Development of a novel solid-state fermentation strategy for the production of poly-3-hydroxybutyrate using polyurethane foams by Bacillus sphaericus NII 0838

The extensive use of synthetic plastics has caused serious waste disposal problems in our environment. Poly-3-hydroxybutyrates (PHB) are eco-friendly bacterial polyesters which are produced under unbalanced nutrient conditions. Few reports are available on PHB production by solid state fermentation (SSF). We have developed a novel SSF bioprocess in which polyurethane foam (PUF) is used as a physical inert support for the production of PHB by Bacillus sphaericus NII 0838. Media engineering for optimal PHB production was carried out using response surface methodology (RSM) adopting a Box–Behnken design. The factors optimized by RSM were inoculum size, pH and (NH₄)₂SO₄ concentration. Under optimized conditions—6.5 % inoculum size, 1.7 % (w/v) (NH₄)₂SO₄ and pH 9.0—PHB production and biomass were 0.169?±?0.03 and 0.4?±?0.002 g/g PUF, respectively. This is the first report on PHB production by SSF using PUF as an inert support. Our results demonstrate that SSF can be used as an alternative strategy for the production of PHB.     

Conidia production by <Emphasis Type="Italic">Beauveria bassiana</Emphasis> (for the biocontrol of a diamondback moth) during solid-state fermentation in a packed-bed bioreactor

Conidia of Beauveria bassiana CS-1, which have the potential for the control of the diamondback moth (Plutella xylostella), were produced by solid-state fermentation (SSF) using a packed-bed bioreactor with rice straw and wheat bran. As the packing density and the bed height were increased, the production of conidia decreased. In a packed-bed bioreactor under no aeration and no addition of polypropylene (PP) foam (control), the total average of conidia was 4.9 × 10⁸ g^-1. The production of conidia was affected more by the addition of PP foam as an inert support than forced aeration and was approx. 23 times higher than that of the control. The total average of conidia produced by B. bassiana was 1.1–1.2 × 10¹⁰ g^-1 . Revisions requested 6 September 2004/2 November 2004; Revisions received 1 November 2004/8 December 2004     

Isolation of entomopathogenic fungi from Northern Thailand and their production in cereal grains

Spore productivity in six entomopathogenic fungal strains isolated from insect cadavers at four locations in Chiang Mai province was evaluated in five cereal grains: white-rice, wheat, rye, corn and sorghum. According to sequence analysis of the internal transcribed spacer regions of these isolates, they were closely related to Beauveria bassiana (2 isolates), Metarhizium flavoviride (1 isolate), Metarhizium anisopliae (1 isolate), Paecilomyces lilacinus (1 isolate) and Isaria tenuipes (1 isolate). Among all fungal isolates, the maximum amount of spores (530.0?×?10⁹ conidia/g) was yielded P. lilacinus CMUCDMT02 on sorghum grain followed by white-rice (399.3?×?10⁹ conidia/g). Moreover, the highest number of spore in M. flavoviride was 102.8?×?10⁹ conidia/g sorghum whereas white-rice yielded the greatest amount of spore for B. bassiana CMUCDMF03 (141.0?×?10⁹ conidia/g) after 60?days incubation. The fungal growth rate was found highest in corn for all strains and rye showed the lowest with the exception of P. lilacinus CMUCDMT02 among the tested grains. Spore viability was over 80?% for all isolates that had been inoculated for 60?days. Fungal conidia suspension of P. lilacinus obtained highest virulence against Bactrocera spp. at a concentration of 1?×?10⁶ spore/ml. The strains isolated, exhibited good production of conidia suggesting a promising strategy for the mass production of inoculum as biocontrol agents with low production cost.     

