Bioethanol

Bioethanol

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Nanocellulose and Bioethanol Production from Orange Waste using Isolated Microorganism

August 2013
Journal of the Brazilian Chemical Society 24(9):1385-1543

DOI:
10.5935/0103-5053.20130195

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Citrus processing waste from oranges (CPWO) was explored for the production of nanocellulose and bioethanol. After the isolation of 20 microorganisms from CPWO, their fermentation abilities were screened and two microorganisms identified as Candida parapsilosis strains IFM 48375 and NRRL Y(-1)2969 (ATCC 22019) were selected for a further fermentation. The CPWO was steam distilled for the isolation of essential oil (1.5% g g(-1) of dry CPWO) and converted into a mixture of fermentable sugars (40% g g(-1) of dry CPWO) using acid or enzymes hydrolyses. Hydrolyzates were fermented with three different yeast strains, the two Candida sp. and Saccharomyces cerevisiae. Candida parapsilosis strain IFM 48375 accomplished excellent results in ethanol production (21% g g(-1) of dry CPWO) from CPWO, higher when compared to other strains. Nanocellulose (2.5% g g(-1) dry CPWO) and nanofibers (0.5% g g(-1) dry CPWO) were isolated from solid residues obtained from enzymatically treated and fermented CPWO. To the best of our knowledge, this work reports for the first time the nanocellulose production from CPWO.

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Page 1

Article

J. Braz. Chem. Soc.

, Vol. 24, No. 9, 1537-1543, 2013.

0103 - 5053 $6.00+0.00

http://dx.doi.org/10.5935/0103-5053.20130195

*e-mail: ljubica@iqm.unicamp.br

Nanocellulose and Bioethanol Production from Orange Waste using

Isolated Microorganisms

Junko Tsukamoto,

Nelson Durán

b,c

and Ljubica Tasic*

Departamento de Química Orgânica and

Departamento de Físico-Química,

Laboratório de Química Biológica, Instituto de Química,

Universidade Estadual de Campinas, 13083-970 Campinas-SP, Brazil

Centro de Ciências Naturais e Humanas, Universidade Federal de ABC, Santo André-SP, Brazil

A biomassa procedente do processamento de laranjas (CPWO) foi utilizada na produção de

nanocelulose e do etanol da segunda geração. Primeiramente, vinte micro-organismos foram

isolados da CPWO e suas capacidades de fermentação foram testadas. Dois destes, identificados

como

Candida parapsilosis

IFM 48375 e NRRL Y-12969 (ATCC 22019), foram selecionados

para a posterior fermentação. A biomassa foi destilada a vapor para o isolamento do

óleo

essencial

(1,5% g g

-1

de CPWO seco) e convertida em uma mistura de açúcares fermentáveis (40% g g

-1

CPWO seco) usando hidrólise

ácid

a ou enzimática. Os hidrolisados foram fermentados utilizando

micro-organismos isolados e a

Saccharomyces cerevisiae

. A levedura

Candida parapsilosis

IFM 48375 foi a mais eficiente na fermentação dos açúcares obtidos desta biomassa e os maiores

rendimentos de bioetanol (21% g g

-1

de CPWO seco) foram alcançados. A nanocelulose (2,5% g g

-1

de CPWO seco) e as nanofibras (0,5%

g g

-1

de CPWO seco) foram isoladas partindo de bioresíduos

vindo da hidrólise enzimática e da fermentação alcoólica e este resultado

o primeiro do gênero.

Citrus processing waste from oranges (CPWO) was explored for the production of

nanocellulose and bioethanol. After the isolation of 20 microorganisms from CPWO, their

fermentation abilities were screened

and two microorganisms identified as

Candida parapsilosis

strains IFM 48375 and NRRL Y-12969 (ATCC 22019) were selected for a further fermentation.

The CPWO was steam distilled for the isolation of essential oil (1.5%

g g

-1

of dry CPWO)

and

converted into a mixture of fermentable sugars (40% g g

-1

of dry CPWO) using acid or enzymes

hydrolyses. Hydrolyzates were fermented with three different yeast strains, the two

Candida

sp. and

Saccharomyces cerevisiae

Candida parapsilosis

strain IFM 48375 accomplished excellent results

in ethanol production (21%

g g

-1

of dry CPWO) from CPWO, higher when compared to other

strains. Nanocellulose (2.5% g g

-1

dry CPWO) and nanofibers (0.5% g g

-1

dry CPWO) were isolated

from solid residues obtained from enzymatically treated

and fermented CPWO. To the best of our

knowledge, this work reports for the first time the nanocellulose production from CPWO.

