Investigation on alpha-galactosidase production by Streptomyces griseoloalbus in a forcefully aerated packed-bed bioreactor operating in solid-state fermentation condition

Solid-state fermentation (SSF) was carried out for the production of extracellular alpha-galactosidase by Streptomyces griseoloalbus. Soybean flour was the best solid substrate for alpha-galactosidase production. Packed-bed bioreactor performed well in enhancing the enzyme yield and resulted in a highest yield of 197.2 +/- 0.02 U/gds with a forced aeration of 2 vvm, which was approximately twofold the yield obtained in flasks. The alpha-galactosidase production was growth-associated, and the highest enzyme yield was obtained after 96 h of incubation. It is significant that this is the first report on alpha-galactosidase production by a filamentous bacterium in SSF using packed-bed bioreactor.     

Effect of operating conditions on biomass growth during citric acid production by solid-state fermentation

The effect of aeration, agitation, and bed loading on biomass and product concentration during citric acid biosynthesis by solid-state fermentation was investigated. For this purpose, Aspergillus niger S was cultivated on sugar beet pulp in a 4.5 dm³ horizontal rotating drum bioreactor. The results suggest that the parameters examined have a remarkable effect on the quantity of biomass being formed and on the product concentration. The maximum citric acid production (about 150 g per kg of the substrate dry matter) was obtained under the following conditions: aeration rate 0.2 dm³ kg⁻¹ min⁻¹, mixing (periodical) 1 min once an hour, and bed loading 30 % of the bioreactor working volume. However, these values did not favour biomass formation. Moreover, it was found that accumulation of the product reached its maximum when the amount of biomass was minimal (approximately 252-29 g per kg of the substrate dry matter) under the conditions involved. Presented at the 34th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 21–25 May 2007.     

Development of a bionematicide with Paecilomyces lilacinus to control Meloidogyne incognita

Root-knot disease caused by Meloidogyne incognita is a matter of grave concern because it affects several economically important crop plants. The use of solid-state fermentation (SSF) may help to elaborate efficient formulations with fungi to be employed in the biologic control of nematodes. Attempts were made to select low-cost substrates for spore production of a strain of Paecilomyces lilacinus with known nematicide capacity. Coffee husks, cassava bagasse, and defatted soybean cake were utilized as substrates, and sugarcane bagasse was used as support. Fermentations were carried out in flasks covered with filter paper at 28°C for 10 d. The products obtained by SSF were evaluated for their nematicide activity in pot experiments containing one seedling of the plant Coleus inoculated with the nematode M. incognita. The plants were evaluated 2 mo after inoculation. Fermented products showed a reduction in the number of nematodes. The best results were obtained with defatted soybean cake, which showed almost 100% reduction in the number of nematodes; the reduction with coffee husk was 80% and with cassava bagasse was about 60%.     

Simultaneous saccharification and fermentation of cassava bagasse for l-(+)-lactic acid production using Lactobacilli

Saccharification and fermentation of cassava (Manihot esculenta) bagasse was carried out in a single step for the production of L-(+)-lactic acid by Lactobacillus casei and Lactobacillus delbrueckii. Using 15.5% w/v of cassava bagasse as the raw material, a maximum starch to lactic acid conversion of 96% was obtained with L. casei with a productivity rate of 1.40mg/mL·h and maximum yield of 83.8 mg/mL. It was 94% with L. delbrueckii with a productivity rate of 1.36 mg/mL·h. and maximum yeild of 81.9 mg/mL. Supplementation of bagasse with 0.01% w/v MnCl₂ showed positive influence on the lactic acid production by L. casei.     

