Science of the Total Environment 690 (2019) 956–964

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Science of the Total Environment

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Waste valorization: Identification of an ethanol tolerant bacterium

Acetobacter pasteurianus SKYAA25 for acetic acid production from
apple pomace
Alokika Vashisht, Karnika Thakur, Baljinder S. Kauldhar, Vinod Kumar, Sudesh Kumar Yadav ⁎
Center of Innovative and Applied Bioprocessing (CIAB), Sector-81, Knowledge City, Mohali 140306 (PB), India

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Isolated an adapted mesophilic bacteria

that tolerates upto 14% ethanol and 42
°C
• Phenotypic and genotypic characteriza-
tion identified isolate as A. pasteurianus
• The isolate produced acetic acid from
apple pomace to the level of
52.4 g/100 g DM
• Developed simple fermentative process
to convert apple pomace into acetic acid

a r t i c l e i n f o a b s t r a c t

Article history: In present study, a potential bacterial isolate Acetobacter pasteurianus SKYAA25 was found to be very effective in
Received 8 May 2019 the bioconversion of apple pomace to acetic acid. The isolated strain was tolerant to high ethanol concentrations
Received in revised form 2 July 2019 of upto 14% and temperature of 42 °C. Fermentation of apple pomace alone in presence of brewing yeast pro-
Accepted 5 July 2019
duced 7.3% of bio-ethanol which was further used for acetic acid production. Apple pomace in combination
Available online 5 July 2019
with cane molasses produced 14% of bio-ethanol. The fermented bio-ethanol was used as medium for acetic
Editor: Huu Hao Ngo acid production which yielded 52.4 g of acetic acid/100 g of DM (Dry Matter) of apple pomace. Hence, an
ecofriendly process has been developed that is entirely based on microbial processing of apple pomace to pro-
Keywords: duce acetic acid without involving commercial enzymes. The present bio-conversion will prove to be beneficial
Acetobacter pasteurianus for utilizing food and beverage industrial waste in the production of acetic acid.
Apple pomace © 2019 Elsevier B.V. All rights reserved.
Bio-ethanol
Acetic acid
Fermentation

1. Introduction

Acetic acid (ethanoic acid) is a high commercial importance and a

⁎ Corresponding author. key intermediate for value added products in textile industry, food in-
E-mail address: [email protected] (S.K. Yadav). dustry, paint and adhesives (Pal and Nayak, 2017, Raspor and

https://doi.org/10.1016/j.scitotenv.2019.07.070
0048-9697/© 2019 Elsevier B.V. All rights reserved.
 A. Vashisht et al. / Science of the Total Environment 690 (2019) 956–964 957

