Submerged cultivation of a strain ofHumicola lutea 72 producing acid protease

Summary It is demonstrated for the first time that a species from the genusHumicola is a potential source of acid protease. A strain was classified by morphological investigations asHumicola lutea. The influence of constituents of the culture medium on the growth and acid protease production ofH. lutea 72 in submerged cultivation in flasks was investigated. An improved medium was devised for future studies. The optimal aeration rate, inoculum level and cultivation time were determined. A maximal proteolytic activity of 670 g tyrosine liberated from casein ml^–1 culture filtrate min^–1 at pH 3.0 was obtained.     

Survival and colonization of Escherichia coli O157:H7 on spinach leaves as affected by inoculum level and carrier, temperature and relative humidity

Aims: To determine survival and colonization of Escherichia coli O157:H7 on spinach leaves as affected by inoculum level and carrier, temperature and relative humidity (r.h.). Methods and Results: Spinach leaves were inoculated with suspensions of E. coli O157:H7 in distilled water (DW) and 0·1% peptone water (PW) and incubated at 4, 12 and 25°C and 43, 85 and 100% r.h. The number of E. coli O157:H7 on leaves (5·6 or 1·9 log CFU per leaf) inoculated using DW as a carrier medium increased significantly at 25°C and 100% r.h. within 120 h but remained constant or decreased significantly under other test conditions. E. coli O157:H7 on leaves (5·4 log CFU per leaf) inoculated using PW as a carrier increased significantly within 72 and 24 h, respectively, at 12 or 25°C and 100% r.h.; counts using a low inoculum (2·2 log CFU per leaf) increased significantly within 24 h at 25°C. Conclusions: Escherichia coli O157:H7 can colonize on spinach leaves at 12 or 25°C in a 100% r.h. environment. Organic matter in the inoculum carrier may provide protection and nutrients which enhance survival and colonization. Significance and Impact of the Study: Colonization of E. coli O157:H7 on spinach leaves as affected by organic matter in the inoculum, temperature and r.h. was determined. These observations will be useful when developing strategies to prevent growth of E. coli O157:H7 on pre‐ and postharvest spinach.     

Fate of Escherichia coli O157:H7 in ground beef following high‐pressure processing and freezing

Aims: The purposes of this study were to evaluate the efficacy of high pressure to inactivate Escherichia coli O157:H7 in ground beef at ambient and subzero treatment temperatures and to study the fate of surviving bacteria postprocess and during frozen storage. Methods and Results: Fresh ground beef was inoculated with a five‐strain cocktail of E. coli O157:H7 vacuum‐packaged, pressure‐treated at 400 MPa for 10 min at ?5 or 20°C and stored at ?20 or 4°C for 5–30 days. A 3‐log CFU g^?1 reduction of E. coli O157:H7 in the initial inoculum of 1 × 10⁶ CFU g^?1 was observed immediately after pressure treatment at 20°C. During frozen storage, levels of E. coli O157:H7 declined to <1 × 10² CFU g^?1 after 5 days. The physiological status of the surviving E. coli was affected by high pressure, sensitizing the cells to pH levels 3 and 4, bile salts at 5% and 10% and mild cooking temperatures of 55–65°C. Conclusions: High‐pressure processing (HPP) reduced E. coli O157:H7 in ground beef by 3 log CFU g^?1 and caused substantial sublethal injury resulting in further log reductions of bacteria during frozen storage. Significance and Impact of the Study: HPP treatment of packaged ground beef has potential in the meat industry for postprocess control of pathogens such as E. coli O157:H7 with enhanced safety of the product.     

Characterization and flocculation properties of biopolymeric flocculant (glycosaminoglycan) produced by Cellulomonas sp. Okoh

Aims

Bioflocculant production potential of an actinobacteria isolated from a freshwater environment was evaluated and the bioflocculant characterized.

