Environmental Technology

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Isolation and characterization of novel bacterial

strain present in a lab scale hybrid UASB reactor
treating distillery spent wash

R. Thiyagu & P. Sivarajan

To cite this article: R. Thiyagu & P. Sivarajan (2018): Isolation and characterization of novel
bacterial strain present in a lab scale hybrid UASB reactor treating distillery spent wash,
Environmental Technology, DOI: 10.1080/09593330.2018.1473499

To link to this article: https://doi.org/10.1080/09593330.2018.1473499

Accepted author version posted online: 04

May 2018.
Published online: 15 May 2018.

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ENVIRONMENTAL TECHNOLOGY
https://doi.org/10.1080/09593330.2018.1473499

Isolation and characterization of novel bacterial strain present in a lab scale

hybrid UASB reactor treating distillery spent wash
R. Thiyagu and P. Sivarajan
Department of Civil Engineering, Annamalai University, Annamalai Nagar, India

ABSTRACT ARTICLE HISTORY

Recalcitrant compounds degrading novel bacteria was isolated from lab scale hybrid UASB reactor Received 22 September 2017
treating distillery spent wash, enriched with biomass activity. The granules were subjected to SEM Accepted 29 April 2018
analysis to classify and isolate the bacterial strains. The strain was ubiquitous, mesophilic, gram-
KEYWORDS
negative, motile, non-spore forming and cultivable optimally at 30°C at pH 7. Most potential isolates 16S rRNA gene sequence;
from the strains were subjected to 16S rRNA sequencing and branded as a member of diverse distillery spent wash; hybrid
genera, gamma-proteobacteria, Stenotrophomonas sp. Based on 16S rRNA sequencing, genomic UASB reactor; recalcitrant
DNA extraction, PCR amplification, and phylogeny analysis the gram-negative bacteria was compounds;
identified as Stenotrophomonas maltophilia and found to degrade distillery spent wash. The hybrid Stenotrophomonas
UASB reactor was operated for 360 days with 24 h HRT and has an optimum COD removal maltophilia
efficiency of 83.87% at an organic loading rates (OLRs) ranging within 0.25–27.40 kg COD/m3 d.

Abbreviations: 16S rRNA: 16S ribosomal RNA; COD: Chemical oxygen demand; DNA: Deoxyribonucleic
acid; dS/m: deciSiemens per metre; g/L: gram per litre; HRT: Hydraulic retention time; mg/L: milligram
per litre; OLR: Organic loading rate; PCR: Polymerase chain reaction; RNA: Ribonucleic acid; UASB:
Upflow anaerobic sludge blanket; VFA: Volatile fatty acid; VSS: Volatile suspended solid

Introduction
colouring compounds such as melanoidins, metal sul-
Distillery industries release approximately about 8–15 L phides and phenolics which are mainly responsible for
of spent wash for the production of each litre of the dark brown colour of distillery wastewater [4]. The
alcohol. They are more complex, organic and are recalci- colour of wastewater does not allow penetration of
trant compounds which leads to pollution when dis- sunlight, causes deleterious effects on aquatic flora
charged into the surroundings [1,2], poses a strong and fauna [5,6] and causes serious soil and water pol-
environmental risk to flora and fauna in the ecosystem lution by inducing colouration and eutrophication pro-
and may impact biota and benthic organisms [3]. It is blems in aquatic environments. This in turn decreases
highly acidic in nature and has a diversity of recalcitrant both photosynthetic activity and dissolved oxygen

CONTACT R. Thiyagu [email protected]; P. Sivarajan [email protected] Department of Civil Engineering, Annamalai University,
Annamalai Nagar, 608002, India
© 2018 Informa UK Limited, trading as Taylor & Francis Group
2 R. THIYAGU AND P. SIVARAJAN

