain stability of this social-ecological system in the new national park era. We used an intensive social survey to investigate tea’s role, perception of ecosystem services and impacts on tea cultivation from consistent conservation policies. Results showed that tea cultivation brought major household income and associated with multiple culture services. Protected area management affected land use and conservation outcomes were more obvious to farmers than economic and social ones. From the perspective of a social-ecological system, tea cultivation in national should be conservation-compatible activities from which the potentially lost economic value is remedied by ecological and cultural valorisation. To sustain the resilience of the social-ecological system, we proposed a three-scale management framework to regulate biophysical elements at land plot scale, to link production and market at the mountain level, and to secure tenure and encourage community participation at the landscape level.
CONCEPT PAPER | doi:10.20944/preprints202108.0422.v1
Subject: Biology, Ecology Keywords: Rainforest; recovery; indicators; logging; grassland; shifting cultivation; forestry; national parks; traditional rights
Online: 20 August 2021 (19:02:09 CEST)
Recovery of forest after logging can be tested in many ways: the presence of particular species of fauna or flora, the similarity of the biodiversity of the recovering forest to that on neighbouring areas of undisturbed forest; or the characteristics of soils and streams whose conditions may have drastically changed during logging. Three cases of rainforest recovery after logging and clearance from Australia and Borneo exhibit different starting and different goals for recovery. Faunal indicators of recovery vary with size and with species dependence of the rainforest. Endemic forest species may have difficulty in recovering. Tree species richness and abundance may recover in two decades, but canopy closure takes longer. Compacted soils may retain low infiltration capacities for many decades. This diversity in recovery rates is confirmed when compared with those used elsewhere. Because the starting points for recovery vary, from damage by tropical cyclones and landslides, to clearance for shifting cultivation, pasture or agriculture, to post-logging conditions, universal indicators may be inappropriate. The desired endpoints of recovery also range from a “wilderness” state to a National Park for human enjoyment, biodiversity preservation, safeguarding rights of traditional forest-dwelling peoples, or a second round of selective logging.
ARTICLE | doi:10.20944/preprints202110.0409.v1
Subject: Earth Sciences, Atmospheric Science Keywords: Air Pollutant Emissions; Rice Cultivation; Agricultural Machinery; Tier 1 Methodology; Geographic Information System
Online: 27 October 2021 (13:22:08 CEST)
In Korea, rice is a major staple grain and is mainly cultivated using various agricultural machinery. Air pollutants emitted from agricultural machinery have their origins mainly from the exhaustion of internal combustion engines. In this study, emission characteristics of five main air pollutants by European Environment Agency's Tier 1 method for rice cultivation was analyzed. Diesel is a main fuel for agricultural machinery and gasoline is generally used only for rice transplanters as a fuel in Korea. Tractors consume 46% of total fuel consumption and 56% of diesel fuel consumption. Gasoline used for rice transplanters accounts for 17% of total fuel consumption each year. Tractors and rice transplanters are emitting 82% of all total pollutants. From 2011 to 2019, the total amount of air pollutant emissions was decrease by 15%. That accounted for the reduction of rice cultivation fields in those periods. Rice transplanting operation was in charge of 42% of total emissions. Then, harrowing, harvesting, tilling, leveling, and pest control operations generated 10%, 10%, 8%, 8% and 7% of total emissions, respectively. The contribution of each air pollutant held 54% of CO, 39% of NOx, 5% of NMVOC, and 2% of TSP from the total emission inventory. The three major regions emitting air pollutants from mechanized agricultural practices were Jeollanam-do, Chungcheongnam-do, and Jeollabuk-do, which consume 55% of total fuel usage in rice farming. The total amount of air pollutant emissions from rice cultivation practices in 2019 was calculated as 8,448 Mg in Korea.
ARTICLE | doi:10.20944/preprints201710.0101.v1
Subject: Materials Science, Polymers & Plastics Keywords: single-polymer composite; bioreactor; textile reactor; fungal cultivation; economic analysis; polyamide; polyvinyl chloride
Online: 16 October 2017 (06:08:20 CEST)
All-polyamide composite coated fabric (APCCF) materials were developed and used as the material of construction of textile bioreactors as a replacement of traditional costly steel-/concreate-based bioreactors. Then, APCCF-bioreactor was used to cultivate filamentous fungus Neurospora intermedia to produce ethanol and fungal biomass. The results showed similar performance of the fungus cultivated in stain-less steel bioreactors. Techno-economical analysis for a 5000-m3 APCCF bioreactor for fermentation facility would lead to a reduction of the annual production cost of the facility by $128,000,000 compared to similar stainless-steel processes. The comparative analyses (including mechanical and morphological analyses, density measurements and techno-economical analysis) revealed that the APCCF is a better candidate for material of construction of the textile bioreactor. The APCCF is a 100% recyclable single polymer composite which was prepared from textile production line waste.
