Production of multiple extracellular enzyme activities by novel submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis> for ethanol production from raw cassava flour

Cassava is a starch-containing root crop that is widely used as a raw material in a variety of industrial applications, most recently in the production of fuel ethanol. In the present study, ethanol production from raw (uncooked) cassava flour by simultaneous saccharification and fermentation (SSF) using a preparation consisting of multiple enzyme activities from Aspergillus kawachii FS005 was investigated. The multi-activity preparation was obtained from a novel submerged fermentation broth of A. kawachii FS005 grown on unmilled crude barley as a carbon source. The preparation was found to consist of glucoamylase, acid-stable α-amylase, acid carboxypeptidase, acid protease, cellulase and xylanase activities, and exhibited glucose and free amino nitrogen (FAN) production rates of 37.7 and 118.7 mg/l/h, respectively, during A. kawachii FS005-mediated saccharification of uncooked raw cassava flour. Ethanol production from 18.2% (w/v) dry uncooked solids of raw cassava flour by SSF with the multi-activity enzyme preparation yielded 9.0% (v/v) of ethanol and 92.3% fermentation efficiency. A feasibility study for ethanol production by SSF with a two-step mash using raw cassava flour and the multi-activity enzyme preparation manufactured on-site was verified on a pilot plant scale. The enzyme preparation obtained from the A. kawachii FS005 culture broth exhibited glucose and FAN production rates of 41.1 and 135.5 mg/l/h, respectively. SSF performed in a mash volume of about 1,612 l containing 20.6% (w/v) dry raw cassava solids and 106 l of on-site manufactured A. kawachii FS005 culture broth yielded 10.3% (v/v) ethanol and a fermentation efficiency of 92.7%.     

Direct fermentation of raw starch using a Kluyveromyces marxianus strain that expresses glucoamylase and Alpha‐amylase to produce ethanol

Raw starch and raw cassava tuber powder were directly and efficiently fermented at elevated temperatures to produce ethanol using the thermotolerant yeast Kluyveromyces marxianus that expresses α‐amylase from Aspergillus oryzae as well as α‐amylase and glucoamylase from Debaryomyces occidentalis. Among the constructed K. marxianus strains, YRL 009 had the highest efficiency in direct starch fermentation. Raw starch from corn, potato, cassava, or wheat can be fermented at temperatures higher than 40°C. At the optimal fermentation temperature 42°C, YRL 009 produced 66.52 g/L ethanol from 200 g/L cassava starch, which was the highest production among the selected raw starches. This production increased to 79.75 g/L ethanol with a 78.3% theoretical yield (with all cassava starch were consumed) from raw cassava starch at higher initial cell densities. Fermentation was also carried out at 45 and 48°C. By using 200 g/L raw cassava starch, 137.11 and 87.71 g/L sugar were consumed with 55.36 and 32.16 g/L ethanol produced, respectively. Furthermore, this strain could directly ferment 200 g/L nonsterile raw cassava tuber powder (containing 178.52 g/L cassava starch) without additional nutritional supplements to produce 69.73 g/L ethanol by consuming 166.07 g/L sugar at 42°C. YRL 009, which has consolidated bioprocessing ability, is the best strain for fermenting starches at elevated temperatures that has been reported to date. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:338–347, 2014     

Production of ethanol from raw cassava starch by a nonconventional fermentation method

Raw cassava root starch was transformed into ethanol in a one-step process of fermentation, in which are combined the conventional processes of liquefaction, saccharification, and fermentation to alcohol. Aspergillus awamori NRRL 3112 and Aspergillus niger were cultivated on wheat bran and used as Koji enzymes. Commercial A. niger amyloglucosidase was also used in this experiment. A raw cassava root homogenate–enzymes–yeast mixture fermented optimally at pH 3.5 and 30°C, for five days and produced ethanol. Alcohol yields from raw cassava roots were between 82.3 and 99.6%. Fungal Koji enzymes effectively decreased the viscosity of cassava root fermentation mashes during incubation. Commercial A. niger amyloglucosidase decreased the viscosity slightly. Reduction of viscosity of fermentation mashes was 40, 84, and 93% by commercial amyloglucosidase, A. awamori, and A. niger enzymes, respectively. The reduction of viscosity of fermentation mashes is probably due to the hydrolysis of pentosans by Koji enzymes.     