Genetically engineered Escherichia coli FBR5: Part I. Comparison of high cell density bioreactors for enhanced ethanol production from xylose

Five reactor systems (free cell batch, free cell continuous, entrapped cell immobilized, adsorbed cell packed bed, and cell recycle membrane reactors) were compared for ethanol production from xylose using Escherichia coli FBR5. In the free cell batch and free cell continuous reactors (continuous stirred tank reactor‐CSTR) productivities of 0.84 gL^?1 h^?1 and 1.77 gL^?1 h^?1 were achieved, respectively. A cell recycle membrane reactor resulted in the highest productivity of 55.56 gL^?1 h^?1, which is an increase of 66‐fold (e.g., 6614%) over the batch reactor. Calcium alginate gel CSTR resulted in a productivity of 2.04 gL^?1 h^?1 whereas adsorbed cell packed bed reactor resulted in a productivity of 4.39 gL^?1 h^?1. In the five reactor systems, ethanol concentrations ranged from 18.9 to 40.30 gL^?1 with metabolic yields from 0.44 to 0.51. Published 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Survivability and storage stability of Trichoderma atroviride TRS40 preserved by fluidised bed drying on various agriculture by-products

Agriculture by-products were applied to proliferate biomass of Trichoderma atroviride TRS40 in solid state fermentation (SSF) cultures. The culture media overgrown with mycelium together with conidia were preserved by fluidised bed drying at various temperatures (50°C, 60°C and 70°C) and the received biopreparations were stored for 12 months. In order to determine the suitability of TRS40 in the production of biopreparations, the influence of preservation process and storage time on their survivability was examined. The three-component mixture proved more effective in the SSF cultures, ensuring TRS40 count at 6.07?×?10^9?CFU/g?dm, which was ca. 6 times higher than in the mono-component medium. TRS40 survivability after preservation at various temperatures ranged from 40.4% to 100%, regardless of carrier type. In turn, after 12-month storage of the biopreparations produced on the three-component medium, regardless of drying temperature, the number of viable cells ranged from 2.43?×?10⁸ to 2.49?×?10^8?CFU/g?dm. Furthermore, selected parameters of growth kinetics in the Bioscreen C system were determined. The storage time of biopreparations had various effects on growth kinetic parameters. In addition, the preserved preparations based on the TRS40 retained their capability for biosynthesis of hydrolases, even after 12 months of storage.     

High gradient magnetic separation versus expanded bed adsorption: a first principle comparison

A robust new adsorptive separation technique specifically designed for direct product capture from crude bioprocess feedstreams is introduced and compared with the current bench mark technique, expanded bed adsorption. The method employs product adsorption onto sub-micron sized non-porous superparamagnetic supports followed by rapid separation of the loaded adsorbents from the feedstock using high gradient magnetic separation technology. For the recovery of Savinase® from a cell-free Bacillus clausii fermentation liquor using bacitracin-linked adsorbents, the integrated magnetic separation system exhibited substantially enhanced productivity over expanded bed adsorption when operated at processing velocities greater than 48 m h^–1. Use of the bacitracin-linked magnetic supports for a single cycle of batch adsorption and subsequent capture by high gradient magnetic separation at a processing rate of 12 m h^–1 resulted in a 2.2-fold higher productivity relative to expanded bed adsorption, while an increase in adsorbent collection rate to 72 m h^–1 raised the productivity to 10.7 times that of expanded bed adsorption. When the number of batch adsorption cycles was then increased to three, significant drops in both magnetic adsorbent consumption (3.6 fold) and filter volume required (1.3 fold) could be achieved at the expense of a reduction in productivity from 10.7 to 4.4 times that of expanded bed adsorption.     

Utilizing the macroporous packed bed for insect cell/baculovirus expression

Due to their high porosity and biocompatibility, polyurethane foam (PUF) and cellulose foam were adopted for insect cell immobilization and baculovirus expression. Spodoptera frugiperda (SF-21) cells were grown within the macroporous matrix and then infected by Autographa californica nuclear polyhedrosis virus (AcNPV) which was encoded with human interleukin-5 (hIL-5) gene. An appropriate initial cell loading density and medium circulation velocity determined from the previous study were applied in this actual cell cultivation experiments to obtain a uniform initial and final axial cell distribution. The growth of insect cells and the expression of baculovirus were successful in the macroporous packed bed systems used. The final average cell density in cellulose foam achieved was 5.2×10⁷?cells/cm³ and 4.3×10⁷?cells/cm³ in PUF. Under the conditions of sufficient nutrition and oxygen supplement, the average productivity of hIL-5 in cellulose foam packed bed bioreactor reached 7.2×10⁷ unit/l-day. With 50% fresh medium replacement after viral infection, the average productivity of hIL-5 in PUF packed bed reached 8.4×10⁷ unit/l-day, about two fold than that without any fresh medium replacement at infection.     