ARTICLE | doi:10.20944/preprints202107.0477.v1
Subject: Keywords: Agriculture Economics; Agriculture land-holdings; Minimum Support Price (MSP); Poverty; Poverty Line; farmers; Maize cultivation
Online: 21 July 2021 (10:03:52 CEST)
The research probes the relationship between the Minimum Support Price (MSP) price and its income impact on the farmers with different agricultural land-holdings. It examines maize crop cultivation income under three conditions if a farmer is getting crop production value at MSP, above 20% of MSP and above 40% of MSP. It then evaluates the farmers belonging to marginal, small, semi-medium, medium and large categories position in terms of poverty criteria suggested by the World bank. For analysis purposes, it uses an economic-model approach and suggest the concept of survival income for agricultural farmers. Its findings prove that marginal and small farmers require benefits beyond the MSP rate to push them above the poverty line.
ARTICLE | doi:10.20944/preprints201904.0094.v1
Subject: Social Sciences, Other Keywords: Keywords: vegetable cultivation; health; physical health; mental health; social well-being; qualitative study; adult; Japan
Online: 8 April 2019 (12:49:04 CEST)
The present short communication aimed to determine the effects of vegetable cultivation on health. We conducted a qualitative study using responses to an open-ended question that was part of a survey on regional differences on vegetable intake. Participants in this study were residents of Gunma Prefecture, Japan, and were aged between 20–74 years. In the questionnaire, we asked: “Aside from increasing vegetable intake, how does vegetable cultivation affect your health?” We were able to categorize the answers into six subcategories related to physical health, mental health, and social well-being.
ARTICLE | doi:10.20944/preprints202109.0367.v1
Subject: Biology, Agricultural Sciences & Agronomy Keywords: daily light integral (DLI) maps; LED grow lights; greenhouse integrated PV; adaptive lighting control; year-round cultivation; agrivoltaics
Online: 21 September 2021 (14:31:23 CEST)
High efficacy LED lamps combined with adaptive lighting control and greenhouse integrated photovoltaics (PV) could enable the concept of year-round cultivation and become a feasible option even in the harsh climate of the Nordic countries. Meteorological satellite data of this region was analyzed in a parametric study to evaluate the potential of these technologies. The generated maps showed monthly average temperatures fluctuating from -20°C to 20°C throughout the year. The natural photoperiod and light intensity also changed drastically, resulting in monthly average daily light integral (DLI) levels ranging from 45-50 mol·m-2·d-1 in summer and contrasting with 0-5 mol·m-2·d-1 during winter. To compensate, growth room cultivation independent from outdoor conditions could be used in winter. Depending on the efficacy of the lamps, the electricity required for sole-source lighting at 300 µmol·m-2·s-1 for 16 hours would be between 1.4 and 2.4 kWh·m-2·d-1. Greenhouses with supplementary lighting could help start the cultivation earlier in spring and extend it further into autumn. The energy required for lighting highly depends on several factors such as the natural light transmittance, the light threshold settings and the lighting control protocol, resulting in electric demands between 0.6 and 2.4 kWh·m-2·d-1. Integrating PV on the roof or wall structures of the greenhouse could offset some of this electricity, with specific energy yields ranging from 400 to 1120 kWh·kWp-2·yr-1 depending on the region and system design.
ARTICLE | doi:10.20944/preprints202103.0403.v1
Subject: Engineering, Biomedical & Chemical Engineering Keywords: non-canonical branched chain amino acids; scale-down; strain screening; mixed-acid fermentation; pyruvate pulse; norvaline; norleucine; fed-batch cultivation; bioreactor; Enpresso; Enbase
Online: 16 March 2021 (09:11:34 CET)
Insufficient mixing in large-scale bioreactors, provokes gradient zones of substrate, dissolved oxygen, pH and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and -methyl-norleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids, but also that pyruvate pulses produce the same effect. Therefore we combined the enzyme based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions, produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor. To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.