Goranovič, 2008). Demand of acetic acid is increasing globally. Accord- SKYAA25. The identified bacterial strain was used to develop a simple
ing to the recent report, global acetic acid market is expected to increase process for conversion of bio-ethanol derived from apple pomace to
from CAGR (compound annual growth rates) of 4.30% to 6.8% (Pal and acetic acid.
Nayak, 2017). The Asia pacific region will soon lead the world market
for acetic acid as its demand is rapidly growing in the global food and 2. Materials and methods
textile sector. Approximately, 75% of the acetic acid is produced by syn-
thetic route through carbonylation of methanol and only 10% of world 2.1. Materials
production is achieved via biological route (De Roos and De Vuyst,
2018). The disadvantage of these chemical methods is generation of 2.1.1. Procurement of biomass
by-products and the direct discharge of wastes, leading to severe envi- Apple pomace used in this entire study was collected from The Unati
ronmental pollution. Additionally, the raw materials required for these cooperative marketing cum processing unit, Punjab, India. Apple pom-
processes such as methanol, acetaldehyde or hydrocarbons are derived ace collected was used from one single batch in order to minimize any
from petroleum. Expensive catalysts, downstream separation involving potential interference due to residual composition variation. Cane mo-
evaporation and condensation units to obtain pure acetic acid has fur- lasses used was obtained from Karnal Sugar Industry, Haryana, India.
ther increased its cost of production. Residual chemicals in the final ace-
tic acid are also a major concern among the consumers. 2.1.2. Microorganisms
Although, the production of acetic acid at large scale is achieved Red Star dry yeast and Lalvin yeast EC-1118, two commercially avail-
through synthetic routes involving chemicals, recently production able brewing yeasts were used for conversion of apple pomace to bio-
based upon microbial fermentation is considered to be a potential ethanol.
clean alternative. Furthermore, these fermentation processes have
some fundamental advantages such as potential of using agri/biowaste 2.1.3. Chemicals
as carbon sources, easy and simple process and doesn't produce toxic All the media components were procured from HiMedia and
by-products. The leftovers from the fermentation may either be used chemicals were procured from Sigma-Aldrich (ACS grade). Water used
as manure or cattle feed depending upon the substrate used. One of in all the experiments was purified by Surepro Prefiltration System
the widely used methods for acetic acid production is submerged fer- (Merck Millipore).
mentative process known as German method (Pal and Nayak, 2017).
However, the later process has disadvantage of slow speed and requires 2.2. Sample collection
pure substrate to produce quality acetic acid. While an efficient process
involves a cheap substrate and robust micro-organisms capable of uti- Samples were collected in and around different regions of Punjab,
lizing substrate to produce acetic acid in less time and cost. Earlier at- Haryana and Himachal Pradesh states of India. In total, 26 samples
tempts have been made to find the best cocktail of microbial species were collected which included soil, fruit waste, spoiled fruit juices,
for the production of acetic acid (Mateo et al., 2014; Chen et al., 2016). spoiled pickles and waste from juice industry (Table 1). From the
Acetic acid bacteria are classified into 19 genera in the family established juice vendors, samples were collected from middle layer of
Acetobacteriaceae (Gomes et al., 2018). Most of its members are obligate landfill having waste dump. Whereas, in case of the waste from food &
aerobes and are found in sugar and alcohol enriched environments beverage industry, samples were collected from topmost layer as well
(Mamlouk and Gullo, 2013). Acetic acid bacteria have a distinct metab- as the innermost layer, in order to examine the diversity and microbial
olism that differentiates them from all other bacteria. They possess high population. The soil sample collected from jaggery production unit was
capability to oxidize sugars, sugar alcohols and ethanol to produce ace- taken by slight scrapping the upper layer of the soil. The samples were
tic acid (Gomes et al., 2018). Members of Acetobacter, Gluconobacter, collected using sterile zip lock bags, spatula and falcon tubes. After col-
Gluconoacetobacter are commonly used for acetic acid production at in- lection they were stored at 4 °C until processed.
dustrial scale (Chen et al., 2016).
Some of the effective substrates for acetic acid production at low cost 2.3. Isolation of acetic acid producing microorganism
are agro−/fruit-waste, corn steep liquor, hydrolyzed soy flour, ultra-
filtered ethanol silage and whey lactose (Tiwari et al., 2014, Pal and Screening of 26 samples obtained from different sugar rich niches
Nayak, 2017). India is among the largest producers of fruits like apples was performed for isolation of potential AAB for the production of acetic
and therefore, numerous apple-processing industries are found acid (Table 1). For identification and isolation of AAB different
(Gulhane et al., 2015). The most demanding products from apple in mediaused were GYE (glucose (2–5%), yeast extract (1–3%), ethanol
global market are apple concentrate, apple juice and apple cider vinegar (1–2%)), SM Media (glucose (2%), yeast extract (2%), peptone (1.5%),
(Vendruscolo et al., 2008). Production of these high value products from ethanol (1–2%), acetic Acid (1–2%)), GYC (Glucose (3%), yeast extract
apple leads to production of high amount of waste or pomace. Pomace (3%), calcium carbonate (2%)). Ten grams of sample was taken in
consists of 30% of the original fruit including seed and peal (Bhushan 500 mL flask containing 100 mL of above mentioned media. Flasks
et al., 2008). It is a rich source of dietary fibers, carbohydrates, pectins, with different media and samples were incubated for 15 days. Ethanol
natural oxidants and minerals (Skinner et al., 2018; Shalini and Gupta, was added after every 3 days interval in order to increase the load of
2010). Apple pomace is reported as substrate for production of enzymes acetic acid bacteria. One mL from each flask was further spreaded on
such as pectinases and β-glycosidase, aroma compounds, phenolic com- GYC agar plates. Ten isolates showing halozone on GYC media were se-
pounds, fruity compounds, single cell proteins, heteropolysaccharides, lected and inoculated in 10 mL of Carr media (yeast extract 2%,
citric acid and ethanol (Vendruscolo et al., 2008; Hegde et al., 2018).; bromocresol green 0.02% and ethanol 2%) and incubated at 37 °C for
Pomace is considered to be a potential substrate for production of com- 48 h with 150 rpm. After incubation in liquid media, 100 μL of the inoc-
mercially valuable products because of the availability of high carbohy- ulated media was dissolved in 900 μL of autoclaved water and was
drate and fiber content in the form of cellulose, hemicellulose, pectin, β- spread on Carr media plate.
glucans, gums, and lignin (Skinner et al., 2018). Among these, produc-
tion of acetic acid from apple pomace is one of the potential and bene- 2.4. Estimation of acetic acid production
ficial products. Apple processing industries are unable to fully tap/
capture the potential of apple pomace for the production of acetic acid. The starter culture of each isolated isolate was prepared by incubat-
In view of this, a new potential bacterial strain was identified and ing in GYE medium at 37 °C for 18 h at 150 rpm. The sterilized produc-
characterized as an ethanol tolerant bacterium Acetobacter pasteurianus tion medium containing 20 g/L glucose, 20 g/L yeast extract and 20 mL/L
958 A. Vashisht et al. / Science of the Total Environment 690 (2019) 956–964