Enhanced production of raw starch degrading enzyme using agro-industrial waste mixtures by thermotolerant Rhizopus microsporus for raw cassava chip saccharification in ethanol production

In the present study, solid-state fermentation for the production of raw starch degrading enzyme was investigated by thermotolerant Rhizopus microsporus TISTR 3531 using a combination of agro-industrial wastes as substrates. The obtained crude enzyme was applied for hydrolysis of raw cassava starch and chips at low temperature and subjected to nonsterile ethanol production using raw cassava chips. The agro-industrial waste ratio was optimized using a simplex axial mixture design. The results showed that the substrate mixture consisting of rice bran:corncob:cassava bagasse at 8?g:10?g:2?g yielded the highest enzyme production of 201.6?U/g dry solid. The optimized condition for solid-state fermentation was found as 65% initial moisture content, 35°C, initial pH of 6.0, and 5?×?10⁶ spores/mL inoculum, which gave the highest enzyme activity of 389.5?U/g dry solid. The enzyme showed high efficiency on saccharification of raw cassava starch and chips with synergistic activities of commercial α-amylase at 50°C, which promotes low-temperature bioethanol production. A high ethanol concentration of 102.2?g/L with 78% fermentation efficiency was achieved from modified simultaneous saccharification and fermentation using cofermentation of the enzymatic hydrolysate of 300?g raw cassava chips/L with cane molasses.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.     

Original properties of ropy strains of Lactobacillus plantarum isolated from the sour cassava starch fermentation

C. FIGUEROA, A.M. DAVILA AND J. POURQUIÉ 1997. Sour cassava starch is the result of a lactic fermentation of raw cassava starch followed by sun drying. Lactobacillus plantarum strains are commonly isolated from this fermentation. Among them, a particular group of strains has been characterized by a strong ropy phenotype, the production of a thickening factor under submerged cultures conditions, a low nutritional requirement for organic nitrogen and an absence of amylolytic activity. However, these strains have been shown to thrive on starch, through commensalistic interactions with amylolytic lactic acid bacteria. These results explain the frequent occurrence of ropy, non-amylolytic strains in sour starch fermentation, and support the hypothesis of exopolysaccharides production during this fermentation.     

Extracellular amylase production by cassava-fermenting bacteria

Summary Fermentation of cassava tubers was accompanied by a gradual decrease in pH, increased amylase activity in the steep liquor, and increased microbial load and lactic acid concentration. Amylase-producing bacterial strains associated with cassava fermentation were isolated and identified asBacillus subtilis, Bacillus licheniformis andBacillus cereus. The pH optima for the partially purified enzymes of these organisms were 7.0, 5.5 and 7.5, whilst their temperature optima were 30, 37 and 80°C. There was no significant difference in amylase activities when starch, dextrin, amylopectin, glucose and maltose were used as growth substrates.     

Diversity and technological properties of predominant lactic acid bacteria from fermented cassava used for the preparation of Gari, a traditional African food

Traditional fermentation of cassava is dominated by a lactic acid bacteria (LAB) population. Fermentation is important for improving product flavour and aroma as well as safety, especially by reduction of its toxic cyanogenic glucosides. The production of Gari from cassava in Benin typically occurs on a household or small industrial scale, and consequently suffers from inconsistent product quality and may not always be safe for consumption. Therefore, the diversity of LAB from a typical cassava fermentation for the preparation of Gari, and their technologically relevant characteristics were investigated with a view towards selection of appropriate starter cultures. A total of 139 predominant strains isolated from fermenting cassava were identified using phenotypic tests and genotypic methods such as rep-PCR and RAPD-PCR. DNA-DNA hybridisation and sequencing of the 16S rRNA genes were done for selected strains. Lactobacillus plantarum was the most abundantly isolated species (54.6% of isolates), followed by Leuconostoc fallax (22.3%) and Lactobacillus fermentum (18.0%). Lactobacillus brevis, Leuconostoc pseudomesenteroides and Weissella paramesenteroides were sporadically isolated. The L. plantarum strains were shown to be better acid producers and capable of faster acid production than the L. fallax or L. fermentum strains. The incidence of beta-glucosidase (linamarase) activity was also highest among strains of this species. Production of antagonistic substances such as H2O2 and bacteriocins, however, was more common among L. fallax and L. fermentum strains. Strains of all three species were capable of utilising the indigestible sugars raffinose and stachyose. Therefore, a starter culture containing a mixture of strains from all three species was recommended.     

Design and evaluation of a Lactobacillus manihotivorans species-specific rRNA-targeted hybridization probe and its application to the study of sour cassava fermentation

Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.     