Scale-up and application of a cyclone reactor for fermentation processes

A cyclone reactor for microbial fermentation processes was developed with high oxygen transfer capabilities. Three geometrically similar cyclone reactors with 0.5?l, 2.5?l and 15?l liquid volume, respectively, were characterized with respect to oxygen mass transfer, mixing time and residence time distribution. Semi-empirically correlations for prediction of oxygen mass transfer and mixing times were identified for scale-up of cyclone reactors. A volumetric oxygen mass transfer coefficient k _L a of 1.0?s^?1 (available oxygen transfer rate with air: 29?kg?m^?3?h^?1) was achieved with the cyclone reactor at a volumetric power input of 40?kW?m^?3 and an aeration gas flow rate of 0.2?s^?1. Continuous methanol controlled production of formate dehydrogenase (FDH) with Candida boidinii in a 15?l cyclone reactor resulted in more than 100% improvement in dry cell mass concentration (64.5?g?l^?1) and in about 100% improvement in FDH space-time yield (300?U?l^?1?h^?1) compared to steady state results of a continuous stirred tank reactor.     

Enhanced inorganic carbon uptake by Chlorella sp. IMMTCC-2 under autotrophic conditions for lipid production and CO2 sequestration

To achieve sustainable production of biofuel from microalgae, a well-optimized and sustained biomass production is prerequisite. The major factor determining the higher productivity of algae is the availability and uptake of CO₂ for biomass growth. In this study, an improved CO₂ sequestration method leading to improved biomass yields has been investigated. The ability of OH^? ions in fixing dissolved CO₂ in form of HCO ₃ ^? in algal growth medium was studied using a Chlorella sp. and scaled-up in a photobioreactor. It was observed that a critical concentration of 0.005?M OH^? is required for HCO ₃ ^? formation and utilization by algae. HCO ₃ ^? uptake was enhanced by 70.8% (in presence of 0.01?M NaOH) with a sixfold increase in growth rate compared with only CO₂ system. In mineral carbon systems such as NaHCO₃ and Na₂CO₃, increase in HCO ₃ ^? uptake was enhanced by 65.4% and 63.4%, respectively. The maximum rate of CO₂ fixation of 6.6?mg?L^?1?h^?1 was obtained with 0.01?M NaOH which was 1.5 times compared with mineral carbon sources. The biomass from scale-up experiment contained 16.3% lipid (by weight) of which 75% is unsaturated fatty acids (in total lipids). This supports the idea that fixing the dissolved CO₂ in the form of bicarbonate using alkali helps in increased biomass productivity rather than CO₂ itself, forms a precursor for biodiesel, and increases CO₂ sequestration in a cyclic process.     

Fipronil enhanced natural occurrence of Fusarium solani (Hypocreales: Nectriaceae) on building infesting termite Heterotermes indicola Wasmann (Blattodea: Rhinotermitidae)

The Southeast Asian subterranean termite, Heterotermes indicola Wasmann (Blattodea; Rhinotermitidae), is recognized as a building infesting lower termite species in urban environment. The extensive use of chemical termiticides against aerial mud tubes and underground nests of H. indicola beneath the buildings could not suppress its infestation; however, it enhanced the environmental contamination and insecticide resistance. In the present study, we tried to control termites using naturally occurring entomopathogenic fungi Fusarium solani (Mart.) Sacc., along with sublethal concentrations of termiticide fipronil in no-choice feeding pathogenecity bioassay for 20?days. Termite mortality after 20?days of continuous exposure to highest fungal treatment 1?×?10⁹?conidia/mL was 10% exclusively, whereas 100% mortality was calculated just after 16?days of concurrently exposure to 5?ppm of fipronil and highest rate of fungus 1?×?10⁹?conidia/mL. These results indicated that insecticidal stress declined the immune response of termites and reduced the repellency of termites against fungal conidia by breaching the primary defense mechanism (allogrooming). This co-application of F. solani at suitable sublethal concentrations of fipronil showed the promising potential against termites that may reduce the selection pressure of pesticides and resistance risk by targeted pests, but further investigations are necessary for developing field trials.     