Table 1
Sample collection sites.

Sample Location Source Sample Type Production of Carr Amount of acetic acid
No. halozone Media produced
on GYC media (g/100 mL of GYE)

S1 Chandigarh, India Fruit Vendor, Sector 36 Fruit waste – – –

S2 Chandigarh, India Fruit Vendor, Sector 36 Soil sample Yes – –
S3 Chandigarh, India Fruit Vendor, Sector 14 Fruit waste – – –
S4 Chandigarh, India Fruit Vendor, Sector 14 Soil sample – – –
S5 Chandigarh, India Fruit Vendor, Sector 7 Fruit waste Yes – –
S6 Chandigarh, India Fruit Vendor, Sector 7 Soil sample – – –
S7 Chandigarh, India Whole sale vegetable/fruit market, Rotten lemon – – –
Sector 26
S8 Chandigarh, India Whole sale vegetable/fruit market, Citrus fruits waste Yes – –
Sector 26
S9 Chandigarh, India Whole sale vegetable/fruit market, Vinegar – – –
Sector 26
S10 Chandigarh, India Jaggery processing Unit Leftover spoiled juice – – –
S11 Chandigarh, India Jaggery processing Unit Leftover spoiled juice – – –
S12 Chandigarh, India Jaggery processing Unit Soil sample – – –
S13 Chandigarh, India Jaggery processing Unit Soil sample Yes – –
S14 Chandigarh, India Jaggery processing Unit Soil sample Yes Yes 0
S15 Solan, Himachal HPMC apple juice factory outlet Soil sample Yes – –
Pradesh, India
S16 Solan, Himachal HPMC apple juice factory outlet Fruit waste dump Yes Yes 0.2
Pradesh, India
S17 Solan, Himachal HPMC apple juice factory outlet Soil sample – – –
Pradesh, India
S18 Solan, Himachal Fruit/vegetable market Soil sample Yes Yes 0.73
Pradesh, India
S19 Solan, Himachal Fruit/vegetable market Soil sample Yes Yes 13
Pradesh, India
S20 Solan, Himachal Fruit/vegetable market Soil sample – – –
Pradesh, India
S21 Solan, Himachal Fruit/vegetable market Soil sample – – –
Pradesh, India
S22 Chandigarh, India Mango pulp Spoiled by keeping it aside till it started – – –
producing foul smell
S23 Chandigarh, India Orange pulp Spoiled by keeping it aside till it started – – –
producing foul smell
S24 Chandigarh, India Tomato juice Spoiled by keeping it aside till it started – – –
producing foul smell
S25 Chandigarh, India Sugar cane Juice Spoiled by keeping it aside till it started – – –
producing foul smell
S26 Chandigarh, India Mango pickle Spoiled pickle – – –