Characterization and distribution of lactic acid bacteria in cassava fermentation during fufu production

One hundred and thirty four lactic acid bacterial strains isolated during the 96-h period of cassava fermentation for fufu production were identified. The spectrum and proportion of the strains include Lactobacillus plantarum , 81%; Leuconostoc mesenteroides , 16%; Lact. cellobiosus , 15%; Lact. brevis , 9%; Lact, coprophilus , 5%; Lact. lactis , 4%; Leuc. lactis , 3% and Lact. bulgaricus , 1%. The isolates were characterized into strains. The succession among the lactic isolates was established. Lactobacillus plantarum was identified as the most dominant lactic acid bacterial strain involved in the fermentation.     

Biodiesel production from algal oil using cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) as feedstock

As a potential source of biomass supplies, cassava (Manihot esculenta Crantz) has been studied for bioethanol production, but not for the production of biodiesel. In this study, we used cassava hydrolysate as an alternative carbon source for the growth of microalgae (Chlorella protothecoides) which accumulated oil in vivo, with high oil content up to 53% by dry mass under a 5-L scale fermentation condition. The oils were extracted and converted into biodiesel by transesterification. The biodiesel obtained consisted of mainly unsaturated fatty acids methyl ester (over 82%), cetane acid methyl ester, linoleic acid methyl ester, and oleic acid methyl ester. This work suggests the feasibility of an alternative choice for producing biodiesel from cassava by microalgae fermentation. We report herewith the optimized condition for the fermentation and for the hydrolysis of cassava as the carbon source.     

Ethanol production by <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> CHZ2501 from industrial starch feedstocks

The optimal conditions of ethanol fermentation process by Zymomonas mobilis CHZ2501 were investigated. Brown rice, naked barley, and cassava were selected as representatives of the starch-based raw material commercially available for ethanol production. Considering enzyme used for saccharification of starch, the ethanol productivity with complex enzyme was higher than glucoamylase. With regards to the conditions of saccharification, the final ethanol productions of simultaneous saccharification and pre-saccharified process for 1 h were not significantly different. The result suggested that it is possible for simultaneous saccharification and fermentation as a cost-effective process for ethanol production by eliminating the separate saccharification. Additionally, the fermentation rate in early fermentation stage was generally increased with increase of inoculum volume. As the result, optimal condition for ethanol production was simultaneous saccharification and fermentation with complex enzyme and 5% inoculation. Under the same condition, the volumetric productivities and ethanol yields were attained to 3.26 g/L·h and 93.5% for brown rice, 2.62 g/L·h and 90.4% for naked barley, and 3.28 g/L·h and 93.7% for cassava, respectively.     

Design and Evaluation of a Lactobacillus manihotivorans Species-Specific rRNA-Targeted Hybridization Probe and Its Application to the Study of Sour Cassava Fermentation

Based on 16S rRNA sequence comparison, we have designed a 20-mer oligonucleotide that targets a region specific to the species Lactobacillus manihotivorans recently isolated from sour cassava fermentation. The probe recognized the rRNA obtained from all the L. manihotivorans strains tested but did not recognize 56 strains of microorganisms from culture collections or directly isolated from sour cassava, including 29 species of lactic acid bacteria. This probe was then successfully used in quantitative RNA blots and demonstrated the importance of L. manihotivorans in the fermentation of sour cassava starch, which could represent up to 20% of total lactic acid bacteria.     

L-Lactic acid production from raw cassava starch in a circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrica)

l-Lactic acid was produced from raw cassava starch, by simultaneous enzyme production, starch saccharification and fermentation in a circulating loop bioreactor with Aspergillus awamori and Lactococcus lactis spp. lactis immobilized in loofa sponge. A. awamori was immobilized directly in cylindrical loofa sponge while the L. lactis was immobilized in a loofa sponge alginate gel cube. In the loofa sponge alginate gel cube, the sponge serves as skeletal support for the gel with the cells. The alginate gel formed a hard outer layer covering the soft porous gel inside. By controlling the rate and frequency of broth circulation between the riser and downcomer columns, the riser could be maintained under aerobic condition while the downcomer was under anaerobic condition. Repeated fed-batch l-lactic acid production was performed for more than 400 h and the average lactic acid yield and productivity from raw cassava starch were 0.76 g lactic acid g^–1 starch and 1.6 g lactic acid l^–1 h^–1, respectively.     