concentration, affecting the normal life cycle. On land it Nutrients

causes a reduction in soil alkalinity, inhibition of seed
The nutrients for biomass growth contained the sub-
germination and damage to vegetation [7–9]. The treat-
sequent composition (g/L) of NH4Cl - 0.5, K2HPO4 -
ment of distillery wastewater by physical or chemical
0.25, MgCl2 6H2O - 0.3, CaCl2-0.005, CuCl2 - 0.0105 was
methods is not feasible due to the high cost and gener-
added to the substrate to maintain a C:N:P ratio of
ation of secondary pollutants. Moreover, it is also
100:5:1 [21]. All chemicals were of analytical grade.
complex to treat it by conventional biological methods
[8,10], as melanoidins have antioxidant properties that
are toxic to aquatic macro and microorganisms [7]. Physico-chemical characteristics
Therefore, a complementary treatment process is
required to remove the colour and the residual COD. The characteristics of distillery spent wash, spent wash
So far, there has been a limited success in the exploration diluted with 75% water and 50% water were studied.
of bacteria and fungi which can able to degrade recalci- The samples were examined for various physico-chemi-
trant compounds in order to reduce the colour and cal parameter [22] such as odour, colour, pH, conduc-
organic load of anaerobically digested molasses spent tivity, chemical oxygen demand (COD), calcium,
wash [11]. [12] reported that the isolated strain of chlorides, magnesium, phosphate, potassium, sulphates,
Nostoc muscorum, belonging to the phylum Cyanobac- total solids (TS), total dissolved solids (TDS), total sus-
teria found in the distillery wastewater could be possibly pended solids (TSS), and total hardness etc.
used for the biological treatment. [13] reported that
Paracoccus pantotrophus SAG1 strain could effectively Batch process
decolourize the distillery spent wash. Moreover various
bacterial groups, such as Lactobacillus Plantarum [14], Batch experiment was carried out in a 1000 ml saline bottle
Bacillus licheniformis, Bacillus sp., Alcaligenes sp. [4], Kleb- with a working volume of 500 ml. The effect of dilution (dis-
siella oxytoca, Serratia marcescens, Citrobacter sp. [15], tilled water:distillery spent wash) at various mixing ratios of
Pseudomonas aeruginosa PAO1, Proteus mirabilis [16], 50:50, 60:40, 70:30, 80:20, and 90:10 were studied. The pH
and Pseudomonas putida, Aeromonas sp., are reported value was maintained within 6.5 to 7.5 in the reactor using
as potential organisms to treat distillery wastewater 1N HCl or 1N NaOH. Anaerobic digested sludge of 234 g
[17]. However, studies on the molecular identification VSS/L was inoculated under aseptic conditions and incu-
of the microbial communities in anaerobic treatment bated at 30 ± 5°C. Nutrients and glucose were added
processes based on cloning and analysis of the nucleo- initially to enhance the biomass growth [23].
tide sequences of genes encoding the 16S ribosomal
RNA (16S rRNA) are quite limited [18]. Hence, there is a Experimental study on hybrid UASB reactor
requirement to discover a techno-economically feasible
solution with the knowledge of the importance of pure bac- Experiment was also performed using a hybrid UASB reactor
terial cultures and their probable use in the treatment of for treating distillery wastewater at various OLR and the
industrial wastewater [19]. The purpose of the present extent of degradation was monitored by % COD removal
report is to isolate and characterize a novel bacterial strain and VSS Concentration in the reactor. The bacteria respon-
by 16S rRNA gene sequencing based on nucleotide hom- sible for degradation based on the % COD removal and cor-
ology and phylogenetic analysis from the anaerobically responding biomass growth was isolated and identified.
digested sludge of distillery industry and could be probably
used in the treatment of distillery industrial wastewater.
Isolation of bacteria
Media preparation
Materials and methods Nutrient agar medium for culture of bacteria containing
Peptone 5.0 g/L, Sodium chloride5.0 g/L, Beef extract
Source of sample
1.5 g/L, Yeast extract 1.5 g/L and Agar 15.0 g/L
Distillery industry waste, both aqueous effluent and thoroughly mixed in 1000 ml pure distilled water main-
sludge samples were collected as per standard pro- tained at pH 7.4 was prepared and sterilized at 121°C
cedure [20] from an alcohol manufacturing industry, with 15 lbs pressure for 15 min in an autoclave [24].
Cuddalore, India and stored in containers at 4°C. The
anaerobic sludge of a lab-scale hybrid UASB reactor Serial dilution and agar plating
treating aqueous distillery effluent was also collected Isolation of bacterial strains degrading distillery spent
from the reactor and stored as per norms. wash was carried by serial dilution method. Sample
 ENVIRONMENTAL TECHNOLOGY 3