ethanol and pH 5.5 was inoculated with 5% of starter culture and incu- (Kumar et al., 2018). The evolutionary history was inferred by using
bated at 37 °C for 48 h at 150 rpm. The cell culture was then centrifuged the Maximum Likelihood method and Kimura 2-parameter model
at 4000 g for 10 min at 4 °C. Supernatant obtained was then titrated (Kimura, 1980).
against NaOH as described by Sharafi et al. 2010, in order to determine
the acetic acid production. The potential isolate was selected on basis of 2.6. Standardization of substrate concentration
titration method and was maintained on GYE media with glucose 2%,
yeast extract 2% and ethanol 4%. Apple pomace was dried in oven at 60 °C for 2 days, grounded and
screened to the particle size between 1.4 mm to 1.8 mm. Five grams of
2.5. Phenotypic and genotypic characterization of strain grounded apple pomace used in entire study was also examined for dif-
ferent physical and chemical parameters. In order to standardize the op-
The potential strain designated as SKYAA25 was characterized on timum substrate concentration, different concentrations of apple
the basis of various morphological, physiological and biochemical tests pomace and ethanol were used. To obtain fine slurry, pomace with vary-
using HiMedia identification kits(HiAcinetobacter Identification Kit ing concentration of 2–16% was taken in 250 mL of conical flask and dis-
(KB014), HiCarbo Kit (KB009A)).SKYAA25 strain was characterized ge- solved/diluted with distilled water. Resultant slurry was sterilized by
notypically through 16S rDNA gene sequencing. Total genomic DNA of autoclaving at 121 °C, 15 psi for 20 min and allowed to cool to room tem-
the strain was extracted using MagListo™ 5 M Genomic DNA extraction perature. Bio-ethanol with varying concentration of 2–16% was added to
kit (Bioneer Corporation, Republic of Korea) following manufacturer's the medium before inoculating it with 1 mL of primary culture of
protocol. 16S rDNA region was amplified using primers Forward: 5’ isolatedA.pasteurianusSKYAA25strain. Flasks were further incubated at
AGAGTTTGATCCTGGCTAG 3′; and Reverse: 5’ ACGGCTACCTTGTTAC 37 °C. Samples were taken after every 24 h and estimation of acetic acid
GACCT 3′ as reported earlier (Sharafi et al. 2010). Final product obtained and residual sugar was done. Acetic acid production was measured by
from PCR was analyzed on 1% w/v agarose gel electrophoresis. Amplified using titration method as described earlier (Sharafi et al. 2010).
product was eluted from gel using GenElute™ Gel Extraction Kit
(Sigma-Aldrich,St. Louis, Missouri, United States). The amplified prod- 2.7. Fermentative synthesis of direct acetic acid from pomace
uct was sequenced using Sanger method and sequence obtained was
then subjected to BLAST analysis against NCBI nr database (Altschul For direct conversion of apple pomace to acetic acid using A.
et al., 1990). Evolutionary analyses were conducted in MEGA X pasteurianusSKYAA25, 12% of apple pomace in distilled water was
 A. Vashisht et al. / Science of the Total Environment 690 (2019) 956–964 959

