PMLA Production Research Articles

Using Machine Learning Methods to Predict the ß-Poly (L-Malic Acid) Production by Different Substrates Addition and Secondary Indexes in Strain Aureobasidium melanogenum

ß-poly (L-malic acid) (PMLA) is a polyester ligated by malate subunits. It has a wide prospective application as an anti-cancer drug carrier, and its malate subunits have a great application in the food industry. The strain Aureoabsidium melanogenum could produce a high amount of PMLA during fermentation, and different substrates addition could influence the production. In this study, we directly added potassium acetate, corn steep liquor, MgSO4, MnSO4, vitamin B1, vitamin B2, and nicotinamide as the fermentation substrate to the basic fermentation medium based on a generated random matrix that represented the added value. The PMLA production and four secondary indexes, pH, biomass, osmotic pressure, and viscosity were measured after 144 h fermentation. Finally, a total of 212 samples were collected as the dataset, by which the machine learning methods were deployed to predict the PMLA production by different substrates’ concentrations and the secondary indexes. The results indicated that PMLA production was negatively correlated with corn steep liquor and betaine and positively correlated with potassium acetate. The PMLA production could be predicted using all different substrates’ concentrations with a Mean Absolute Error (MAE) of 4.164 g/L and with an MAE of 6.556 g/L by different secondary indexes. Finally, the convolutional neural network (CNN) was applied to predict the PMLA production by fermentation medium images, in which the collected images were categorized into three groups, 0–20 g/L, 21–40 g/L, and >41 g/L, based on the PMLA production. The CNN model could predict the production with high accuracy. The methods and results presented in this study provided new insight into evaluating different substrates concentration on PMLA production and demonstrating the possibility of using the convolutional neural network model in the PMLA fermentation industry.

Electrolytic stimulation in aid of poly(β-L-malic acid) production by Aureobasidium melanogenum ipe-1

Poly (β-L-malic acid) (PMLA) is attracting industrial interest for its potential application in medicine and other industries. In this study, electrolytic stimulation assisted PMLA production was developed. Firstly, it was found that the pentavalent nitrogen source (i.e., NO3−) was more suitable for PMLA production. Secondly, a usual single-chamber bioelectric-fermentation system (BES) cannot improve PMLA production, which can only promote cell growth. Then, a new single-chamber BES with an external circulation was developed, where the PMLA metabolism was further intensified. Finally, the integration of NO3− addition and electrolytic stimulation mode (c) showed a positive synergy on the PMLA production. Compared to the case without NO3− addition and electrolytic stimulation, the PMLA production was increased by 22.9 % using the integrated process. Moreover, compared to the case without the electrolytic stimulation mode (c), it was revealed that the different genes involved in 12 metabolic subsystems using the integrated process, where 31 and 177 genes were up-regulated and down-regulated, respectively. The up-regulated genes were mainly participated in melanin metabolic process, catalase activity, and oxidoreductase activity. Hence, the integration of electrolytic stimulation represents a novel approach to improve PMLA production.

Efficient poly(β-L-malic acid) production from cassava hydrolysate by cell recycle of Aureobasidium pullulans.

Poly(β-L-malic acid) (PMLA) is a water-soluble, biodegradable, and biocompatible polymer with broad prospective applications and can be hydrolyzed to produce widely used acidulant L-malic acid. In order to meet an increasing demand of PMLA, we employed two effective cell-recycling strategies to produce PMLA from raw cassava hydrolysate by Aureobasidium pullulans ZD-3d. In fed-batch fermentation with raw cassava hydrolysate, 101.9g/L PMLA was obtained with the productivity and yield of 0.77g/L/h and 0.40g/g, respectively. Further, three times of membrane filtration-based cell recycling fermentation was carried out, with a high productivity and yield of 1.04-1.64g/L/h and 0.5-0.84g/g achieved, respectively. While harnessing centrifugation-based cell recycling fermentation for five times, the productivity and yield approached 0.98-1.76g/L/h and 0.78-0.86g/g, respectively. To our knowledge, the processes showed the highest average PMLA productivity compared with others using low-cost biomass, which offered efficient and economical alternatives for PMLA production. KEY POINTS: • PMLA production from raw cassava hydrolysate by Aureobasidium pullulans was studied • High PMLA productivity and yield were obtained via two cell recycling strategies • The highest average PMLA productivity from low-cost biomass to date was achieved.