Solid-State Fermentation of Palm Kernel Cake with <Emphasis Type="Italic">Aspergillus flavus</Emphasis> in Laterally Aerated Moving Bed Bioreactor

Solid-state fermentation (SSF) was employed to enhance the nutritive values of palm kernel cake (PKC) for poultry feeding. Aspergillus flavus was isolated from local PKC and utilized to increase the mannose content of PKC via the degradation of β-mannan in PKC; evaluation was done for batch SSF in Erlenmeyer flasks and in a novel laterally aerated moving bed (LAMB) bioreactor. The optimum condition for batch SSF in flasks was 110% initial moisture content, initial pH 6.0, 30 °C, 855 μm particle size, and 120 h of fermentation, yielding 90.91 mg mannose g⁻¹ dry PKC (5.9-fold increase). Batch SSF in the LAMB at the optimum condition yielded 79.61 mg mannose g⁻¹ dry PKC (5.5-fold increase) within just 96 h due to better heat and mass transfer when humidified air flowed radially across the PKC bed. In spite of a compromise of 12% reduction in mannose content when compared with the flasks, the LAMB facilitated good heat and mass transfer, and improved the mannose content of PKC in a shorter fermentation period. These attributes are useful for batch production of fermented PKC feed in an industrial scale.     

Continuous Ethanol Production from Cassava Through Simultaneous Saccharification and Fermentation by Self-Flocculating Yeast Saccharomyces Cerevisiae CHFY0321

In this study, a fermentor consisting of four linked stirred towers that can be used for simultaneous saccharification and fermentation (SSF) and for the accumulation of cell mass was applied to the continuous production of ethanol using cassava as the starchy material. For the continuous process with SSF, the pretreated cassava liquor and saccharification enzyme at total sugar concentrations of 175 g/L and 195 g/L were continuously fed to the fermentor with dilution rates of 0.014, 0.021, 0.031, 0.042, and 0.05 h⁻¹. Considering the maximum saccharification time, the highest volumetric productivity and ethanol yield were observed at a dilution rate of 0.042 h⁻¹. At dilution rates in the range of 0.014 h⁻¹ to 0.042 h⁻¹, high production rates were observed, and the yeast in the first to fourth fermentor showed long-term stability for 2 months with good performance. Under the optimal culture conditions with a feed sugar concentration of 195 g/L and dilution rate of 0.042 h⁻¹, the ethanol volumetric productivity and ethanol yield were 3.58 g/L∙h and 86.2%, respectively. The cell concentrations in the first to fourth stirred tower fermentors were 74.3, 71.5, 71.2, and 70.1 g dry cell/L, respectively. The self-flocculating yeast, Saccharomyces cerevisiae CHFY0321, developed by our group showed excellent fermentation results under continuous ethanol production.     

Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6

The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases. In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of 8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature, medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase productivity by Aspergillus niger C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme production and also for enzyme characterization.     

Early phase process scale-up challenges for fungal and filamentous bacterial cultures

Culture pelleting and morphology has a strong influence on process productivity and success for fungal and filamentous bacterial cultures. This impact is particularly evident with early phase secondary metabolite processes with limited process definition. A compilation of factors affecting filamentous or pelleting morphology described in the literature indicates potential leads for developing process-specific control methodologies. An evaluation of the factors mediating citric acid production is one example of an industrially important application of these techniques. For five model fungal and filamentous bacterial processes in an industrial fermentation pilot plant, process development strategies were developed and effectively implemented with the goal of achieving reasonable fermentation titers early in the process development cycle. Examples of approaches included the use of additives to minimize pelleting in inoculum shake flasks, the use of large-volume frozen bagged inoculum obtained from agitated seed fermentors, and variations in production medium composition and fermentor operating conditions. Results were evaluated with respect to productivity of desired secondary metabolites as well as process scalability. On-line measurements were utilized to indirectly evaluate the cultivation impact of changes in medium and process development. Key laboratory to pilot plant scale-up issues also were identified and often addressed in subsequent cultivations.     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

Research on the Solid State Fermentation of Jerusalem Artichoke Pomace for Producing <Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">R</Emphasis>-2,3-Butanediol by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> ZJ-9