used to make fine slurry in a conical flask. The resultant slurry was ster- 3.1. Isolation of acetic acid producing bacteria
ilized using autoclave (121 °C, 15 psi for 20 min) and allowed to cool to
room temperature. Bio-ethanol (8%) was added to the medium before Acetic acid bacteria can utilize glucose, ethanol, lactate or glycerol as
inoculating it with 1 mL of primary culture of A. pasteurianus energy sources and have been isolated from various alcoholic beverages,
SKYAA25. Flasks were incubated at 37 °C at 180 rpm for 24 h. Acetic vinegar, fruits, sugarcane juices, soil and water (Sharafi et al. 2010). In
acid production was measured by using titration method (Sharafi the present study, acetic acid bacterium was isolated from agro/fruit
et al. 2010). waste and soil samples where fruit waste was dumped, considering
the fact that the microorganisms isolated from such wastes might
2.8. Bio-ethanol production using apple pomace have potential to utilize it more efficiently. Samples were collected
from soil, fruit waste, spoiled fruit juices, pickles and waste from juice
For conversion of apple pomace to acetic acid via bio-ethanol pro- industry. Routine acid production by bacteria in the presence of ethanol
duction, apple pomace (15%) with or without cane molasses (5%) was was shown as a capability of bacteria to produce clear halozone around
used to first produce bio-ethanol using commercial brewing yeasts, in the medium containing calcium carbonate or by the change in color of
Red Star dry yeast and Lalvin yeast EC-1118. Powdered yeast (3%) was indicator bromocresol green in the medium from green to yellow. In-
rehydrated in distilled water and 1% of glucose was added. Incubated order to increase the load of ethanol-tolerant acetic acid bacteria, sam-
at 40 °C for 20 min and was then transferred to MGYP medium (Malt ex- ples were initially incubated in GYC medium supplemented with etha-
tract (0.3%), yeast extract (0.3%), peptone (0.5%), glucose (1%)) at nol at regular intervals as selective stress. During isolation and
130 rpm and 37 °C for 24-48 h. This was further used to inoculate enrichment process, regular and irregular bubbled colonies after 24 h
apple pomace/cane molasses slurry in dried and grounded form as men- of incubation were observed for creamish, yellow, and white color. Of
tioned above. Four different reactions were performed at 30 °C without these, 10 halozone forming colonies on GYC agar plate were selected
any agitation (Supplementary Table 1). Samples were taken after every as candidate for further screening. Halozone formation on GYC medium
24 h for 4continuous days. Collected samples were centrifuged at is one of the most basic and dominant characteristic of acetic acid bacte-
8000 rpm for 15 min and the supernatant was stored at 4 °C until proc- ria (Sengun and Karabiyikli, 2011). Gluconobacter sp. and Acetobacter sp.
essed further. The samples were analyzed on Agilent 1260 Infinity have been reported to produce halozone on GYC media by the acid hy-
HPLC-Chip/MS system using Agilent Hi-Plex H, 7.7 × 300 mm, 8 μm drolysis of CaCO3 present in GYC medium. However, presence of
(p/n PL1170–6830) column for the separation of carbohydrates and or- halozone alone is not completely reliable confirmation method for ace-
ganic acids. The conditions used for HPLC analyses were: column tem- tic acid production as some lactic acid producing bacteria could also
perature 50 °C, detector temperature 30 °C, run time 25 min, isocratic form halozones. Therefore, the selected colonies were further inocu-
flow rate 0.6 mL/min with 0.005 N sulphuric acid as mobile phase/ lated in Carr media which differentiated two genera of Acetobactereacea
solvent. i.e. Gluconobacter and Acetobacter. Carr media is constituted of
bromocresol green and acts as an indicator of acid production (Gomes
et al., 2018). Oxidation of ethanol was resulted in the production of
2.9. Fermentative synthesis of acetic acid via bio-ethanol production acid which has turned the color of medium from green to yellow.
Over-oxidation of acetic acid after prolonged incubation turned color
For converting apple pomace to acetic acid via bio-ethanol produc- back to green. Four isolates (S14, S16, S18 and S19) were found to pro-
tion, apple pomace slurry (12%) was incubated with commercial yeast duce acid as they had converted the color of bromocresol green to yel-
Red Star (3%; as described above) at 30°Cwithout any agitation for4 low after incubation (Table 1). Out of these four isolates, one isolate
days. The supernatant from fermentation of these samples having bio- from Solan (S19), Himachal Pradesh, India (Fig. 2) was selected based
ethanol was inoculated with 1% of A.pasteurianusSKYAA25strain and in- on its high potential to produce acetic acid in GYE media and named
cubated at 37 °C with 180 rpm. Samples were taken at regular interval SKYAA25. The strain A. pasteurianus SKYAA25 was isolated from soil
and analyzed for acetic acid production. Change in various parameters sample where discarded/spoiled apples were thrown. As the strain
such as pH, and amount of acetic acid produced was determined at reg- was isolated from apple pomace enriched soil, it possessed the ability
ular intervals. to utilize apple pomace as a substrate for growth which could be further
used to derive other metabolites/products.
3. Results and discussions
3.2. Characterization of acetic acid producing bacteria
Production strategies are experiencing a shift in favor of green pro-
cesses often involving waste valorization appears imminent. Growing 3.2.1. Physiological characterization of acetic acid producing strain
environmental awareness, implementation of stringent product devel- SKYAA25
opment regulations all over the world and need to utilize waste are During the fermentation process, ethanol resistance and tempera-
the major deriving factors of this paradigm shift. Apple pomace is a po- ture tolerance are two crucial rate limiting factors. Also, high concentra-
tential substrate for the production of various products, but still is tion of ethanol has been reported to negatively affect the growth of
underutilized and often results as waste in open landfill sites causing se- acetic acid producing bacteria. To examine, isolated strain was subjected
rious environmental issues. Acetic acid is widely used in various seg- to ethanol stress ranging from 2 to 16%. The growth profile of the strain
ments of food and beverage industries along with other industries. was determined through spectrophotometric analysis at 600 nm at
Demand of food grade acetic acid as preservative from biological route every 6 h for 2 days. SKYAA25 strain had the ability to grow at a very
is expected to grow exponentially. Apple processing industries are un- high rate when grown at high concentration of ethanol (14%). Earlier,
able to fully tap/capture the potential of apple pomace for the produc- the maximum resistance to ethanol has been reported for an ethanol-
tion of acetic acid as an additional source of income because of the tolerant AAB4 to the extent of 12% (Chen et al., 2016). As the fermenta-
complicated processes which usually involves addition of commercial tion progresses, rise in temperature has been observed due to rapid heat
enzymes. Here, in this study an effort was made to isolate a potential accumulation during the process which was more than the optimal
ethanol tolerant bacterial strain for production of acetic acid from temperature of AAB (Matsushita et al., 2016). To overcome this, the
waste agro-biomass. A simple, clean and green process was developed strain should be thermo-tolerant and be able to survive beyond the op-
for the production of acetic acid from apple pomace by utilizing an iden- timal temperature. In order to find the thermo-tolerance level of
tified thermotolerant A. pasteurianus SKYAA25. Roadmap of the devel- A. pasteurianus SKYAA25, it was grown with varying temperatures
oped process is shown in Fig. 1. from 25 °C to 47 °C. The A. pasteurianus SKYAA25 strain was observed
960 A. Vashisht et al. / Science of the Total Environment 690 (2019) 956–964