Keywords

: citrus processing waste from oranges, nanocellulose, bioethanol, fermentation

Introduction

Recycling of agricultural and agro-industrial waste

is growing in importance as a way to diminish the

environmental impact caused by industrial and urban

activities. Many recycling projects have been performed

worldwide and also in Brazil. Among them, stand out

the reuse of sugarcane bagasse and paper.

1,2

Orange juice

is one of the most popular juices in the world and Brazil

produces annually approximately 22 million tons of oranges,

with the state of Sao Paulo generating alone more than

17 million tons.

After juice extraction, around half of the

fruit weight becomes citrus processing waste from oranges

(CPWO), a very interesting low cost material already used

for producing 1G-bioethanol.

4-8

Botanically, CPWO is

composed of peel, rag (segment membranes and cores),

juice sacs, and seeds. Chemically, despite being a food

industry residue, CPWO contains high value compounds

like soluble sugars, pectin, proteins, appreciable amounts

of fiber (cellulose and hemicelluloses), and lignin. CPWO

can also be an important source of limonene that can be

isolated as a by-product in relatively high yield.

CPWO

Page 2

Nanocellulose and Bioethanol Production from Orange Waste using Isolated Microorganisms

J. Braz. Chem. Soc.

1538

is a good material for 2G-bioethanol production through

either an acidic or an enzymatic hydrolysis followed by

subsequent fermentation with

Saccharomyces cerevisiae

10,11

For example, in a fermentation of ground orange peels

avoiding any pre-treatment it was found that the submerged

fermentation provided superior ethanol yields compared to

the solid-state fermentation.

4,7,10-12

The maximum ethanol

yields from CPWO in a submerged fermentation method

were 1.3% (v v

-1

) and 1.4% (v v

-1

) when

Saccharomyces

cerevisiae

and

Candida albicans

were applied, respectively.

Hydrolysis of CPWO with enzymes (cellulase,

pectinase, and

-glucosidase) followed by fermentation

with

Saccharomyces cerevisiae

and some bacteria or

genetically modified microorganisms gave excellent

quantities of ethanol.

13-16

Using orange peel waste in a novel

lab-scale direct steam injection apparatus, depolymerization

with a commercial cellulase, and fermentation with a

Saccharomyces cerevisiae

strain led to the high glucose and

ethanol yields (50% m m

-1

Also, a scale up experiment

resulted in an excellent ethanol productivity (6 g L

-1

The final overall process yield (mass balance) at the

bench-reactor scale of 140 L of bioethanol per metric ton

of dry orange peel waste was reported.

Although efficient processes starting from acid

hydrolyzates of CPWO are already used for bioethanol

production in the USA,

18-21

there are some serious

fermentation problems still unsolved. Yeast cultures such

Saccharomyces cerevisiae

can be inhibited in some

cases.

For example, when very high sugar content, nutrient

deficiency, very high or low temperature

and inhibitors

23,24

are present there is inefficient production of ethanol. Even

if these limiting factors are absent, fermentation may cease

prematurely due to substances produced by yeast during the

normal course of fermentation,

like the ethanol obtained

during fermentation.

Therefore, a search for the new

microorganism strains and process improvement are still

an open field of investigation.

Some interesting results, not directly linked to the

CPWO, have also been published on nanocellulose

isolation from the solid residues of the cellulosic biomass

remaining after processing for bioethanol production.

27,28

Also, bio-residues from wood fermentation to ethanol

were used as raw material for industrial production

of cellulose nanowhiskers,

and orange was used for

obtaining microcrystalline cellulose.

Production of

nanocellulose fibers and application of nanocellulose

for composite materials have brought attention to

valuable nanocellulose properties, such as high strength,

light weight, unique morphology, biodegradability and

renewability.

For example, nanocellulose has great

applicability when used in composite materials because of

its high stiffness as reinforcing material.