Effects of corn steep liquor on \u03b2-poly(l-malic acid) production in Aureobasidium melanogenum

β-poly(l-malic acid) (PMLA) is a water-soluble biopolymer used in medicine, food, and other industries. However, the low level of PMLA biosynthesis in microorganisms limits its further application in the biotechnological industry. In this study, corn steep liquor (CSL), which processes high nutritional value and low-cost characteristics, was selected as a growth factor to increase the PMLA production in strain, Aureobasidium melanogenum, and its metabolomics change under the CSL addition was investigated. The results indicated that, with 3 g/L CSL, PMLA production, cell growth, and yield (Yp/x) were increased by 32.76%, 41.82%, and 47.43%, respectively. The intracellular metabolites of A. melanogenum, such as amino acids, organic acids, and key intermediates in the TCA cycle, increased after the addition of CSL, and the enrichment analysis showed that tyrosine may play a major role in the PMLA biosynthesis. The results presented in this study demonstrated that the addition of CSL would be an efficient approach to improve PMLA production.

A sustainable pH shift control strategy for efficient production of β-poly(L-malic acid) with CaCO3 addition by Aureobasidium pullulans ipe-1.

β-poly(L-malic acid) (PMLA) has attracted industrial interest for its potential applications in medicine and other industries. For a sustainable PMLA production, it requires replacing/reducing the CaCO3 usage, since the residual CaCO3 impeded the cells' utilization, and a large amount of commercially useless gypsum was accumulated. In this study, it was found that more glucose was converted into CO2 using soluble alkalis compared with CaCO3 usage. Moreover, since the high ion strength and respiration effect of soluble alkalis also inhibited PMLA production, they could not effectively replace CaCO3. Furthermore, comparing the fermentations with different neutralizers (soluble alkali vs. CaCO3), it was found that the differential genes are mainly involved in the pathway of starch and sucrose metabolism, pentose and glucuronate interconversions, histidine metabolism, ascorbate and aldarate metabolism, and phagosome. In detail, in the case with CaCO3, 562 genes were downregulated and 262 genes were upregulated, and especially, those genes involved in energy production and conversion were downregulated by 26.7%. Therefore, the irreplaceability of CaCO3 was caused by its effect on the PMLA metabolic pathway rather than its usage as neutralizer. Finally, a combined pH shift control strategy with CaCO3 addition was developed. After the fermentation, 64.8 g/L PMLA and 38.9 g/L biomass were obtained with undetectable CaCO3 and less CO2 emission. KEY POINTS: • The effect of CaCO3 on PMLA metabolic pathway resulted in its irreplaceability. • A pH shift control strategy with CaCO3 addition was developed. • Undetectable CaCO3 and less CO2 emission were detected with the new strategy. Graphical abstract.

Membrane-assisted β-poly(L-malic acid) production from bagasse hydrolysates by Aureobasidium pullulans ipe-1

Membrane-assisted β-poly(L-malic acid) (PMLA) production from bagasse hydrolysates was developed. For the first time, it was found that mixing the acid and enzyme hydrolysates was unfavorable for PMLA production because too high hexose: pentose ratio and glucose concentration in the mixed sugar could inhibit the assimilation of pentose. 120 g/L sugar concentrations in the acid hydrolysate was suitable for PMLA production with 23.2 g/L PMLA and 34.7 g/L biomass. Moreover, an integrated membrane process consisting of ultrafiltration, nanofiltration and reverse osmosis membranes could concentrate sugars and adjust acetic acid concentration prior to fermentation of lignocellulosic sugars. Meanwhile, it was found that 1.46 g/L acetic acid was preferred for PMLA production from enzyme hydrolysate or sole glucose which respectively increased PMLA production and cell growth by 25.4% and 5.9% from sole glucose, while it showed no significant enhancement in PMLA production with a higher cell growth and productivity from acid hydrolysate.