Fig. 1. A simple plan for the production of acetic acid from apple pomace. Process involves yeast fermentation of apple pomace and the bio-ethanol produced was converted into acetic acid
by the Acetobacter pasteurianus SKYAA25.

to tolerate upto 42 °C. Whereas, the maximum resistance to tempera- 3.3. Standardization of physiochemical parameters for acetic acid
ture exhibited by AAB4 was 43 °C (Chen et al., 2016). Preliminary iden- production
tification based on biochemical tests showed that the strain was positive
for methyl red test, an established biochemical test for detection of acid The rate of acetic acid production is dependent on initial ethanol
production. Whereas, no activity for β-galactosidase, lysine decarboxyl- concentration, temperature, pH, availability of oxygen, inoculums
ation, orthinine decarboxylation, urease, phenylalanine deamination, amount and concentration of product i.e. acetic acid. Careful selection
nitrate reduction, citrate utilization, acetoin production, indole and and regulation of above parameters can improve acetic acid yield.
H2S production were observed (Supplementary Table 2). This biochem- Therefore, the isolated strain was grown in medium containing pure
ical analysis has confirmed that the isolated strain was non-pathogenic substrate glucose and was examined at varying range of pH, tempera-
in nature and a major character of Acetobacter sp. The isolated strain was ture, time and agitation. The product was analyzed by normal titration
also found to be capable of growing aerobically on different sugars like method. The strain showed maximum production at pH 5.5 (Fig. 4a),
xylose, mannose, melibiose, dextrose and glucose. While, the strain whereas at pH more than 6 acetic acid production was decreased. Tem-
was incapable of utilizing lactose, fructose, galactose, raffinose, treha- perature is one of the significant factors in fermentation; thermo-
lose, sucrose and L-arabinose (Supplementary Table 3). tolerant strains reduce the cooling expenses of fermentation system.
The optimum temperature for most of the AAB for acid production is
30 °C (Chen et al., 2016). Heat accumulation during fermentation was
3.2.2. Molecular characterization of acetic acid producing strain SKYAA25 observed to easily increase this temperature to 37 °C. For strain
As phenotypic characterization alone are not reliable methods for SKYAA25, the optimum temperature for maximum acetic acid produc-
bacterial identification, different molecular techniques such as DNA: tion was 37 °C (Fig. 4b) and could tolerate upto 42 °C while still produc-
DNA hybridization, RFLP or PCR amplified 16S rDNA are routinely ing acetic acid. Acetobacter sp. are strictly aerobic in nature and hence
employed to identify the bacteria. Therefore, 16S rDNA gene sequence agitation plays a major role in the fermentation as well. With
analysis was conducted to characterize this strain. Nucelotide- A. pasteurianus SKYAA25, acetic acid production was found to be maxi-
nucelotide BLAST analysis of sequence obtained showed more than mum at 180 rpm (Fig. 4c) and at 18 h (Fig. 4d).
99% sequence similarity with A. pasteurianus suggesting that the iso- Concentration of different media components and substrate used
lated bacterium belongs to Acetobacter genus and pasteurianus species. such as ethanol, apple pomace glucose and yeast extract concentration
16S rDNA sequence of the strain was then subjected to phylogenetic was also standardized for the maximum production of acetic acid
analysis with the typed strains of other acetic acid producing bacteria (Fig. 5). For this, the strain was grown in glucose and yeast extract sep-
(Fig. 3). The strain formed a separate clade with different strains of arately with varying concentration from 0.5% to 3.5%. Maximum acetic
A. pasteurianus indicating that it belongs to A. pasteurianus. 16S rDNA se- acid production was observed at 2% glucose (Fig. 5a) and at 2.5% yeast
quence of the newly isolated strain has been submitted in NCBI under extract (Fig. 5b). Ethanol is a substrate for acetic acid production but
the accession no: MK592751. This strain has also been deposited in Mi- at high concentration it inhibits the growth of AAB and limits the acid
crobial Type Culture Collection (MTCC) at Institute of Microbial Tech- production. Ethanol is oxidized by AAB in the presence of oxygen to
nology, Chandigarh, India, with accession number MTCC 25206. synthesize acetic acid. A respiratory chain consisting of alcohol