Methods and Results

16S rDNA nucleotide sequence and BLAST analysis was used to identify the actinobacteria and fermentation conditions, and nutritional requirements were evaluated for optimal bioflocculant production. Chemical analyses, FTIR, 1H NMR spectrometry and SEM imaging of the purified bioflocculant were carried out. The 16S rDNA nucleotide sequences showed 93% similarities to three Cellulomonas species (strain 794, Cellulomonas flavigena DSM 20109 and Cellulomonas flavigena NCIMB 8073), and the sequences was deposited in GenBank as Cellulomonas sp. Okoh (accession number HQ537132 ). Bioflocculant was optimally produced at an initial pH 7, incubation temperature 30°C, agitation speed of 160 rpm and an inoculum size of 2% (vol/vol) of cell density 1·5 × 10⁸ cfu ml^?1. Glucose (88·09% flocculating activity; yield: 4·04 ± 0·33 g l^?1), (NH₄)₂NO₃ (82·74% flocculating activity; yield: 4·47 ± 0·55 g l^?1) and MgCl₂ (90·40% flocculating activity; yield: 4·41 g l^?1) were the preferred nutritional source. Bioflocculant chemical analyses showed carbohydrate, protein and uronic acids in the proportion of 28·9, 19·3 and 18·7% in CPB and 31·4, 18·7 and 32·1% in PPB, respectively. FTIR and 1H NMR indicated the presence of carboxyl, hydroxyl and amino groups amongst others typical of glycosaminoglycan. SEM imaging revealed horizontal pleats of membranous sheets closely packed.

Conclusion

Cellulomonas sp. produces bioflocculant predominantly composed of glycosaminoglycan polysaccharides with high flocculation activity.

Significance and Impact of the Study

High flocculation activity suggests suitability for industrial applications; hence, it may serve to replace the hazardous flocculant used in water treatment.     

A New Fungal Metabolite and Root Growth-stimulating Activity of Its Methyl Ester

Escherichia coli rodA mutant AOS151 grows as round cells at 30 and 42°C (H. Matsuzawa, K. Hayakawa, T. Sato, and K. Imahori, J. Bacteriol., 115, 436–442 (1973)). The mutant was found to be resistant to mecillinam at both temperatures. lip⁺ transductants were prepared by Pl phage transduction via strain AOS151, the cotransduction frequency of round morphology (Rod^?) at 42°C with the lip gene being about 90%. At 42°C all 54 Rod^? transductants tested were resistant to mecillinam. At 30°C all but two of these Rod^? (at 42°C)-type transductants were rod-shaped, and all were sensitive to mecillinam; the two strains grew as ovoid cells. The original rodA mutant AOS151 probably involves an additional mutation(s), that expresses the round cell shape at lower temperature, whereas the rodA51 mutation alone seems to result in temperature-sensitive expression of round cell morphology and mecillinam resistance. rodA mutant cells cultured at either 30 or 42°C had wild-type penicillin-binding protein 2, judging from penicillin-binding activity, electrophoretic mobility, and thermosensitivity.     

Improved ethanol production of a newly isolated thermotolerant Saccharomyces cerevisiae strain after high-energy-pulse-electron beam

Aims: To isolate thermotolerant Saccharomyces cerevisiae with high‐energy‐pulse‐electron (HEPE) beam, to optimize the mutation strain fermentation conditions for ethanol production and to conduct a preliminary investigation into the thermotolerant mechanisms. Methods and Results: After HEPE beam radiation, the thermotolerant S. cerevisiae strain Y43 was obtained at 45°C. Moreover, the fermentation conditions of mutant Y43 were optimized by L3³ orthogonal experiment. The optimal glucose content and initial pH for fermentation were 20% g l^?1 and 4·5, respectively; peptone content was the most neglected important factor. Under this condition, ethanol production of Y43 was 83·1 g l^?1 after fermentation for 48 h at 43°C, and ethanol yield was 0·42 g g^?1, which was about 81·5% of the theoretical yield. The results also showed that the trehalose content and the expression of the genes MSN2, SSA3 and TPS1 in Y43 were higher than those in the original strain (YE0) under the same stress conditions. Conclusions: A genetically stable mutant strain with high ethanol yield under heat stress was obtained using HEPE. This mutant may be a suitable candidate for the industrial‐scale ethanol production. Significance and Impact of the Study: High‐energy‐pulse‐electron radiation is a new efficient technology in breeding micro‐organisms. The mutant obtained in this work has the advantages in industrial ethanol production under thermostress.     