R,R-2,3-butanediol (R,R-2,3-BD) was produced by Paenibacillus polymyxa ZJ-9, which was capable of utilizing inulin without previous hydrolysis. The Jerusalem artichoke pomace (JAP) derived from the conversion of Jerusalem artichoke powder into inulin extract, which was usually used for biorefinery by submerged fermentation (SMF), was utilized in solid state fermentation (SSF) to produce R,R-2,3-BD. In this study, the fermentation parameters of SSF were optimized and determined in flasks. A novel bioreactor was designed and assembled for the laboratory scale-up of SSF, with a maximum yield of R,R-2,3-BD (67.90 g/kg (JAP)). This result is a 36.3% improvement compared with the flasks. Based on the same bath of Jerusalem artichoke powder, the total output of R,R-2,3-BD increased by 38.8% for the SSF of JAP combined with the SMF of inulin extraction. Overall, the utilization of JAP for R,R-2,3-BD production was beneficial to the comprehensive utilization of Jerusalem artichoke tuber.     

Improvement on Citric Acid Production in Solid-state Fermentation by <Emphasis Type="Italic">Aspergillus niger</Emphasis> LPB BC Mutant Using Citric Pulp

Citric acid (CA) production has been conducted through a careful strain selection, physical–chemical optimization and mutation. The aim of this work was to optimize the physical–chemical conditions of CA production by solid-state fermentation (SSF) using the Aspergillus niger LPB BC strain, which was isolated in our laboratory. The parental and mutant strain showed a good production of CA using citric pulp (CP) as a substrate. The physical–chemical parameters were optimized and the best production was reached at 65% moisture, 30 °C and pH 5.5. The influence of the addition of commercial and alternative sugars, nitrogen sources, salts, and alcohols was also studied. The best results (445.4 g of CA/kg of CP) were obtained with sugarcane molasses and 4% methanol (v/w). The mutagenesis induction of LPB BC was performed with UV irradiation. Eleven mutant strains were tested in SSF where two mutants showed a higher CA production when compared to the parental strain. A. niger LPB B3 produced 537.6 g of CA/kg of CP on the sixth day of fermentation, while A. niger LPB B6 produced 616.5 g of CA/kg of CP on the fourth day of fermentation, representing a 19.5% and 37% gain, respectively.     

Tannase production by solid state fermentation of cashew apple bagasse

The ability of Aspergillus oryzae for the production of tannase by solid state fermentation was investigated using cashew apple bagasse (CAB) as substrate. The effect of initial water content was studied and maximum enzyme production was obtained when 60 mL of water was added to 100.0 g of CAB. The fungal strain was able to grow on CAB without any supplementation but a low enzyme activity was obtained, 0.576 U/g of dry substrate (g(ds)). Optimization of process parameters such as supplementation with tannic acid, phosphorous, and different organic and inorganic nitrogen sources was studied. The addition of tannic acid affected the enzyme production and maximum tannase activity (2.40 U/g(ds)) was obtained with 2.5% (w/w) supplementation. Supplementation with ammonium nitrate, peptone, and yeast extract exerted no influence on tannase production. Ammonium sulphate improved the enzyme production in 3.75-fold compared with control. Based on the experimental results, CAB is a promising substrate for solid state fermentation, enabling A. oryzae growth and the production of tannase, with a maximum activity of 3.42 U/g(ds) and enzyme productivity of 128.5x10(-3) U x g(ds)(-1) x h(-1).     

Immunomodulatory Properties of Filamentous Fungi Cultivated through Solid-State Fermentation on Rapeseed Meal

Water extracts from solid-state fermentation (SSF) on rapeseed meal using filamentous fungi exhibit interesting immunomodulatory activities in vitro. Immunomodulation was determined by the capacity of the compounds to activate blood neutrophils and to influence cytokine production in human peripheral blood mononuclear cells (PBMC) and mouse bone marrow-derived macrophages (BMDM). Among the strains tested, Aspergillus sojae mycelium and SSF extracts were the most promising in terms of enhancing the immune response. The filamentous fungus was also successfully cultivated in a pre-pilot bioreactor with forced aeration. The results indicated that the extracts not only activated blood neutrophils but also significantly modulated IL-1β cytokine levels with lipopolysaccharide (LPS)-stimulated PBMC and BMDM without any cytotoxicity in immune cells. IL-1β was down-regulated in a dose-dependent manner in the presence of A. sojae crude mycelium and SSF extract with PBMC, which indicated that there was an anti-inflammatory activity, whereas IL-1β secretion was up-regulated in the presence of stimulated BMDM with the highest concentration that was tested (100 μg/mL). The non-fermented rapeseed had no effect at the same concentration. SSF culture, as a natural product, may be a good source for the development of functional feed with an immunostimulating effect or could potentially be used in medicinal applications.     