solution were serially diluted and were aseptically trans- Sequencing of 16S rRNA
ferred via sterile pipettes into nutrient agar plates, the 16S rRNA amplified fragments were purified using QIA
processes were performed in a laminar air flow to quick gel extraction kit to agarose gel in a sequence
sustain a sterile environment. Nutrient agar bacterial using automated ABI PRISM 3730 genetic analyzer.
plates were prepared through pour plate and streak The partial sequences were subjected to BLAST analy-
plate techniques. The agar plates were made airtight sis using the online option available at www.ncbi.nlm.
with covering its sides by means of a parafilm and nih.gov/BLAST [28] suggesting the identity of the
were incubated for 24 h at room temperature so as to isolates [29].
obtain colonies. The colonies were streaked onto the
plates containing nutrient agar media so as to promote Phylogeny analysis
the growth of species predominantly. The plates were The molecular characterization of isolated bacterial strain
incubated for 24 h at 32°C in a sterile environment [25]. was studied by the method of 16S rRNA sequencing
technique. The amplified 16S rDNA sequences were
Isolation of pure bacterial cultures compared with the nucleotide sequences present in
The bacterial colonies on the agar plate having excellent the Gene Bank using the standard BLASTN site at NCBI
growth were morphologically characterized and purified server (http://www.ncbi.nlm.nih.gov/BLAST) [30]. The
by repeated culturing. The medium was incubated at 32° alignments of the sequences were done using the CLUS-
C, so as to nurture the bacteria [26]. TALW programme (version 1.8.3). Distances Kimura’s two
parameter correction, phylogeny tree by neighbour
joining, bootstrap analysis and 1000 replications are
Identification of bacterial strain carried out [31].
Potent growth colonies from the plate were diluted in
Phosphate buffered saline and centrifuged to obtain Results and discussion
pellets, extraction of bacterial genomic DNA was
carried by cell lysis from these pellets and a single Physico-chemical characteristics
DNA strand was mined by incubation, 5 μl of the The characteristics of distillery spent wash, spent wash
freshly extracted DNA on 3 μl of gel loading dye was diluted with 75% water and 50% water were studied.
loaded on top of 1% agarose gel and subjected to Table 1 highlights the results of the analysis of various
electrophoresis. After electrophoresis the samples characteristics of raw spent wash and diluted spent
were mixed and loaded into the column, the process wash at various levels. As the dilution ratio increases
was repeated until a single DNA sample was com- there was no effect on colour and still the presence of
pleted [26]. brown pigmentation existed. It reduces light penetration
in water bodies as lakes, rivers or lagoons which in turn
PCR amplification analysis affect the aquatic life [32]. The pH value of raw and
Purified DNA was subjected to PCR amplification for diluted spent wash was acidic. The lower pH of the efflu-
magnifying a specific fragment of DNA of interest by a ent may be due to the presence of weak organic acids
series of successive cycles. PCR amplification was [33]. The electrical conductivity, TDS, TSS and TS of
carried out as, in a total volume of 50 µl of a 0.2 ml
PCR tubes. Amplification was followed by the condition Table 1. Characteristics of distillery spent wash and diluted spent
of an initial denaturation for 2 min at 94°C sufficient for wash.
most amplicons from pure DNA templates. For longer Parameters Dilution 75% Dilution 50% Spent wash
cycles denaturation at 94°C for 30 s, annealing at 58°C Colour Light brown Brown Dark brown
for 30 s, extension at 72°C for 1 min 30 s, final extension Odour Offensive Offensive Unpleasant
pH 4.0 3.9 3.8
at 72°C for 5 min and finally hold up to 4°C in a thermo- Conductivity dS/m 18.1 29.2 45.5
cycler [27]. The PCR products obtained from DNA extrac- Total solids mg/L 33558 74220 140260
Total dissolved solids mg/L 26542 57840 112400
tion from samples were first analysed by electrophoresis Total suspended solids mg/L 7016 16380 27860
on 1% agarose gel and was stained with ethidium Total hardness mg/L 5500 14400 23400
COD mg/L 92650 114480 162000
bromide and visualized under short-wavelength UV Calcium mg/L 684 1692 2975
light. Amplification of the 16S rRNA gene was performed Chlorides mg/L 3954 7806 10650
Magnesium mg/L 788 1436 2380
using the primers. Forward primer: 5′ -AGAGTTT- Potassium mg/L 3823 7120 10820
GATCCTGGCTCAG-3′ and Reverse primer: 5′ -ACGGC- Phosphate mg/L 840 1400 2100
TACCTTGTTACGACTT-3′ . Sulphates mg/L 690 1785 3015
4 R. THIYAGU AND P. SIVARAJAN

spent wash was high. The concentration of solids in

spent wash decreased with increased dilution rate. The
high conductivity of the effluent may be due to the pres-
ence of high concentration of dissolved salts [34]. COD
concentration of raw effluent was high and decreased
with dilution. High values of COD might be due to the
presence of higher amounts of organic compounds in
distillery effluents [34]. The total hardness, calcium, mag-
nesium chloride and phosphate concentration of the
spent wash was high and decreased with dilution.
Higher concentration of calcium and magnesium has
also been reported by [35]. Higher concentration of
chloride and phosphate has also been reported by [36].
The potassium of the spent wash was very high as
10820 mg/L. Higher concentration of potassium has Figure 2. Influence of OLR on biomass growth.
also been reported by [37]. The raw distillery effluent
also contains a high concentration of sulphates. [38]
reported the higher concentration of sulphates in
distillery effluent.