Enhanced S-adenosyl-l-methionine production in Saccharomyces cerevisiae by spaceflight culture, overexpressing methionine adenosyltransferase and optimizing cultivation

Aims: S‐adenosyl‐l ‐methionine (SAM) is an important biochemical molecule with great potential in the pharmacological and chemotherapeutic fields. In this study, our aims were to enhance SAM production in Saccharomyces cerevisiae. Methods and Results: Through spaceflight culture, a SAM‐accumulating strain, S. cerevisiae H5M147, was isolated and found to produce 86·89% more SAM than its ground control strain H5. Amplified fragment length polymorphism (AFLP) analysis demonstrated that there were genetic variations between strain H5M147 and its ground control. Through recombinant DNA technology, the heterologous gene encoding methionine adenosyltransferase was integrated into the genome of strain H5M147. The recombinant strain H5MR83 was selected because its SAM production was increased by 42·98% when compared to strain H5M147. Furthermore, cultivation conditions were optimized using the one‐factor‐at‐a‐time and Taguchi methods. Under optimal conditions, strain H5MR83 yielded 7·76 g l^?1 of SAM in shake flask, an increase of 536·07% when compared to the strain H5. Furthermore, 9·64 g l^?1 of SAM was produced in fermenter cultivation. Conclusions: A new SAM‐accumulating strain, S. cerevisiae H5MR83, was obtained through spaceflight culture and genetic modification. Under optimal conditions, SAM production was increased to a relative high level in our study. Significance and Impact of the Study: Through comprehensive application of multiple methods including spaceflight culture, genetic modification and optimizing cultivation, the yield of SAM could be increased by 6·4 times compared to that in the control strain H5. The obtained S. cerevisiae H5MR83 produced 7·76 g l^?1 of SAM in the flask cultures, a significant improvement on previously reported results. The SAM production period with S. cerevisiae H5MR83 was 84 h, which is shorter than previously reported results. Saccharomyces cerevisiae H5MR83 has considerable potential for use in industrial applications.     

Comparison of mutant and parent strains of Clostridium acetobutylicum: butyrate uptake at different temperatures

A high butanol producing mutant strain of Clostridium acetobutylicum ATCC 4259 was obtained by chemical mutagenesis. Both mutant and parent strains were evaluated for butyrate uptake using the culture effluents of solventogenic fermentor-2 of the two-fermentor continuous system. Batch incubation of fermentor-2 culture effluents at 37?°C indicated lower butyrate uptake rates for mutant and parent strain, at 0.05 and 0.03?g?l^?1?h^?1, respectively. Increased butyrate uptake rates of 0.33 and 0.26 g l^?1 h^?1 for mutant and parent strain, respectively, were observed when effluents were batch incubated at lower temperature of 30?°C. Butyrate conversion efficiency, at 5?±?0.1 g l^?1 of externally added butyrate, were 98.8% and 96.9% for mutant and parent strain, respectively. Butyrate up to the externally added concentration of 11.4 g l^?1 did not inhibit butyrate uptake. The maximum butyrate consumption at a slightly reduced uptake rate was seen at 10.2 g l^?1 butyrate concentration at 27?°C. Based on the results under different temperatures, the electron flow pattern has been computed and the mechanism for butyrate uptake has been hypothesized.     

Optimization of Fermentation Conditions for Phytase Production by Two Strains of Bacillus licheniformis (LF1 and LH1) Isolated from the Intestine of Rohu, Labeo rohita (Hamilton)

The culture conditions for extracellular production of phytase by two strains of Bacillus licheniformis (LF1 and LH1) isolated from the proximal and distal intestine of rohu (Labeo rohita) were optimized to obtain maximum level of phytase. Both the strains were cultured TSA broth for 24 h at 37 ± 2 °C, when average viable count of 9.75 × 10⁷ cells ml^?1 culture broth was obtained. This was used as the inoculum for the production medium. Sesame (Sesamum indicum) oilseed meal was used as the source of phytic acid (substrate). The effects of moisture, pH, temperature, fermentation period, inoculum size, different nitrogen sources, vitamins and surfactants on phytase production by these two strains were evaluated. Phytase yield was highest (1.87 U in LF1 and 1.57 U in LH1) in solid-state fermentation. Enzyme production in both the isolates increased in an optimum pH range of 5.5–6.5. Minimum phytase production was observed at 50 °C, while maximum production was obtained at 40 °C. To standardize the fermentation period for phytase production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 72 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points for the strain LF1 and up to 2.0 % for the strains LH1. Ammonium sulphate as the nitrogen source was most effective in LF1, while beef extract proved useful to maximize enzyme production by LH1.     