Efficient β-poly(l-malic acid) production from Jerusalem artichoke by Aureobasidium pullulans ipe-1 immobilized in luffa sponge matrices

β-poly(l-malic acid) (PMLA) production by Aureobasidium pullulans ipe-1 using Jerusalem artichoke tuber (JA) hydrolysate as a low cost carbon source was developed. The PMLA production was favored by JA pretreated with 0.06 M nitric acid without adding exogenous nitrogen sources into fermentation medium. With an initial 130 g/L total sugar of the JA hydrolysate, the highest PMLA productivity 0.52 g/L·h was achieved, which was increased by 2.0 folds compared to that with sole glucose case. To further enhance PMLA productivity, the cells were immobilized in luffa sponge matrices, and repeated batch culture was carried out for 4 cycles. The resulting PMLA productivity was further enhanced by 50% compared with the batch culture. The cost of PMLA production in the JA case was only 5.4% of that in the glucose case. The outcomes of this work provided a strategy of PMLA production on a commercial scale.

Evaluation of surfactant effect on β-poly(L-malic acid) production by Aureobasidium pullulans

β-poly(L-malic acid) (PMLA) is a highly water-soluble biopolymer used in medicine and other industries. PMLA is usually biosynthesized by Aureobasidium pullulans, but in low concentrations, which has restricted its further large-scale production. Here effects of surfactants on PMLA production were investigated. Of various surfactants examined, the addition of Tween 80 at the start of cell growth increased mycelial growth, PMLA production, PMLA productivity and the ratio of PMLA to cell biomass concentration (Yp/x) of A. pullulans. Transmission electron microscopy and flow cytometry showed that cell membrane permeability was increased, improving the secretion of PMLA and uptake of nutrients and oxygen. Proteomic analysis showed that key pathways of PMLA synthesis, along with cellular energy metabolism, were enhanced. Moreover, glutathione (GSH) synthesis and anti-oxidation capacity were also improved. These results indicated that the addition of Tween 80 is an efficient approach for improving the production of PMLA.

Toward understanding the key enzymes involved in β-poly (L-malic acid) biosynthesis by Aureobasidium pullulans ipe-1.

β-poly (L-malic acid) (PMLA) is a biopolyester which has attracted industrial interest for its potential application in medicine and other industries. A high dissolved oxygen concentration (DO) was beneficial for PMLA production, while the mechanisms of DO in PMLA biosynthesis by Aureobasidium pullulans are still poorly understood. In this work, the amount of PMLA was first compared when A. pullulans ipe-1 were cultured under a high DO level (70% saturation) and a low DO level (10% saturation). Meanwhile, the key enzymes involved in different pathways of the precursor L-malic acid biosynthesis were studied. The results revealed that the activities of glucose-6-phosphate dehydrogenase (G6PDH) and phosphoenolpyruvate carboxylase (PEPC) were positively correlated with cell growth and PMLA production, while the activities of phosphofructokinases (PFK), pyruvic carboxylase (PC) and citrate synthetase (CS) did no show such correlations. It indicated that the Pentose Phosphate Pathway (PPP) may play a vital role in cell growth and PMLA biosynthesis. Moreover, the precursor L-malic acid for PMLA biosynthesis was mainly biosynthesized through phosphoenolpyruvic acid (PEP) via oxaloacetate catalyzed by PEPC. It was also found that low concentration of sodium fluoride (NaF) might impel carbon flux flow to the oxaloacetate through PEP, but inhibit the flux to the oxaloacetate via pyruvic acid.

Poly(β-L-malic acid) (PMLA) from Aureobasidium spp. and its current proceedings.

Poly(β-L-malic acid) is one natural biopolymer that has the outstanding features of biocompatibility, biodegradability, water solubility, and non-immunogenicity, and it is easily chemically modified. So poly(β-L-malic acid) (PMLA) and its derivatives may have a great potential application as a novel drug delivery system and in the production of advanced biomaterials which have attracted so much research attention. The fungi of Aureobasidium spp. have been discovered to be the most suitable candidates for PMLA production in large quantities which satisfy the demand of either research or industry. In this review, we will give an overall summary about the PMLA produced by Aureobasidium spp. based on related research in the last decades and the elaboration of this PMLA producer will also be accomplished. More importantly, the latest proceedings will be specified and some suggestions to the elucidation of a PMLA biosynthesis pathway which remains undefined up to date will be proposed. Finally, through this review, the further exploitation for the application of PMLA from Aureobasidium spp. can be emphasized and promoted.