Conversion of sugarcane bagasse to carboxylic acids using a mixed culture of mesophilic microorganisms

Using the MixAlco process, biomass can be converted into carboxylic acids, which can be chemically converted into mixed alcohol fuels. This study focused on the use of countercurrent fermentation to anaerobically convert sugarcane bagasse and chicken manure to mixed carboxylic acids using a mixed culture of mesophilic microorganisms from terrestrial and marine sources. Bagasse was pretreated with lime to increase digestibility. The continuum particle distribution model (CPDM) simulated continuous fermentors based on data collected from batch experiments. This model saves considerable time in determining optimum operating conditions. For an 80% bagasse/20% chicken manure fermentation with terrestrial inoculum at a volatile solids loading rate (VSLR) of 7.36 g/(L of liquid·d) and a liquid residence time (LRT) of 8.88 d, total carboxylic acid productivity, total acid selectivity, and yield were 2.49 g/(L of liquid·d), 0.581 g of total acid/g of VS digested, and 0.338 g of total acid/g of VS fed, respectively, at a concentration of 18.7 g of total acid/L. At the same VSLR and LRT, fermentation with marine inoculum gave higher total acid productivity, total acid selectivity, and yield than fermentation with terrestrial inoculum. For an 80% bagasse/20% chicken manure fermentation with marine inoculum at a VSLR of 3.83 g/(L of liquid·d) and an LRT of 12.1 d, total carboxylic acid productivity, total acid selectivity, and yield were 1.38 g/(L of liquid·d), 0.667 g of total acid/g of VS digested, and 0.359 g of total acid/g of VS fed, respectively, at a concentration of 16.2 g of total acid/L.     

Production of lovastatin by wild strains of Aspergillus terreus

A wild fungal strain of Aspergillus terreus, labeled as PM3, was isolated by using the Candida albicans bioassay and confirmed by 18S r DNA analyses. Lovastatin was produced by submerged and solid state fermentations. Of the 30 isolated fungal strains, 11 showed lovastatin production with Aspergillus terreus PM3 being the best with a yield of 240 mg/L at the 10th day of submerged fermentation. Carboxymethylcellulose had a stimulatory effect on lovastatin production. It restricted uncontrolled filamentous growth, induced pellet formation and, thereby, improved lovastatin yield. In solid state fermentation (SSF), of the agro wastes from five crops (bran of wheat and rice, husks of red gram and soybean, and green gram straw), wheat bran showed maximum lovastatin production (12.5 mg/g of dry substrate) at pH 7.1 and a temperature of 30 +/- 2 degrees C. Development of a lovastatin production process based on wheat bran as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Production of hyaluronic acid by Streptococcus

The effects of the addition of lysozyme and forced aeration on the rheological properties and production of hyaluronic acid by Streptococcus zooepidemicus were investigated. Lysozyme was added to the culture broth in two pulses during the exponential and stationary phases of a fermentation carried out in a rotary shaker (150 rpm), using 200 mL Erlenmeyer flasks. The effect of aeration was evaluated by feeding air into a 2.5 L fermentor at a 2 vvm rate. The effects were analyzed in terms of concentration, viscosity, viscoelasticity, and molecular weight of the hyaluronic acid produced.     

Production of coconut aroma by fungi cultivation in solid-state fermentation

The production of 6-pentyl-α-pyrone (6-PP), an unsaturated d-lactone with a strong coconut-like aroma was studied and compared with liquid and solid substrates. A fungi strain that produces coconut aroma compound was selected. The liquid medium of the submerged culture was used to impregnate a solid support of sugarcane bagasse in SSF (Solid State Fermentation). This substrate was adequate for growth and aroma production; the concentration obtained using SSF was higher than using liquid fermentation process. In the present work, it is demonstrated that, by solid-state-fermentation process, it is possible to produce 6-PP. The amount of 6-PP produced using a solid state substrate, following a 5 d culture, was 3 mg/g dry matter. Therefore, the amount of 6-PP produced during solid-state-fermentation process is higher than that reported in literature for submerged process.     