A B

Fig. 2. A) Isolates showing halozone on GYC medium were considered as positive and were selected for further screening on Carr media. B) Halozone producing colonies were streaked on
Carr media. The positive isolate convert the bottle green color of Carr media to sharp yellow color which indicates the production of acid and also differentiates Gluconobacter sp. from
Acetobacter sp.
 A. Vashisht et al. / Science of the Total Environment 690 (2019) 956–964 961

A1

G1
A2

A3

Fig. 3. 16S rDNA gene based phylogenetic analysis of isolated Acetobacter pasteurianus SKYAA25. 16S rDNA gene of typed strains of acetic acid producing bacteria were subjected to
phylogenetic analysis using MEGA X. Evolutionary history was inferred by using the Maximum Likelihood method and Kimura 2-parameter model. The analysis revealed that isolated
Acetobacter pasteurianus SKYAA25 (Acession no: MK592751) showed maximum similarity to other Acetobacter pasteurianus strains (clade A2).

dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and terminal this study was to produce acetic acid by utilizing apple pomace as car-
oxidase localized in the cell membrane of AAB is involved in the produc- bon source. This was achieved by two processes in this study i.e. direct
tion of acetic acid from ethanol. First ethanol is oxidized by membrane and via bio-ethanol production. For direct conversion, apple pomace
bound pyrroloquinoline quinine (PQQ)-dependent alcohol dehydroge- slurry (12%) was taken as sole carbon source for the growth of
nase (ADH) to acetaldehyde. Acetaldehyde is immediately oxidized by A. pasteurianus SKYAA25 to which bio-ethanol (8%) was added. Isolated
membrane bound aldehyde dehydrogenase (ALDH) to acetate. Ethanol strain A. pasteurianus SKYAA25 was able to utilize apple pomace as sub-
concentration in the medium during initial fermentation is also a crucial strate and produced acetic acid upon incubated at 37 °C for 18 h with ag-
factor. Although the A. pasteurianus SKYAA25 strain showed ethanol tol- itation of 180 rpm.
erance upto 14%, but maximum acetic acid production was observed at In the other process, first bio-ethanol was produced using apple
8% and was used further in the study. Additionally, glucose and yeast ex- pomace as substrate for Saccharomyces. Various efforts have been
tract at 2% could be used as an alternative for production of acetic acid made in the past to produce bio-ethanol from apple pomace. These ei-
by isolated A. pasteurianus SKYAA25. ther involved addition of enzymatic cocktails to make the carbohydrate
available for fermentation (Magyar et al., 2016, Parmar and Rupasinghe,
3.4. Fermentative synthesis of acetic acid 2013, Mahawar et al., 2013), physical treatment or co-culturing with
other organism that facilitate the uptake of carbohydrates from pomace
Apple pomace is an abundant, easily available and renewable natural (Evcan and Tari, 2015). Solid state fermentation is one of the widely
resource with immense commercial potential in bio-ethanol/acetic acid used processes to produce bio-ethanol (Hang et al., 1982). Here in this
production. The use of agro-industrial waste to produce bio ethanol/ study, a simple process was adopted using only commercially available
acetic acid have advantage as it does not compete with food crops rather yeast strains, Red Star dry yeast and Lalvin yeast EC-1118 without in-
reduces the environmental pressure caused by the disposal of such volvement of hydrolytic enzymes. Four different types of reactions
wastes. As the strain A. pasteurianus SKYAA25 was isolated from soil were examined for the production of ethanol from apple pomace. Reac-
sample where discarded/spoiled apples were thrown, it possessed the tion 1 and 2 involved the use of apple pomace (15%) and commercial
ability to utilize apple pomace as a substrate. Apple pomace has 84.7% brewing yeasts LalvinEC-1118 and Red Star respectively. While reaction
of carbohydrate and reducing sugars amounting to 10.8% to 15.0% db 3 and 4 involved apple pomace (15%) along with cane molasses (5%)
depending upon the variety of apple and processing conditions used. and commercial brewing yeasts LalvinEC-1118 and Red Star respec-
Among these sugars, the most abundant is fructose (23.6%), followed tively. Molasses was added in addition to apple pomace to bring the re-
by glucose (22.7%), arabinose (14% to 23%), galactose (6% to 15%) and ducing sugar in right proportion. Reaction type 4 produced maximum
xylose (1.1%). These sugars can be converted to bio-ethanol using amount of ethanol to the level of 14% in 48 h (Supplementary Fig. 1). Al-
yeast which could further be used to produce acetic acid. The aim of though maximum ethanol production was achieved in reaction 4, still it
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Fig. 4. Acetic acid production by Acetobacter pasteurianus SKYAA25 strain examined at varying range of pH, temperature, time and agitation A) Acetic acid production at varying pH range
(4–7). Maximum acetic acid production was observed at pH 5.5. B) Acetic acid production at varying temperature range (25 °C -47 °C). Maximum acetic acid production was observed at 37
°C . C) Acetic acid production at varying agitation (100 rpm −200 rpm). Maximum acetic acid production was observed at 180 rpm. D) Acetic acid production at different timepoints in
hours (0 h- 48 h). Maximum acetic acid production was observed at 24 h.