Further Studies on Cynara Rhabdovirus

Cynara rhabdovirus (CyRV) was isolated from symptomless artichoke plants in southern Italy using Nicotiana langsdorffii as susceptible host and immune serum to artichoke latent virus to eliminate this virus from inoculum. CyRV can infect several solanaceous species, has thermal inactivation point of 40-45°C, dilution end point between 10^?2 and 10^?5 and longevity in vitro at 4 and 20°C of 4-5 days and 6-24 h, respectively. It was purified and used for preparing antisera with homologous titre varying from 1: 16 to 1: 64. In decoration tests, the virus did not react against antisera to eggplant mottled dwarf virus (EMDV) and its antiserum did not decorate ivy vein clearing virus (IVCV). Ultrastructural aspects of CyRV infection in Datura stramonium and Nicotiana glutinosa were very like those described for the same virus in the past except for presence of longer virions which were often encountered in infected cells during this study.     

Extracellular acid phosphatase as a minor enzyme during the production of proteinases by Humicola lutea 120–5

Extracellular acid phosphatase was studied as a minor enzyme of the fungal strain Humicola lutea 120–5 having a clear relation to the secretion of acid proteinases. A medium lacking in mineral orthophosphates ensured a fivefold higher yield of phosphatase while the proteinase production was reduced. An acid phosphatase fraction free of proteinase activity was isolated demonstrating a maximum hydrolysis of 4-nitrophenyl-phosphate at a pH of 4.0 and 50°C. The phosphatase catalyzed a partial dephosphorylation of up to 30% of casein at a pH of 3.0 causing a complete substrate precipitation. Both proteinase and phosphatase biosynthesis increased twofold when natural casein was replaced by partially dephosphorylated casein in the cultivation medium.     

Mycelium of Alternaria alternata as a potential biological control agent for Eupatorium adenophorum

The fungus, Alternaria alternata (Fr.) Keissler Strain 501, has been evaluated as a bioherbicide for control of Eupatorium adenophorum Spreng., but the biology of the pathogen–host interaction and the optimal environmental conditions for disease development and effective weed control are unknown. Disease development of A. alternata Strain 501 mycelia on E. adenophorum was assessed under several factors including pathogen inoculum concentration, plant age, dew period duration, post-dew temperature, storage temperature and duration. The minimum inoculum concentration required to kill E. adenophorum seedlings was 3.2×10⁶ mycelial fragment mL^?1. E. adenophorum seedlings at the four-leaf-pair stage were more susceptible than the older plants, especially those at the older than seven-leaf-pair stage. With a dew period of at least 14 h, 100% mortality occurred. The optimal post-dew temperature for disease development was 18–25°C. Storage at <4°C maintained the infectivity of A. alternata strain 501 mycelia on E. adenophorum longer. Using light and scanning electron microscopy to examine the infection process of A. alternata Strain 501 mycelia, it was shown that the time from initiation to completion of infection with mycelia was much shorter (14 h) than with conidia (72 h). It was further shown that mycelial infection occurred predominately through direct penetration at intercellular junctions, while conidial infection occurred predominately through stomatal penetration. This suggests that mycelia are more suitable as infection propagules for A. alternata strain 501 in a bioherbicide for the control of E. adenophorum.     

Factors affecting incidence and severity of leaf spot disease on lettuce caused by Septoria birgitae

In the 1990s during wet seasons a new disease causing brown leaf spots on lettuce (Lactuca sativa) was found for the first time in many lettuce‐growing areas of Austria and Germany. The causal agent, a new pathogenic species called Septoria birgitae, may be responsible for total crop loss. To study how temperature, inoculum density and leaf wetness period influence disease incidence and severity of leaf spot on lettuce caused by S. birgitae, we carried out in vivo experiments in growth chambers and in the field. Additionally, we evaluated the relevance of infected plant debris acting as a primary inoculum source in soil for subsequent crops. S. birgitae produces spores over a wide temperature range between 5°C and 30°C, and can infect plants at temperatures between 10°C and 30°C, with an optimum between 20°C and 30°C. Spores of S. birgitae at a density of at least 10³ conidia mL^–1 are essential for disease outbreak on lettuce. Because leaf wetness is crucial for releasing conidia from pycnidia, we studied the impact of leaf wetness duration on disease development under various temperature conditions. For relevant leaf spot disease development on lettuce in vivo, a leaf wetness duration of at least 24 h and temperatures higher than 10°C were necessary. Leaf spot disease development in the field required several leaf wetness periods longer than 20 h at approximately 15°C at the beginning of crop cultivation. Incorporating S. birgitae infected plant debris in soil as a primary inoculum was not relevant for leaf spot disease outbreak in the next year. However, in cases of continuous cropping of lettuce on the same field and in the same season, Septoria‐infected lettuce debris may become more relevant.     