Carbon budget of oligotrophic mire sites in the Southern Taiga of Western Siberia

The results of a long-term (1999–2007) investigation of vegetation productivity and carbon dioxide (CO₂) emission from the surface of an oligotrophic mire in southern taiga in Western Siberia are presented. The studied ecosystems include pine—shrub—sphagnum (PSS) community, a similar community with oppressed (low) tree stand (LPSS), and sedge—sphagnum fen (SSF). Net primary production for PSS, LPSS and SSF are equal to 552, 575, and 561 g m^?2 yr^?1. The mean respiration during the snow-free season determined by chamber method is 165.8, 105.6, 112.4 mgCO₂ m^?2 h^?1 for PSS, LPSS and SSF, respectively. Field measurements of NPP and CO₂ emission in combination with reference data on methane emission, winter CO₂ and CH₄ emissions and carbon export by river run–off were used to develop an overall carbon budget of the mire ecosystems. At present conditions the studied mire ecosystems are net sinks for atmospheric carbon and accumulate peat. Rates of the actual modern carbon accumulation are equal to 21, 112 and 102 g C m^?2 yr^?1 for PSS, LPSS and SSF communities.     

The potential of Isaria spp. as a bioinsecticide for the biological control of Nasutitermes corniger

The termite Nasutitermes corniger is a serious pest infesting urban areas of Brazil and many other countries. Control largely depends on synthetic pesticides whose indiscriminate use can impact the environment and the health of humans and other animals. Alternative strategies against insect pests, such as biological control by entomopathogenic fungi, could be effective while minimising these deleterious effects. We analysed the actions of the entomopathogenic fungi Isaria farinosa, Isaria fumosorosea, and Isaria javanica against the insect N. corniger. Our results indicated that the fungi examined were pathogenic against N. corniger, with I. farinosa ESALQ1355 being the most efficacious strain, resulting in the death of 95% of the workers (LC₅₀ 6.66?×?10⁴ conidia/mL) and 85% of the soldiers (LC₅₀ 6.81?×?10⁴ conidia/mL). This is the first report of the pathogenicity of Isaria spp. on N. corniger. These in vitro results suggest that I. farinosa ESALQ1355 demonstrates a significant biological potential for controlling N. corniger.     

Algal biomass: A sustainable,economical and renewable approach for microbial production of pectinolytic enzymes using submerged and solid state fermentation techniques

In the past decade, algal waste has been used as useful natural resource for production of enormous range of products that have wide economical and commercial importance. Pectinases are group of enzymes that have wide commercial applications. Hence, current study was designed to utilize algal biomass for the production of pectinases using submerged (SmF) and solid state fermentation (SSF) techniques. Different algal sources including brown (Dictyopteris polypodioides, Sargassum wightii and Dictyopteris divaricata) and green algae (Ulva lactuca and Codium tomentosum) were used and U. lactuca was found to be the most suitable substrate. Several bacterial and fungal strains were screened and among them Bacillus licheniformis KIBGE-IB4 was selected based on maximum pectinase production. SmF and SSF were studied utilizing U. lactuca as a substrate and results revealed that enzyme production was favoured by SmF (2457?±?3.31?U?mg^?1) as compared to SSF (1432?±?1.46?U?mg^?1). Parametric optimization of pectinase production indicated that B. licheniformis KIBGE-IB4 requires 10.0?g L^–1 U. lactuca as a biomass in the medium with a pH 7.0 when incubated at 37?°C for 24 hours. Likewise, production of pectinase using algal resource was also compared with that of the conventional agricultural biomass and it was observed that when U. lactuca was used, the selected bacterial isolate produced a higher yield of enzyme than sugarcane bagasse and rice husk. Hence, it is anticipated that algal biomass can be efficiently utilized as an environmental friendly bioresource for the production of industrially important hydrolytic enzymes.     

Impact of Nitrogen Fertilizer on the Mycorrhizal Inoculating Potential and Fungal Community Structure in Rhizosphere of Medicinal Plant Curcuma longa L.