Fungal Pretreatment by Phanerochaete chrysosporium for Enhancement of Biogas Production from Corn Stover Silage

Corn stover silage (CSS) was pretreated by Phanerochaete chrysosporium in solid-state fermentation (SSF), to enhance methane production via subsequent anaerobic digestion (AD). Effects of washing of corn stover silage (WCSS) on the lignocellulosic biodegradability in the fungal pretreatment step and on methane production in the AD step were investigated with comparison to the CSS. It was found that P. chrysosporium had the degradation of cellulose, hemicellulose, and lignin of CSS up to 19.9, 32.4, and 22.6 %, respectively. Consequently, CSS pretreated by 25 days achieved the highest methane yield of 265.1 mL/g volatile solid (VS), which was 23.0 % higher than the untreated CSS. However, the degradation of cellulose, hemicellulose, and lignin in WCSS after 30 days of SSF increased to 45.9, 48.4, and 39.0 %, respectively. Surface morphology and Fourier-transform infrared spectroscopy analyses also demonstrated that the WCSS improved degradation of cell wall components during SSF. Correspondingly, the pretreatment of WCSS improved methane production by 19.6 to 32.6 %, as compared with untreated CSS. Hence, washing and reducing organic acids (such as lactic acid, acetic acid, propionic acid, and butyric acid) present in CSS has been proven to further improve biodegradability in SSF and methane production in the AD step.     

Maximizing the xylitol production from sugar cane bagasse hydrolysate by controlling the aeration rate

Batch fermentations of sugar cane bagasse hemicellulosic hydrolysate treated for removing the inhibitors of the fermentation were performed byCandida guilliermondii FTI20037 for xylitol production. The fermentative parameters agitation and aeration rate were studied aiming the maximization of xylitol production from this agroindustrial residue. The maximal xylitol volumetric productivity (0.87 g/L h) and yield (0.67 g/g) were attained at 400/min and 0.45 v.v.m. (K_La 27/h). According to the results, a suitable control of the oxygen input permitting the xylitol formation from sugar cane bagasse hydrolysate is required for the development of an efficient fermentation process for large-scale applications.     

Ethanol production from corn starch in a fluidized-bed bioreactor

The production of ethanol from industrial dry-milled corn starch was studied in a laboratory-scale fluidized-bed bioreactor using immobilized biocatalysts. Saccharification and fermentation were carried out either simultaneously or separately. Simultaneous saccharification and fermentation (SSF) experiments were performed using small, uniform κ-carrageenan beads (1.5–2.5 mm in diameter) of co-immobilized glucoamylase and Zymomonas mobilis. Dextrin feeds obtained by the hydrolysis of 15% drymilled corn starch were pumped through the bioreactor at residence times of 1.5–4h. Single-pass conversion of dextrins ranged from 54–89%, and ethanol concentrations of 23–36 g/L were obtained at volumetric productivities of 9–15 g/L-h. Very low levels of glucose were observed in the reactor, indicating that saccharification was the rate-limiting step. In separate hydrolysis and fermentation (SHF) experiments, dextrin feed solutions of 150–160 g/L were first pumped through an immobilized-glucoamylase packed column. At 55°C and a residence time of 1 h, greater than 95% conversion was obtained, giving product streams of 162–172 g glucose/L. These streams were then pumped through the fluidized-bed bioreactor containing immobilized Z. mobilis. At a residence time of 2 h, 94% conversion and ethanol concentration of 70 g/L were achieved, resulting in an overall process productivity of 23 g/L-h. Atresidence times of 1.5 and 1 h, conversions of 75 and 76%, ethanol concentrations of 49 and 47 g/L, and overall process productivities of 19 and 25 g/L-h, respectively, were achieved.