This biomaterial

can be used for polymer composites

and plastics, films,

foams and gels, cosmetics, thickener

and emulsion, implant

material, biodegradable tissue scaffold, as a drug delivery

vehicle, filter paper, concrete, for oil recovery, water

treatment, transport, electronics devices, solar panels,

paint pigments and ink, screens and coatings, among other

applications.

31,32

Our study had two aims: to evaluate the potential of

CPWO for production of nanocellulose after acidic

and

enzymatic hydrolyses,

and to investigate the feasibility

of bioethanol production by fermentation of CPWO

using selected microorganisms isolated from CPWO

(

natural habitat

Experimental

Citrus processing waste (CPWO)

Citrus processing waste from oranges (

Citrus sinensis

(L)

osbeck

) as squeezed orange fruit was obtained from

a local restaurant (Valinhos, SP, Brazil). Material was

ground

with a food homogenizer to around 2 mm in

diameter particles and stored at

−

20 ºC until use. From

ground CPWO residues were determined moisture and ash

contents using AOAC methods.

Pectins were extracted and

analyzed according to Sudhakar and Maini method.

Acid

detergent fiber (ADF), neutral detergent fiber (NDF) and

acid detergent lignin (ADL) were determined with an

Ankom 200 fiber analyzer. Acid detergent fiber (ADF)

value refers to the cellulose

and lignin contents. Neutral

detergent fiber comprises ADF fraction plus hemicellulose.

The difference between ADF and ADL was considered as

cellulose, whereas difference between NDF

and ADF was

reported as hemicelluloses. All analyses were performed

in triplicate.

Analytical Methods

Powdered CPWO residues were analyzed using X-ray

diffractometer (XDR 6000, Shimadzu), at a scanning rate

of 2 degree min

-1

, Cu K

radiation (

= 1.54

Å)

in the range

of 2

= 2º-60º with increments of 0.02º. Sugars and ethanol

were quantified using a HPLC system (Waters, USA) with

a refractive index detector (RID), photodiode array detector

(DAD), a pre-column SH-G (6 × 50 mm) and a column

(Shodex 1011, 300 mm × 8 mm) (Showa Denko, Japan).

The column oven was maintained at 50

ºC.

Degassed mobile

phase containing 0.005 mol L

-1

of sulphuric acid was used

at a flow rate of 0.6 mL min

-1

. Sugar peaks were detected

based on the retention times of standards [glucose, fructose,

Page 3

Tsukamoto

et al.

1539

Vol. 24, No. 9, 2013

sucrose, xylose, galactose, 5-hydroxymethylfurfural

(5-HMF), furfural] purchased from Synth and Sigma

Aldrich. Ethanol fermentation broth contains mostly

sugars

and ethanol. Among these, the major components

were identified separately by obtaining chromatograms of

the individual components under the same conditions. The

calibration curves were generated from the chromatograms

of a series of standard mixtures at several concentrations.

The relationship between peak area and the concentration

was linear over the entire concentration range examined.

Peak height vs. concentration was also linear over the same

range.

H and

C NMR spectra were acquired on a Bruker

Avance instrument operating at 250 MHz (

H) and 62.5 MHz

(

C) using a 5 mm sample tube. These spectra were used

to analyze the quality of the bioethanol. Scanning electron

microscopy (SEM) analyses (JSM-6360LV, JEOL, Japan)

were used to examine the bioresidues from the CPWO

fermentation processes, as well to examine the yeast cells.

Isolation of microoganisms

Microorganisms were isolated from CPWO using

solid media isolation procedure where a sample of

CPWO was transferred to a Petri plate with a sterile

solid medium containing yeast extract-peptone-dextrose

(YPD) agar (20.0 g L

-1

dextrose; 10.0 g L

-1

yeast extract;

20.0 g L

-1

peptone; 15.0 g L

-1

agar and 30 mg mL

-1

chloramphenicol)

and incubated at 30

ºC

for 24-48 h. After

incubation, individual cells that have grown separately into

discrete colonies were picked and transferred again to YPD

agar and incubated at 30

ºC

for 24-48 h. We have repeated

this procedure for four times and obtained the colonies of a

single species that were studied separately from all others.

Limonene extraction

Limonene was extracted from CPWO by steam

distillation. Initially grounded CPWO was placed into

100 mL of water

and kept under distillation temperature

of 97-98

ºC.