Reducing chemical fertilizer while remaining high crop yield has become a trend of agricultural practice. The purpose of this study was to investigate the effects of nitrogen (N) input on the structure of the fungal community and mycorrhizal inoculum potential in rhizosphere of Curcuma longa, aiming to minimize the use of chemicals and optimize the effect of biofertilizer for sustainable turmeric farming. For this, a field study was conducted in two successive years 2015 and 2016 with applications of varrying N fertilizer rates N0 (0 kgN ha^?1 y^?1), N150 (150 kgN ha^?1 y^?1), N350 (350 kgN ha^?1 y^?1), and N500 (500 kgN ha^?1 y^?1) in combination with mycorrhizal biofertilizer on turmeric plants in Hung-Yen. Rhizosphere soils were then analyzed for the abundance of predominant fungal taxon based on amplicon sequencing of rDNA internal transcribed spacer regions. Metagenomic results indicated significant effects of N inputs on rhizosphere fungal communities composition, with dominance of ascomyceteous fungi under optimized N levels, whereas basidiomycetous fungi were more abundant under higher N input. Operational taxonomic units relating to the predominantly abundant genus Glomus were found apparently in higher proportion under optimized N inputs (N150). The results demonstrate an enhanced productivity and icreased mycorrhizal inoculating potential by appropriated N fertilizer application in turmeric rhizosphere.     

Evaluation of a granular formulation containing Metarhizium brunneum F52 (Hypocreales: Clavicipitaceae) microsclerotia in controlling eggs of Aedes aegypti (Diptera: Culicidae)

We evaluated the potential of a granular formulation of Metarhizium brunneum F52 containing microsclerotia (MbMSc granules) for control of Aedes aegypti by targeting eggs. MbMSc granules produced infective conidia within 14 days after application to 2.5?g moist potting soil, producing 5.9?×?10⁵, 2.08?×?10⁶ and 6.85?×?10⁶ conidia from 1, 5 and 25?mg MbMSc granules, respectively. Application of MbMSc triggered premature eclosion of eggs (EC_50?=?12?mg) with percentages as high as 31?±?2.9% and 67?±?4.3% of the eggs treated with 5 and 25?mg MbMSc granules, respectively, after 14 days on moist filter paper. Premature eclosion of eggs started at 3 days subsequent to MbMSc granule application and survival of larvae was significantly reduced for granule treated eggs (74?±?2.2%, 39?±?2.0% and 23?±?4.9% larvae survived for 1, 5 and 25?mg granule treatments, respectively, EC₅₀?=?4.9?mg). When MbMSc granules were applied in moist potting soil with mosquito eggs, rates of 1, 5 and 25?mg of MbMSc granules significantly reduced adult emergence with only 81?±?2.1%, 47?±?1.9%, and 34?±?2.1% emergence, respectively (EC₅₀?=?7?mg). Eggs treated with increasing concentrations of fungal conidia enhanced premature eclosion of eggs with an EC₅₀?=?1.6?×?10⁶ conidia/mL. Our results demonstrate that MbMSc granules are a promising candidate for control of A. aegypti and that fermentative production of Mb F52 microsclerotia as the active propagule has the potential for use for mosquito control.     

Spatial distribution of phytoplankton productivity in the Amundsen Sea, Antarctica

To date, no direct measurements of primary production were taken in the Amundsen Sea, which is one of the highest primary productivity regions in the Antarctic. Phytoplankton carbon and nitrogen uptake experiments were conducted at 16 selected stations using a ¹³C–¹⁵N dual isotope tracer technique. We found no statistically significant depletions of major inorganic nutrients (nitrate?+?nitrite, ammonium, and silicate) although the concentrations of these nutrients were markedly reduced in the surface layer of the polynya stations where large celled phytoplankton (>20?μm) predominated (ca. 64?%). The average chl-a concentration was significantly higher at polynya stations than at non-polynya stations (p?<?0.01). Average daily carbon and nitrogen uptake rates by phytoplankton at polynya stations were 2.2?g?C?m^?2?day^?1 (SD?=?±1.4?g?C?m^?2?day^?1) and 0.9?g?N?m^?2?day^?1 (SD?=?±0.2?g?N?m^?2?day^?1), respectively, about 5–10 times higher than those at non-polynya stations. These ranges are as high as those in the Ross Sea, which has the highest productivity among polynyas in the Antarctic Ocean. The unique productivity patterns in the Amundsen Sea are likely due to differences in iron limitation, phytoplankton productivity, the timing of phytoplankton growing season, or a combination of these factors.     