was not preferred to produce the acetic acid in the final process. Since concentration of 52.4 g/100 g DM of apple pomace at temperature 37
the isolated strain was observed to produce maximum acetic acid in °C in 24 h. Additionally, the capability of this strain was also tested to
the presence of 8% ethanol, reaction 2 having apple pomace with Red utilize apple pomace as a carbon substrate for its growth. If industry is
Star was chosen for further study. Ethanol production in reaction 2 already producing bio-ethanol from some other sources then they can
was 7.3% and was close to the optimal concentration of ethanol for ace- utilize that bio-ethanol in the production of acetic acid using apple pom-
tic acid production. Once the concentration of ethanol reached satura- ace. Importantly, the isolated strain A. pasteurianus SKYAA25 was found
tion phase, A. pasteurianus SKYAA25 strain was added in the same to be able to produce acetic acid when grown in 12% apple pomace
medium. Glucose and xylose sugars of apple pomace might be serving slurry supplemented with 8% bio-ethanol.
as energy source for the isolated strain. In order to make the process Though the acetic acid production rate is low compared to chemical
cost efficient for industries and eco-friendly, no other additions were route, but it provides advantage and sustainability due to restricted or
made. Acetic acid production was carried out in fermented broth of no toxic waste disposal and reduced greenhouse gases. The leftover
apple pomace containing ethanol. From 100 g DM (Dry Matter) of after fermentation could be either used as cattle feed or manure. An-
apple pomace, 52.4 g of acetic acid was produced in 24 h. The decrease other advantage of this process over chemical route is ease of acetic
in pH of the medium indicated the conversion of ethanol to acetic acid acid separation and purification from fermentation broth. Acetic acid
by A. pasteurianus SKYAA25. can be easily separated by using membranes without disturbing the mi-
In a similar study, apple pomace was treated with enzymatic cocktail crobes used in the fermentation process. Whereas, acetic acid produced
(cellulase, 43 units; pectinase, 183 units; b-glucosidase, 41 units/g dry through chemical route requires energy-intensive purification. Most of
matter) to make available the fermentable sugars present in the form the earlier reported processes for acetic acid production requires costly
of cellulose and pectin in pomace and to convert these sugars into eth- enzymes, pure substrates and use of micro-organisms incapable to tol-
anol (Parmar and Rupasinghe, 2013). The produced ethanol was further erate high amount of ethanol. Here, a simple and green process was de-
converted to acetic acid using commercially available Acetobacter aceti veloped for acetic acid production from apple pomace using a newly
strain. This bioconversion was resulted in the production of acetic acid identified thermotolerant A. pasteurianus SKYAA25.
at a concentration of 61.4 g/100 g DM at temperature 40 °C in 24 h.
However, the use of enzymatic cocktail might increase the cost of pro- 4. Conclusions
duction of acetic acid. In the present study, a simple process without
the addition of enzymatic cocktail was used to produce acetic acid. A process of bio-conversion of apple pomace to acetic acid has been
Apple pomace was subjected to fermentation without any prior treat- developed using an ethanol–tolerant (upto 8%) and thermo-tolerant
ment, and hence could be cost effective. The bio-ethanol produced dur- (upto 42 °C) A. pasteurianus SKYAA25. Reported strain was isolated
ing this present process was sufficient enough to be converted into from apple pomace rich soil and hence could utilize apple pomace as
acetic acid. In current process, acetic acid was produced to the substrate for growth. Apple pomace (12%) fermented with 3%
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Fig. 5. Optimization of substrate concentration for acetic acid production. A) Acetic acid production at varying glucose concentration (0.5 to 3.5%). Maximum acetic acid production was
observed at 2% glucose concentration. B) Acetic acid production at varying yeast extract concentration (0.5 to 3.5%). Maximum acetic acid production from yeast extract was observed at
2.5%.C) Acetic acid production at varying ethanol concentration (2 to 16%). Maximum acetic acid production was observed at 8% ethanol concentration. D) Acetic acid production at varying
apple pomace concentration (2 to 16%). Maximum acetic acid production from apple pomace was observed at 12%.

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Appendix A. Supplementary data
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