Development of starter culture for improved processing of Lafun,an African fermented cassava food product

Aims: To select appropriate micro‐organisms to be used as starter culture for reliable and reproducible fermentation of Lafun. Methods and Results: A total of 22 cultures consisting of yeast, lactic acid bacteria (LAB) and Bacillus cereus strains predominant in traditionally fermented cassava during Lafun processing were tested as potential starter cultures. In an initial screening, Saccharomyces cerevisiae 2Y48P22, Lactobacillus fermentum 2L48P21, Lactobacillus plantarum 1L48P35 and B. cereus 2B24P31 were found to be the most promising of the cultures and were subsequently tested in different combinations as mixed starter cultures to ferment submerged cassava roots. Saccharomyces cerevisiae, inoculated singly or combined with B. cereus, gave the softest cassava root after 48 h of fermentation according to determination of compression profile and stress at fracture. Overall, sensory quality testing showed that Lafun obtained from S. cerevisiae‐fermented cassava gave the most preferred stiff porridge. Saccharomyces cerevisiae 2Y48P22 showed pectinase production in a model system. Conclusions: The results suggest that S. cerevisiae 2Y48P22 is the most efficient organism for cassava softening during the fermentation. Therefore, it could be combined with LAB and used as starter for Lafun processing. Significance and Impact of the Study: Starter cultures are made available for controlled fermentation of Lafun.     

Bioethanol from fermentation of cassava pulp in a fibrous-bed bioreactor using immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense

There is an increasing worldwide interest in bioethanol production from agricultural, industrial, and urban residues for both ecological and economic reasons. The acid hydrolysis of cassava pulp to reducing sugars and their fermentation to ethanol were evaluated in a fibrousbed bioreactor with immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense. A maximum yield of total reducing sugars of 53.5% was obtained after 8 h of hydrolysis at 85oC in 0.4 mol/L hydrochloric acid with a solid-to-liquid ratio of 1:20, which was optimized by using an orthogonal design based on preliminary experiments. In the FBB, the fed-batch fermentation, using glucose as the sole carbon source, gave a maximum ethanol production of 38.3 g/L with a yield of 0.364 g/g in 100 h; whereas the fed-batch fermentation, using xylose as the sole carbon source, gave 34.1 g/L ethanol with a yield of 0.342 g/g in 135 h. When cassava pulp hydrolysate was used as a carbon source, 39.1 g/L ethanol with a yield of 0.123 g/g cassava pulp in185 h was observed, using the fed-batch fermentation model. In addition, for repeated batch fermentation of cassava pulp hydrolysate carried out in the fibrous-bed bioreactor, long-term operation with high ethanol yield and volumetric productivity were achieved. The above results show that the acid hydrolysate of cassava pulp can be used for ethanol production in a fibrous-bed bioreactor, although some inhibition phenomena were observed during the process of fermentation.     

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.     

Development and evaluation of a starter culture for the industrial production of gari

Gari starter cultures (Gastat) were developed by mixing pure single strains of the organisms that ferment cassava. They were propagated and maintained as granules on dried cocoyam slurry. The cultures were tested for fermentative and acid-producing activity. The acidity produced at 30°C varied from 0.07% to 0.85% lactic acid with maximum levels occurring after 48 h. High levels of reducing sugar were produced during the first 24 h. The amounts produced were about 50% more than those from the self-inoculated cassava. The quality of the gari produced by the starter cultures was good and well accepted. The texture was similar to that produced by natural fermentation. These results highlight the possibility of using starter cultures in the large-scale production of gari.     

Production of free fatty acids from switchgrass using recombinant Escherichia coli

Switchgrass is a promising feedstock to generate fermentable sugars required for the sustainable operation of biorefineries because of their abundant availability, easy cropping system, and high cellulosic content. The objective of this study was to investigate the potentiality of switchgrass as an alternative sugar supplier for free fatty acid (FFA) production using engineered Escherichia coli strains. Recombinant E. coli strains successfully produced FFAs using switchgrass hydrolysates. A total of about 3 g/L FFAs were attained from switchgrass hydrolysates by engineered E. coli strains. Furthermore, overall yield assessments of our bioconversion process showed that 88 and 46% of the theoretical maximal yields of glucose and xylose were attained from raw switchgrass during sugar generation. Additionally, 72% of the theoretical maximum yield of FFAs were achieved from switchgrass hydrolysates by recombinant E. coli during fermentation. These shake‐flask results were successfully scaled up to a laboratory scale bioreactor with a 4 L working volume. This study demonstrated an efficient bioconversion process of switchgrass‐based FFAs using an engineered microbial system for targeting fatty acid production that are secreted into the fermentation broth with associated lower downstream processing costs, which is pertinent to develop an integrated bioconversion process using lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:91–98, 2018