This way, water (steam) was introduced into

the distillation apparatus, providing depression of the

boiling points of limonene and other orange essential oil

components. Then, after distillation, vapour was condensed

yielding a two-phase system of water and limonene

(approximately 80 mL). Quantity of total oil was measured

applying Scott oil analysis by bromated titration.

Acid hydrolysis

To elenmeyer flasks (250 mL) containing thawed

CPWO residues (17.0 g), distilled water and sulphuric acid

(98%) were added to reach an acid concentration of 0.5, 0.1

or 1.5% (m v

-1

) to a final 100 mL volume. Next, samples

were heated in an autoclave at 120

ºC

(15 or 30 min). The

hydrolyzates were analyzed for sugars, furfurals, acetic

acid, and total phenols using HPLC. The treatment that

enabled the highest sugar contents in hydrolyzates was

selected and used for following fermentations. All acid

hydrolysis experiments were carried out in triplicate.

Enzymatic treatment

To erlenmeyer flasks (250 mL) containing thawed

CPWO (17.0 g) or autoclaved residues (17.0 g) and

distilled water (total volume 100 mL) was added a cocktail

of enzymes: pulpzyme HA (Novozyme), celluclast 1.5 L.

(Novozyme) and

-galactosidase from

Aspergillus oryzae

(Sigma). Next, the slurries were incubated at 50

ºC

during

48 h. The collected hydrolyzates were analyzed by HPLC.

The enzymatic treatment that enabled the highest sugar

yields from CPWO was selected as the best

and was used

for subsequent fermentations. Enzyme loadings were:

3.4 mg of pulpzyme HA, 3.4 mg of celluclast 1.5 L and

0.6 mg of

-galactosidase (

A. oryzae

)

per

g of CPWO. The

enzymatic activities were measured as described by Ghose

for celluclast 1.5 L and pulpzyme HA. For

-galactosidase

(

A. oryzae

), the modified method of Sigma from

Kuby and Lardy

and Borooah

et al.

was applied. The

enzymatic treatment procedures were repeated three times.

After being hydrolyzed, the CPWO samples were

heated to 105

ºC

for 15 min to inactivate enzymes and

subsequently fermented to ethanol using three different

types of yeast.

Yeast cells and fermention

The commercial yeast (

Saccharomyces cerevisiae

Sigma Aldrich) and two isolated microorganisms from

CPWO,

Candida parapsilosis

IFM 48375 and

Candida

parapsilosis

NRRL Y-12969, were aseptically inoculated

into sterilized erlenmeyer flasks (150 mL) containing

50 mL of glucose yeast extract broth (GYE), and

chloramphenicol (30 mg mL

-1

). The flasks were incubated

at 30

ºC

for 24 h in a shaker with 100 × g. The inoculum

was aseptically transferred to sterile erlenmeyer flasks

(250 mL) containing 100 mL of GYE broth supplemented

with 30 mg mL

-1

of chloramphenicol, and flasks were

incubated at the same conditions as cited above for 24 h.

The cells were concentrated by centrifugation in sterilized

centrifuge tubes at 10,000

× g and 15

ºC

for 10 min. The

cell count was determined using a Newbauer plate. Cells

were concentrated to the level of 5.8

cells mL

-1

Page 4

Page 5

Page 6

Page 7

Oligonucleotide Array and VITEK Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Species Identification of Blood Yeast Isolates

Article

Full-text available

Jan 2018
Frontiers in Microbiology

We evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry using VITEK MS (IVD database) and an oligonucleotide array based on the internal transcribed spacer-1 (ITS-1) and ITS-2 sequences of rRNA genes for the identification of Candida spp. from blood cultures. Five-hundred and twelve consecutive bloodstream yeast isolates were collected daily and initially identified by the phenotypic automated method (VITEK YBC or VITEK2 YST card). Inconsistent results were confirmed by D1-D2 region of 28S rRNA genes and ITSs. Excluding two unidentified yeast isolates, the oligonucleotide array and VITEK MS correctly identified 99.6% (508) and 96.9% (494) of 510 yeast isolates, respectively. The oligonucleotide array and VITEK MS demonstrated high correct identification rates for four major Candida species (C. albicans 100%, 98.4%; C. glabrata 100%, 100%; C. parapsilosis 100%, 93.3%; C. tropicalis 100%, 97.3%), but lower correct identification rates for other Candida species (91.7 and 87.5%, respectively). In conclusion, the identification performance of the oligonucleotide array is comparable to that of VITEK MS, and can serve as a supplemental tool for the identification of Candida species.