Targeting the click beetle Agriotes obscurus with entomopathogens as a concept for wireworm biocontrol

Applications of Metarhizium brunneum Petch (Ascomycota: Hypocreales: Clavicipitaceae) isolate LRC112 conidia caused high mortality to Agriotes obscurus L. (Coleoptera: Elateridae) click beetles in field trials. Banded conidiated rice (4.4 × 10¹⁴ conidia ha^?1) and conidia dust (5.0 × 10¹³ conidia ha^?1) resulted in 93.3 % ± 7.3 and 91.3 % ± 3.0 mortality after 18 days, while aqueous conidia suspension spray (5.0 × 10¹³ conidia ha^?1) with and without 80 g ha^?1 spinosad resulted in 68.2 % ± 17.7 and 52.6 % ± 17.4 mortality. Differences in results between 2012 and 2013 were attributed to rainfall, with pronounced effects in 2012 (rain beginning 35 h post treatment) and minimal effects in 2013 (rain beginning at 4 h). In another field experiment, beetles dosed with 1.49 × 10⁷ ± 5.08 × 10⁶ conidia per beetle retained 4.6 % of conidia after seven days while conidia viability on beetle bodies remained unchanged. The results inferred opportunities for controlling click beetles using fungal entomopathogens, and for horizontal transmission of the inoculum.     

Hyperactive α-amylase production by Aspergillus oryzae IFO 30103 in a new bioreactor

Aims: To improve the α‐amylase production in solid‐state fermentation (SSF) condition utilizing a new bioreactor (NB) system. Methods and Results: In NB system, 20 g of wheat bran moistened with liquid medium in 1 : 1 ratio (w/v) was taken on the tray present inside the upper vessel and an additional 80 ml medium was supplemented into the lower vessel. Oxygen uptake rate was improved by supplying compressed air that lifted the liquid medium into the upper vessel and touched the substrate bed. This condition probably facilitated the heat transfer to liquid medium, reduce water loss and catabolite repression. With 1% glucose supplementation, maximum α‐amylase activity of 22 317 Ugds^?1 was produced by Aspergillus oryzae IFO 30103 within a very short incubation period (48 h) at 2‐cm bed height with air flow rate of 0·1 l min^?1 g^?1 wheat bran at 32°C and initial medium pH of 6. Conclusions: Within a short incubation period, significantly high α‐amylase activity was obtained and it is higher than those reported to date at bioreactor scale operating with a fungal strain. Significance and Impact of the Study: The reactor is novel and can overcome some of the major problems associated with SSF process. A. oryzae IFO 30103 is reported as the best fungal source for α‐amylase production.     

Acquisition,retention and dispersal of soilborne plant pathogenic fungi by fungus gnats and moth flies

Adult fungus gnats and moth flies were experimentally demonstrated to function as potential above‐ground vectors for three soilborne plant pathogens: Verticillium dahliae, Fusarium acuminatum and Thielaviopsis basicola. The adult insects externally acquired the conidia of the pathogens after exposure to the cultures as confirmed by scanning electron microscope photography. The intestinal contents and frass deposits of larvae exposed to fungal cultures contained viable fungal propagules. Internally infested larvae developed into internally infested pupae; however, the emerging adults were free of fungal structures. Because of the maintenance of a high level of inoculum on the external body surface and the ability of these adult insects to fly, they can be a significant factor in the dispersal of soilborne fungi in greenhouse agriculture. The rate of dispersal of T. basicola by adult fungus gnats was 1.78 cm² h^?1 per insect and by adult moth flies was 1.17 cm² h^?1 per insect. The area over which the pathogen was dispersed by the adult insects increased with the increase in exposure time. The study demonstrated that adult insects are efficient distributors of soilborne plant pathogenic fungal propagules.