Inactivation of Escherichia coli by citral

Aims: The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral. Methods and Results: The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 μl l^?1 of citral at pH 4·0 for 24 h at 20°C caused the inactivation of more than 5 log₁₀ cycles of cells starting at an inoculum size of 10⁶ or 10⁷ CFU ml^?1, whereas increasing the cell concentration to 10⁹ CFU ml^?1 caused <1 log₁₀ cycle of inactivation. Escherichia coli showed higher resistance to citral at pH 4·0 than pH 7·0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild‐type strain. Occurrence of sublethal injury to both the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. Conclusions: This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Significance and Impact of the Study: Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.     

Acid proteinases production by humicola lutea cells immobilized in polyhydroxyethylmethacrylate gel

The spores of Humicola lutea entrapped in polyhydroxyethylmethacrylate gel were precultivated in production medium for mycelial formation. The immobilized mycelium was reused in batch mode for acid proteinases production. The influence of precultivation time, initial inoculum gel volume, and gel particle size on the enzyme activity and proteinases production half-life were studied. After 70 h precultivation of the entrapped spores (10 ml initial inoculum volume, 12–27 mm³ gel particle size) maximum proteinases activity of 100–140% (compared with free cells) was registered in 15 reaction cycles. Under the same condition the half-life time was 18 cycles, while for the free cells it was 5 cycles. The main advantage of the polyhydroxyethylmethacylate immobilized H. lutea was the long acid proteinases production half-life at a low concentration of outgrowing cells in the medium.     

Transfer of ampicillin resistance from Salmonella Typhimurium DT104 to Escherichia coli K12 in food

Aims: To investigate the transfer of antibiotic resistance from a donor Salmonella Typhimurium DT104 strain to a recipient Escherichia coli K12 strain. Methods and Results: Mating experiments were conducted in broth, milk and ground meat (beef) at incubation temperatures of 4, 15, 25 and 37°C for 18 and 36 h. Ampicillin‐resistance transfer was observed at similar frequencies in all transfer media at 25 and 37°C (10^?4 to 10^?5 log₁₀ CFU ml g^?1, transconjugants per recipient) for 18 h. At 15°C, transfer was observed in ground meat in the recipient strain (10^?6, log₁₀ CFU g^?1, transconjugants per recipient), but not in broth or milk. At 4°C, transfer did not occur in any of the examined mediums. Further analysis of the E. coli K12 nal^R transconjugant strain revealed the presence of a newly acquired plasmid (21 kbp) bearing the β‐lactamase gene bla_TEM. Transconjugants isolated on the basis of resistance to ampicillin did not acquire any other resistant markers. Conclusion: This study demonstrates the transfer of antibiotic resistance in food matrices at mid‐range temperatures. Significance and Impact of the Study: It highlights the involvement of food matrices in the dissemination of antibiotic‐resistant genes and the evolution of antibiotic‐resistant bacteria.     

Water loss and metabolic activity in bed bug eggs (Cimex lectularius)

Few studies have evaluated water loss and respiratory activity of insect eggs, particularly insects that are known to live within indoor environments. The present study quantifies water loss and respiratory activity for the eggs of a re‐emerging indoor pest of human environments Cimex lectularius (L.). Water loss is measured gravimetrically and calculated as a function of chorion permeability. For these studies, bed bug eggs are placed at 0% relative humidity and repeatedly weighed over 48 h. Temperature effects and bed bug strain differences on the standard metabolic rate (SMR) and respiratory quotient are measured using closed system respirometry. The SMR (; mL g^?1 h^?1) is measured for two field strain bed bugs and compared with a laboratory strain held at one temperature (25 °C). The standard metabolic rate is measured for Harlan (laboratory) strain bed bug eggs at six different temperatures (15, 20, 25, 30, 35 and 39 °C). Total water loss is not significantly different between all three strains. However, water loss across the chorion (chorion permeability) is significantly different between the Harlan laboratory strain and the two field collected strains. Standard metabolic rates for Harlan (laboratory) strain bed bug eggs increase with temperatures from 15 to 35 °C but decline at 39 °C. Overall, the Harlan bed bug eggs have the largest standard metabolic rates (0.18 ± 0.05 mL g^?1 h^?1) compared with the Epic Center strain eggs (0.14 ± 0.03 mL g^?1 h^?1) and Richmond strain eggs (0.16 ± 0.04 mL g^?1 h^?1), although this difference is not significant.     