A multistep mild process for preparation of nanocellulose from orange bagasse

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Aug 2018
Cellulose

Orange bagasse in natura and industrial orange bagasse were investigated as starting materials for the production of nanocellulose under moderate chemical sequential extraction conditions. The latter accounted for acid (5% v v⁻¹ and 100 °C) and/or alkaline conditions (NaOH 1.6–4.0% m v⁻¹, 120 °C); and bleaching with NaClO2 (1–3% m v⁻¹, 80 °C). Ultrasound treatment yielded very similar cellulose nanofibers with 60–70% of crystallinity and highly pure (over 98%). As seen by field emission scanning electron microscopy, cellulose nanofibers showed mean diameters of 18.4 nm ± 6.0 nm from bagasse in natura, while 20.5 nm ± 7.0 nm mean diameters were observed for the nanofibers isolated from the industrial bagasse. Crystallinity indices were determined using X-ray diffraction and solid-state nuclear magnetic resonance (CP–MAS ¹³C NMR) data. The obtained materials have numerous potential applications and represent a green alternative for the treatment of orange fruit biomass. Graphical abstract Open image in new window

Micafungin Breakthrough Fungemia in Patients with Hematological Disorders

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Limited data are available on micafungin breakthrough fungemia (MBF), fungemia that develops on administration of micafungin, in patients with hematological disorders. We reviewed medical and microbiological records of patients with hematological disorders who developed MBF between January 2008 and June 2015. A total of 39 patients with MBF were identified and Candida (30 strains) and non- Candida (9 strains) fungal species were recognized as causative strains. Among 35 stored strains, C. parapsilosis (14 strains), Trichosporon asahii (7 strains), C. glabrata (5 strains), and other fungal species (9 strains) were identified by sequencing. Neutropenia was identified as an independent predictor of non- Candida fungemia ( P = 0.023). T. asahii was the most common causative strain (7/19) during neutropenia. The 14-day crude mortality rate of patients treated with early micafungin change (EMC) to other antifungal agents was lower than that of the patients not treated with EMC (14% vs. 43%, P = 0.044). Most of the stored causative Candida strains were susceptible (80%) or showed wild-type susceptibility (72%) to micafungin. The minimum inhibitory concentrations (MICs) of voriconazole for T. asahii were low (range, 0.015 to 0.12 μg/mL), whereas MICs of amphotericin B for T. asahii were high (range, 2 to 4 μg/mL). MBF caused by non- Candida fungus should be considered, especially in patients with neutropenia. EMC could improve early mortality. Based on epidemiology and drug susceptibility profiling, empiric voriconazole-containing therapy might be suitable for treating MBF during neutropenia to cover for T. asahii .

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Citrus waste is an attractive lignocellulosic biomass for the production of bioethanol due to the richness in carbohydrates and low lignin content. In this study, sweet lime peel was chosen as the lignocellulosic biomass. To increase the cellulose for enzymatic hydrolysis, the statistical optimization of process parameters namely, solid loading, time of exposure and sulphuric acid concentration for pretreatment of sweet lime peel were accomplished by Taguchi orthogonal array design. The sweet lime peel was exposed to acid catalyzed steam pretreatment for solid loading [10%, 12%, 15% and 17% (w/v)], time of exposure [15min, 30min, 45min and 60min] and sulphuric acid concentration [0.25%, 0.5%, 0.75% and 1% (v/v)]. The cellulose content was found to be an optimum at 35% for 17% (w/v) solid loading and 0.25% (v/v) acid concentration and steam exposure for 60min. With these optimized process parameters, enzymatic hydrolysis of pretreated sweet lime peel was investigated at 50 °C for 48h using in vitro isolated enzymes, viz., cellulase and pectinase from Aspergillus Niger with an activity of 1.7FPU/ml and15IU/ml respectively. 7.09mg of reducing sugar/ml of hydrolysate was released in enzymatic hydrolysis which was estimated by DNS method. For the production of bioethanol, fermentation of hydrolysate was carried out at 30 °C for 72h using baker's yeast. The yield of ethanol was 18%. From this study, it is proved that citrus waste is a promising source for the production of bioethanol.