High molecular weight β-poly(l-malic acid) produced by A. pullulans with Ca2+ added repeated batch culture

β-Poly(malic acid) (PMLA) has attracted increasing attentions because of its potential application in medicine and other industries. In this study, the variation of PMLA molecular weight (Mw) in the batch culture and the strategies to enhance PMLA Mw were studied. Adding exogenous Ca2+ (0.1g/L CaCl2) to the medium caused a significant increase in both PMLA concentration and Mw (11.38% and 26.3%, respectively) when Na2CO3 was used as the neutralizer. The Mw of PMLA during the process of batch culture, which associated with the specific PMLA production per unit cell mass (Yp/x) before glucose was depleted, increased from 12.522KDa to its maximum 18.693KDa and then kept decreasing until the end of the culture. Compared with the results in batch culture, Mw increased by 84.4% (up to 19.51kDa) with a productivity of 1.1gh−1L−1 when the cells were maintained in exponential growth phase during Ca2+ added repeated batch culture. The present work provides an efficient approach to obtain superior quality PMLA product with high Mw.

β‐poly(l‐malic acid) production by fed‐batch culture of Aureobasidium pullulans ipe‐1 with mixed sugars

β‐poly(l‐malic acid) (PMLA) is a biopolyester, which has attracted growing attention due to its potential applications in medicine and other industries. In this study, the biosynthetic pathway of PMLA and the fermentation strategies with mixed sugars were both investigated to enhance PMLA production by Aureobasidium pullulans ipe‐1. Metabolic intermediates and inhibitors were used to study the biosynthetic pathway of PMLA. It showed that exogenous addition of l‐malic acid, succinic acid, TFA, and avidin had negligible effect on PMLA production, while pyruvic acid and biotin were the inhibitors, indicating that PMLA biosynthesis was probably related to phosphoenolpyruvate via oxaloacetate catalyzed by phosphoenolpyruvate carboxylase. Sucrose was suitable for achieving the highest PMLA concentration, while fructose generated a higher yield of PMLA (PMLA produced per biomass). Furthermore, the fed‐batch culture using fed solution with different sugar mixture for PMLA production was implemented. During the fed‐batch culture with mixed solution, fructose could increase PMLA production. Compared with the batch culture, the feeding with mixed sugar (sucrose and glucose) increased PMLA concentration by 23.9%, up to 63.2 g/L, and the final volume of the broth was increased by 25%. These results provide a good reference for process development and optimization of PMLA production.

Control strategy of pH, dissolved oxygen concentration and stirring speed for enhancing β‐poly (malic acid) production by Aureobasidium pullulans ipe‐1

AbstractBACKGROUND: β‐poly(malic acid) (PMLA) can be used as a pro‐drug or for a drug‐delivery system. Effects of pH, dissolved oxygen concentration (DO) and stirring speed were investigated to improve PMLA production by A. pullulans ipe‐1.RESULTS: The strain produced a high PMLA concentration when pH and DO remained at about 6.0 and above 70%, respectively, and the yeast‐like cells were the main PMLA producers. To further promote PMLA production, the cultivation could be divided into three phases. In phase I, cell growth was accelerated by maintaining high DO (>70%) with a constant stirring speed of 800 rpm. In phase II, PMLA production was increased by controlling DO at 70% using the automatically controlled stirring speed. In phase III, PMLA production on per gram of glucose (Yp/s) was enhanced by keeping DO at 70%, and using a low stirring speed to decrease cell growth. Compared with batch cultures, a higher PMLA yield was obtained with this strategy, i.e. PMLA production and Yp/s increased by 15% and 18%, respectively.CONCLUSION: Control strategies for pH, DO and stirring speed provide a good reference for process development and optimization of PMLA production. © 2012 Society of Chemical Industry

Intensification of β-poly(l-malic acid) production by Aureobasidium pullulans ipe-1 in the late exponential growth phase

β-Poly(malic acid) (PMLA) has attracted industrial interest because this polyester can be used as a prodrug or for drug delivery systems. In PMLA production by Aureobasidium pullulans ipe-1, it was found that PLMA production was associated with cell growth in the early exponential growth phase and dissociated from cell growth in the late exponential growth phase. To enhance PMLA production in the late phase, different fermentation modes and strategies for controlling culture redox potential (CRP) were studied. The results showed that high concentrations of produced PMLA (above 40 g/l) not only inhibited PMLA production, but also was detrimental to cell growth. Moreover, when CRP increased from 57 to 100 mV in the late exponential growth phase, the lack of reducing power in the broth also decreased PMLA productivity. PMLA productivity could be enhanced by repeated-batch culture to maintain cell growth in the exponential growth phase, or by cell-recycle culture with membrane to remove the produced PMLA, or by maintaining CRP below 70 mV no matter which kind of fermentation mode was adopted. Repeated-batch culture afforded a high PMLA concentration (up to 63.2 g/l) with a productivity of 1.15 g l(-1) h(-1). Cell-recycle culture also confirmed that PMLA production by the strain ipe-1 was associated with cell growth.