Effect of temperature,cultivars, injury of root and inoculums load of Ralstonia solanacearum to cause bacterial wilt of tomato

Bacterial wilt, caused by Ralstonia solanacearum , is responsible for severe losses in tomato crops in the world. In the present study, the effect of temperature, cultivars of tomato, injury of root system and inoculums load of R. solanacearum to cause bacterial wilt disease under control conditions was undertaken. Three strains UTT-25, HPT-3 and JHT-5 of R. solanacearum were grown at 5–40?°C in vitro to study, the effect of temperature on the growth of bacteria and maximum growth was found at 30?°C after 72?h in all the strains. Twenty-one days old seedlings of two cultivars of tomato i.e. N-5 (moderately resistant) and Pusa Ruby (highly susceptible) were transplanted into the pots and inoculated with R. solanacearum strain UTT-25 (5 × 10⁸?cfu/ml), mechanically injured and uninjured roots of the plant. The plants were allowed to grow at 20, 25, 30 and 35?°C at National Phytotron Facility, IARI, New Delhi to study the effect of temperature on intensity of bacterial wilt disease. Maximum wilt disease intensity was found 98.73 and 95.9 % in injured roots of Pusa Ruby and N-5 cultivars of tomato at 35?°C on 11th days of inoculation, respectively. However, no wilt disease was observed in both the cultivars at 20?°C up to 60?days. For detection of R. solanacearum from asymptomatic tomato plants, hrpB-based sequence primers (Hrp_rs2F and Hrp_rs2R) amplified at 323?bp was used in bio-PCR to detect R. solanacearum from crown, mid part of stem and upper parts of the plant. Another experiment was conducted to find out the inoculum potential of R. solanacearum strain UTT-25 to cause bacterial wilt in susceptible cultivar Pusa Ruby. The bacteria were inoculated at concentration of bacterial suspension 10 to 10¹⁰?cfu/ml in injured and uninjured roots of the plants separately and injured root accelerated wilt incidence and able to cause wilt disease 63.3% by 100?cfu/ml of R. solanacearum, while no disease appeared at 10?cfu/ml on the 11th day of inoculation in injured and uninjured roots of the plant.     

Production of acid proteinase by Humicola lutea 120-5 immobilized in mixed photo-cross-linked polyvinyl alcohol and calcium-alginate beads

Humicola lutea 120-5 spores were immobilized in a mixed photo-crosslinked polyvinyl alcohol and calcium-alginate gel. Maximum enzyme synthesis was established with 1:8 (v:v) gel beads: growth medium inoculum and 48 h duration of one cycle. The free cells were very unstable in replacement fermentations. The operational stability of the immobilized system indicated the possibility of the application of Humicola lutea 120-5 in a semi-continuous process for the production of acid proteinase.     

Quantitative proteomic profiling reveals photosynthesis responsible for inoculum size dependent variation in Chlorella sorokiniana

High density cultivation is essential to industrial production of biodiesel from microalgae, which involves in variations of micro‐environment around individual cells, including light intensity, nutrition distribution, other abiotic stress and so on. To figure out the main limit factor in high inoculum cultivation, a quantitative proteomic analysis (iTRAQ‐on‐line 2‐D nano‐LC/MS) in a non‐model green microalga, Chlorella sorokiniana, under different inoculum sizes was conducted. The resulting high‐quality proteomic dataset consisted of 695 proteins. Using a cutoff of P < 0.05, 241 unique proteins with differential expression levels were identified between control and different inoculum sizes. Functional analysis showed that proteins participating in photosynthesis (light reaction) and Calvin cycle (carbon reaction pathway) had highest expression levels under inoculum size of 1 × 10⁶ cells mL^?1, and lowest levels under 1 × 10⁷ cells mL^?1. Canonical correlation analysis of the photosynthesis related proteins and metabolites biomarkers showed that a good correlation existed between them (canonical coefficient was 0.987), suggesting photosynthesis process greatly affected microalgae biodiesel productivity and quality. Proteomic study of C. sorokiniana under different illuminations was also conducted to confirm light intensity as a potential limit factor of high inoculum size. Nearly two thirds of proteins showed up‐regulation under the illumination of 70–110 µmol m^?2 s^?1, compared to those of 40 µmol m^?2 s^?1. This result suggested that by elegantly adjusting light conditions, high cell density cultivation and high biodiesel production might be achieved. Biotechnol. Bioeng. 2013; 110: 773–784. © 2012 Wiley Periodicals, Inc.