Batch experiment
Based on the analysis of the batch experiment inoculated
with the sludge samples with various ratios of diluted
spent wash viz. 50:50, 60:40, 70:30, 80:20, and 90:10.
Initially, the substrate was maintained in the pH range
in between 6.5 to 7.5 at 30 ± 5°C. The COD removal per-
centage was varying viz. 61, 63, 76, 64 and 63%. The vari-
ation in performance efficiency in the experiment may
be due to higher concentration and low dilution of sub-
strate and because of pH deflection, methanogenic Figure 3. Electron micrographs of sludge granules.
activity were affected [23] and for 70:30 ratio the
removal efficiency was high may be due to optimum
dilution and optimum substrate concentration for the
biomass to have higher methanogenic activity to
degrade the distillery spent wash [39].

Figure 1. Influence of OLR on % COD removal. Figure 4. Microorganism culture on nutrient agar medium.
 ENVIRONMENTAL TECHNOLOGY 5

of 90–95% was achieved during this stage. After the

acclimatization phase, the reactor was operated for 280
days at various OLRs of 3.4, 4, 4.8, 6.3, 8.2, 10.5, 12,
14.8, 17, 20, 22.3, and 27.4 kg COD/m3 d at 24 h HRT.
The average COD removal efficiency of various OLRs
are viz. 88.33, 84.55, 86.15, 80, 79.09, 77.39, 80.92,
74.88, 72.32, 70.98, 70.08 and 70.71. As the OLR has
increased the removal efficiency has also gradually
increased [40] and a further increase in OLR, the
removal percentage slightly decreased. At lower OLRs
the microbes suffered from insufficient organic content
for the conversion to biogas production. Figure 1
shows the influence of organic loading rate on removal
percentage of COD. The stable function of a reactor
due to self-regulation capability is natural in the biologi-
cal system, building it promising for the microbial con-
sortium to acclimate itself to the increased OLRs [41].
Figure 2 shows the influence of OLR on biomass growth.

Figure 5. Isolated bacteria obtained by streak plate technique.

Isolation and identification of microbes from the
effluent
Performance of hybrid UASB reactor
Microscopic examination of granules
A hybrid UASB reactor was used for treating the distillery
Anaerobic digested seed sludge from a hybrid UASB
spent wash. Anaerobically digested sludge collected
reactor was taken and the granules were examined
from a distillery unit was used as inoculum. The efficiency
under SEM to understand the morphology and the
of the reactor was evaluated by COD removal percentage
nature of microbial species [42]. Figure 3 shows the
and biomass growth. Initially, during the acclimatization
observation of granules with 5000 times magnification
stage the reactor was operated at 24 h HRT for 80 days
with cylindrically shaped microbes.
with the organic loading rate ranging between 0.25
to 2.30 kg COD/m3 d. Maximum COD removal efficiency
Cell morphology
Table 2. Morphological characteristics of the bacterial isolate. The cell morphology of the bacterial isolates in nutrient
Morphological DSM 50170T agar plate showed that within 24–48 h of incubation
Colony colour White the bacterial isolate grew to form intense white colonies
Gram nature Negative (2–3 mm in diameter) having entire margin and smooth
Cell morphology Flagellated bacillus
Motility Positive consistency (Figures 4 and 5). Gram staining of bacterial
Colony shape Rod cells revealed the presence of gram-negative bacteria as
Spore formation Negative
shown in Table 2. The bacterial colonies appearing on

Figure 6. Phylogenetic tree of Stenotrophomonas maltophilia.

6 R. THIYAGU AND P. SIVARAJAN

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Acknowledgements [cited 2017 May 1]. Available from: http://linkinghub.
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2015.12.016.
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of molasses based distillery wastewater using a bacterial
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consortium. Sci Asia. 2009;35(4):332–339.
[16] Mohana S, Desai C, Madamwar D. Biodegradation and
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