Beta-poly(L-malate) production by non-growing microplasmodia of Physarum polycephalum. Effects of metabolic intermediates and inhibitors.

The production of beta-poly(L-malate) (PMLA) by non-growing microplasmodia of Physarum polycephalum was investigated. Growth was minimal in culture medium devoid of nitrogen source, but PMLA production occurred at a substantial rate. The addition of metabolic intermediates, malate, fumarate, succinate, and oxaloacetate, and the omission of hematin showed considerable growth inhibition in the presence of the nitrogen source, while PMLA production per unit biomass increased significantly. The results indicated that PMLA production was dissociated from biomass production under these conditions. The stimulating effect of carbonate on PMLA production was independent on growth. Cultivation in the absence of the nitrogen source and hematin or in the presence of the metabolites may be a useful technique for efficient PMLA production at a minimum of biomass production.

β-Poly(L-malate) production by non-growing microplasmodia of Physarum polycephalum: Effects of metabolic intermediates and inhibitors

The production of β-poly(L-malate) (PMLA) by non-growing microplasmodia of Physarum polycephalum was investigated. Growth was minimal in culture medium devoid of nitrogen source, but PMLA production occurred at a substantial rate. The addition of metabolic intermediates, malate, fumarate, succinate, and oxaloacetate, and the omission of hematin showed considerable growth inhibition in the presence of the nitrogen source, while PMLA production per unit biomass increased significantly. The results indicated that PMLA production was dissociated from biomass production under these conditions. The stimulating effect of carbonate on PMLA production was independent on growth. Cultivation in the absence of the nitrogen source and hematin or in the presence of the metabolites may be a useful technique for efficient PMLA production at a minimum of biomass production.

β-Poly( l -malate) production by Physarum polycephalum

β-Poly(l-malate) (PMLA) production in Physarum polycephalum has been followed by using d-[1-13C]glucose and Ca13CO3. Nuclear magnetic resonance studies of PMLA showed that the 13C label from [1-13C]glucose was incorporated in the presence of CaCO3 into positions C-3 (-CH2-) and C-4 (-CO-) of the l-malate repeating unit of PMLA. The 13C label from Ca13CO3 was incorporated into position C-4 and indicated that not only the endogenous CO2 but also the exogenous CO2 from CaCO3 served significantly as a carbon source for PMLA production. In the absence of CaCO3, the 13C labeling pattern of PMLA from d-[1-13C]glucose was almost indistinguishable from that for the natural abundance 13C-NMR spectrum of the polymer. These results indicated that l-malate used for PMLA production is synthesized either via carboxylation of pyruvate and reduction of oxaloacetate in the presence of CaCO3 or via the oxidative tricarboxylic acid (TCA) cycle in the absence of CaCO3. Avidin strongly inhibited the formation of l-malate via carboxylation; the 13C labeling pattern of PMLA in the presence of CaCO3 was almost identical with that for the natural abundance spectrum when avidin was added, indicating that l-malate utilized for PMLA production was supplied under this condition by the oxidative TCA cycle.

Effects of culture conditions on β-poly( l -malate) production by Physarum polycephalum

Culture conditions for the fermentative production of β-poly(l-malate) (PMLA) by microplasmodia of Physarum polycephalum were investigated and optimized. Optimal production was achieved in the presence of CaCO3. For 1.5% (w/v) d-glucose, 1% bactotryptone and 1% CaCO3, a maximum of 1.7 g PMLA/l was secreted in 3 days. For 4.5% glucose and otherwise the same conditions, 2.7 g PMLA/l was produced in 6 days. The contribution of carbonate was inhibited by avidin. PMLA and biomass production were not strictly coupled: PMLA was also synthesized at the beginning of the stationary phase. At pH 5.5 PMLA production was twice that at pH 4.0, while biomass was not changed. Optimal temperatures were 24–28 °C.